U.S. patent application number 13/219440 was filed with the patent office on 2013-01-03 for methods and compositions for synthesis of nucleic acid molecules using multiplerecognition sites.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. Invention is credited to Michael A. Brasch, Devon R.N. Byrd, John Carrino, David Cheo, Jonathan D. CHESNUT, James Fan, Martin A. G. Gleeson, James L. Hartley, Louis Leong, Knut Madden, Gary F. Temple.
Application Number | 20130004996 13/219440 |
Document ID | / |
Family ID | 46300964 |
Filed Date | 2013-01-03 |
United States Patent
Application |
20130004996 |
Kind Code |
A1 |
CHESNUT; Jonathan D. ; et
al. |
January 3, 2013 |
METHODS AND COMPOSITIONS FOR SYNTHESIS OF NUCLEIC ACID MOLECULES
USING MULTIPLERECOGNITION SITES
Abstract
The present invention provides compositions and methods for
recombinational cloning. The compositions include vectors having
multiple recombination sites and/or multiple topoisomerase
recognition sites. The methods permit the simultaneous cloning of
two or more different nucleic acid molecules. In some embodiments
the molecules are fused together while in other embodiments the
molecules are inserted into distinct sites in a vector. The
invention also generally provides for linking or joining through
recombination a number of molecules and/or compounds (e.g.,
chemical compounds, drugs, proteins or peptides, lipids, nucleic
acids, carbohydrates, etc.) which may be the same or different. The
invention also provides host cells comprising nucleic acid
molecules of the invention or prepared according tb the methods of
the invention, and also provides kits comprising the compositions,
host cells and nucleic acid molecules of the invention, which may
be used to synthesize nucleic acid molecules according to the
methods of the invention.
Inventors: |
CHESNUT; Jonathan D.;
(Carlsbad, CA) ; Carrino; John; (San Diego,
CA) ; Leong; Louis; (Mission Viejo, CA) ;
Madden; Knut; (Carlsbad, CA) ; Gleeson; Martin A.
G.; (San Diego, CA) ; Fan; James; (Carlsbad,
CA) ; Brasch; Michael A.; (Gaithersburg, MD) ;
Cheo; David; (Kensington, MD) ; Hartley; James
L.; (Frederick, MD) ; Byrd; Devon R.N.;
(Fredericksburg, VA) ; Temple; Gary F.;
(Washington Grove, MD) |
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
46300964 |
Appl. No.: |
13/219440 |
Filed: |
August 26, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11612445 |
Dec 18, 2006 |
8030066 |
|
|
13219440 |
|
|
|
|
10792035 |
Mar 4, 2004 |
7198924 |
|
|
11612445 |
|
|
|
|
10454793 |
Jun 5, 2003 |
|
|
|
10792035 |
|
|
|
|
10005876 |
Dec 7, 2001 |
7244560 |
|
|
10454793 |
|
|
|
|
10014128 |
Dec 7, 2001 |
7033801 |
|
|
11612445 |
|
|
|
|
09732914 |
Dec 11, 2000 |
7393632 |
|
|
10014128 |
|
|
|
|
60385613 |
Jun 5, 2002 |
|
|
|
60333124 |
Nov 27, 2001 |
|
|
|
60318902 |
Sep 14, 2001 |
|
|
|
Current U.S.
Class: |
435/91.41 ;
435/188; 435/91.5; 536/23.1 |
Current CPC
Class: |
C12N 2800/30 20130101;
C12Y 599/01 20130101; C12N 15/64 20130101; C12N 15/66 20130101;
C12N 2840/20 20130101; C12P 19/34 20130101; C12N 9/90 20130101;
C12N 15/902 20130101; C12N 15/10 20130101; C12N 2800/70 20130101;
C12N 2800/108 20130101 |
Class at
Publication: |
435/91.41 ;
536/23.1; 435/188; 435/91.5 |
International
Class: |
C12P 19/34 20060101
C12P019/34; C12N 9/96 20060101 C12N009/96; C12N 15/66 20060101
C12N015/66; C07H 21/04 20060101 C07H021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 5, 2003 |
US |
PCT/US2003/018036 |
Claims
1. An isolated nucleic acid molecule comprising: (a) one or more
recombination sites; and (b) one or more topoisomerase recognition
sites and/or one or more topoisomerases.
2. The nucleic acid molecule of claim 1, wherein said nucleic acid
molecule is a circular molecule.
3. The nucleic acid molecule of claim 1, wherein said nucleic acid
molecule comprises two or more recombination sites.
4. The nucleic acid molecule of claim 1, wherein said recombination
sites are selected from the group consisting of: (a) attB sites,
(b) attP sites, (c) attL sites, (d) attR sites, (e) lox sites, (f)
psi sites, (g) dif sites, (h) cer sites, (i) frt sites, and
mutants, variants, and derivatives of the recombination sites of
(a), (b), (c), (d), (e), (f), (g), (h) or (i) which retain the
ability to undergo recombination.
5. The nucleic acid molecule of claim 1, wherein said topoisomerase
recognition site is recognized and bound by a type I
topoisomerase.
6. The nucleic acid molecule of claim 5, wherein said type I
topoisomerase is a type IB topoisomerase.
7. An in vitro method of cloning a nucleic acid molecule
comprising: (a) obtaining a first nucleic acid molecule to be
cloned; (b) mixing said first nucleic acid molecule to be cloned in
vitro with a second nucleic acid molecule comprising at least a
first topoisomerase recognition site flanked by at least a first
recombination site, and at least a second topoisomerase recognition
site flanked by at least a second recombination site, wherein said
first and second recombination sites do not recombine with each
other, and at least one topoisomerase; and (c) incubating said
mixture under conditions such that said first nucleic acid molecule
to be cloned is inserted into said second nucleic acid molecule
between said first and second topoisomerase recognition sites,
thereby producing a first product molecule comprising said first
nucleic acid molecule to be cloned between said first and second
recombination sites.
8. The method of claim 7, wherein the second nucleic acid molecule
is a vector.
9. The method of claim 7, wherein said first nucleic acid molecule
to be cloned is a linear nucleic acid molecule.
10. The method of claim 7, further comprising contacting said first
product molecule with at least one third nucleic acid molecule
comprising at least a third and fourth recombination sites that do
not recombine with each other, under conditions favoring
recombination between said first and third and between said second
and fourth recombination sites, thereby producing at least one
second product molecule.
11. The method of claim 10, wherein the third nucleic acid molecule
is a vector.
12. The method of claim 7, wherein said second nucleic acid
molecule comprises at least one additional nucleic acid sequence
selected from the group consisting of a selectable marker, a
cloning site, a restriction site, a promoter, an operator, an
operon, an origin of replication, and a gene or partial gene.
13. The method of claim 10, wherein said third nucleic acid
molecule comprises at least one additional nucleic acid sequence
selected from the group consisting of a selectable marker, a
cloning site, a restriction site, a promoter, an operator, an
operon, an origin of replication, and a gene or partial gene.
14. The method of claim 7, wherein said first and second
recombination sites are selected from the group consisting of: (a)
attB sites, (b) attP sites, (c) attL sites, (d) attR sites, (e) lox
sites, (f) psi sites, (g) dif sites, (h) cer sites, (i) frt sites,
and mutants, variants, and derivatives of the recombination sites
of (a), (b), (c), (d), (e), (f), (g), (h) or (i) which retain the
ability to undergo recombination.
15. The method of claim 10, wherein said third and fourth
recombination sites are selected from the group consisting of: (a)
attB sites, (b) attP sites, (c) attL sites, (d) attR sites, (e) lox
sites, (f) psi sites, (g) dif sites, (h) cer sites, (i) frt sites,
and mutants, variants, and derivatives of the recombination sites
of (a), (b), (c), (d), (e), (f), (g), (h) or (i) which retain the
ability to undergo recombination.
16. The method of claim 7, wherein said topoisomerase is a type I
topoisomerase.
17. The nucleic acid molecule of claim 16, wherein said type I
topoisomerase is a type IB topoisomerase.
18. The method of claim 10, wherein said product nucleic acid
molecule and said third nucleic acid molecule are combined in the
presence of at least one recombination protein.
19. The method of claim 18, wherein said recombination protein is
selected from the group consisting of: (a) Cre; (b) Int; (c) IHF;
(d) X is; (e) F is; (f) Hin; (g) GIn; (h) Cin; (i) Tn3 resolvase;
(j) TndX; (k) XerC; and (l) XerD.
20. A kit comprising the isolated nucleic acid molecule of claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 10/792,035 filed Mar. 4, 2004, which is a
continuation of U.S. application Ser. No. 10/454,793, filed Jun. 5,
2003, which claims the benefit of the filing date of U.S.
provisional patent application No. 60/385,613, filed Jun. 5, 2002.
This application U.S. application Ser. No. 10/454,793 also is a
continuation-in-part of U.S. application Ser. No. 10/005,876, filed
Dec. 7, 2001, which claims the benefit of the filing dates of U.S.
provisional application Nos. 60/333,124, filed Nov. 27, 2001, and
60/318,902, filed Sep. 14, 2001, This application also is a
continuation-in-part of U.S. application Ser. No. 10/014,128, filed
Dec. 7, 2001, and of U.S. application Ser. No. 09/732,914, filed
Dec. 11, 2000. The disclosures of all of the above-referenced
applications are specifically incorporated herein by reference in
their entireties.
[0002] U.S. patent application Ser. No. 09/935,280, filed Aug. 21,
2001, and U.S. provisional patent application No. 60/326,092, filed
Sep. 28, 2001, 60/291,972, filed May 21, 2001, 60/254,510, filed
Dec. 8, 2000, and 60/226,563, filed Aug. 21, 2000, also are
specifically incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to the fields of biotechnology
and molecular biology. In particular, the present invention relates
to joining multiple nucleic acid molecules containing one or more
recombination sites and/or one or more topoisomerase recognition
sites. The present invention also relates to cloning such joined
nucleic acid molecules using recombinational cloning methods such
as those employing topoisomerase and/or recombination proteins. The
invention also relates to joining multiple peptides, and
combinations of peptides and nucleic acid molecules through the use
of recombination sites and/or topoisomerase recognition sites.
Other molecules and compounds or combinations of molecules and
compounds may also be joined through recombination sites and/or
topoisomerase recognition sites according to the invention.
[0005] Such peptides, nucleic acids and other molecules and/or
compounds (or combinations thereof) may also be joined or bound
through recombination reactions and/or through topoisomerase
joining reactions to one or a number of supports or structures in
accordance with the invention.
[0006] 2. Related Art
[0007] Site-Specific Recombinases
[0008] Site-specific recombinases are proteins that are present in
many organisms (e.g. viruses and bacteria) and have been
characterized as having both endonuclease and ligase properties.
These recombinases (along with associated proteins in some cases)
recognize specific sequences of bases in a nucleic acid molecule
and exchange the nucleic acid segments flanking those sequences.
The recombinases and associated proteins are collectively referred
to as "recombination proteins" (see, e.g., Landy, A., Current
Opinion in Biotechnology 3:699-707 (1993)).
[0009] Numerous recombination systems from various organisms have
been described. See, e.g., Hoess, et al., Nucleic Acids Research
14(6):2287 (1986); Abremski, et al., J. Biol. Chem. 261(1):391
(1986); Campbell, J. Bacteriol. 174(23):7495 (1992); Qian, et al.,
J. Biol. Chem. 267(10:7794 (1992); Araki, et al., J. Mol. Biol.
225(1):25 (1992); Maeser and Kahnmann, Mol. Gen. Genet.
230:170-176) (1991); Esposito, et al., Nucl. Acids Res. 25(18):3605
(1997). Many of these belong to the integrase family of
recombinases (Argos, et al., EMBO J. 5:433-440 (1986); Voziyanov,
et al., Nucl. Acids Res. 27:930 (1999)). Perhaps the best studied
of these are the Integrase/att system from bacteriophage (Landy, A.
Current Opinions in Genetics and Devel. 3:699-707 (1993)), the
Cre/loxP system from bacteriophage P1 (Hoess and Abremski (1990) In
Nucleic Acids and Molecular Biology, vol. 4. Eds.: Eckstein and
Lilley, Berlin-Heidelberg: Springer-Verlag; pp. 90-109), and the
FLP/FRT system from the Saccharomyces cerevisiae 2.mu. circle
plasmid (Broach, et al., Cell 29:227-234 (1982)).
[0010] Recombination Sites
[0011] Whether the reactions discussed above are termed
recombination, transposition or integration and are catalyzed by a
recombinase, transposase or integrase, they share the key feature
of specific recognition sequences, often termed "recombination
sites," on the nucleic acid molecules participating in the
reactions. These recombination sites are sections or segments of
nucleic acid on the participating nucleic acid molecules that are
recognized and bound by the recombination proteins during the
initial stages of integration or recombination. For example, the
recombination site for Cre recombinase is loxP which is a 34 base
pair sequence comprised of two 13 base pair inverted repeats
(serving as the recombinase binding sites) flanking an 8 base pair
core sequence. See FIG. 1 of Sauer, B., Curr. Opin. Biotech.
5:521-527 (1994). Other examples of recognition sequences include
the attB, attP, attL, and attR sequences which are recognized by
the recombination protein (Int. attB is an approximately 25 base
pair sequence containing two 9 base pair core-type Int binding
sites and a 7 base pair overlap region, while attP is an
approximately 240 base pair sequence containing core-type Int
binding sites and arm-type Int binding sites as well as sites for
auxiliary proteins integration host factor (IHF), FIS and
excisionase (Xis). See Landy, Curr. Opin. Biotech. 3:699-707
(1993).
[0012] Conventional Nucleic Acid Cloning
[0013] The cloning of nucleic acid segments currently occurs as a
daily routine in many research labs and as a prerequisite step in
many genetic analyses. The purpose of these clonings is various,
however, two general purposes can be considered: (1) the initial
cloning of nucleic acid from large DNA or RNA segments
(chromosomes, YACs, PCR fragments, mRNA, etc.), done in a relative
handful of known vectors such as pUC, pGem, pBlueScript, and (2)
the subcloning of these nucleic acid segments into specialized
vectors for functional analysis. A great deal of time and effort is
expended both in the transfer of nucleic acid segments from the
initial cloning vectors to the more specialized vectors. This
transfer is called subcloning.
[0014] The basic methods for cloning have been known for many years
and have changed little during that time. A typical cloning
protocol is as follows:
(1) digest the nucleic acid of interest with one or two restriction
enzymes; (2) gel purify the nucleic acid segment of interest when
known; (3) prepare the vector by cutting with appropriate
restriction enzymes, treating with alkaline phosphatase, gel purify
etc., as appropriate; (4) ligate the nucleic acid segment to the
vector, with appropriate controls to eliminate background of uncut
and self-ligated vector; (5) introduce the resulting vector into an
E. coli host cell; (6) pick selected colonies and grow small
cultures overnight; (7) make nucleic acid minipreps; and (8)
analyze the isolated plasmid on agarose gels (often after
diagnostic restriction enzyme digestions) or by PCR.
[0015] The specialized vectors used for subcloning nucleic acid
segments are functionally diverse. These include but are not
limited to: vectors for expressing nucleic acid molecules in
various organisms; for regulating nucleic acid molecule expression;
for providing tags to aid in protein purification or to allow
tracking of proteins in cells; for modifying the cloned nucleic
acid segment (e.g., generating deletions); for the synthesis of
probes (e.g., riboprobes); for the preparation of templates for
nucleic acid sequencing; for the identification of protein coding
regions; for the fusion of various protein-coding regions; to
provide large amounts of the nucleic acid of interest, etc. It is
common that a particular investigation will involve subcloning the
nucleic acid segment of interest into several different specialized
vectors.
[0016] As known in the art, simple subclonings can be done in one
day (e.g., the nucleic acid segment is not large and the
restriction sites are compatible with those of the subcloning
vector). However, many other subclonings can take several weeks,
especially those involving unknown sequences, long fragments, toxic
genes, unsuitable placement of restriction sites, high backgrounds,
impure enzymes, etc. One of the most tedious and time consuming
type of subcloning involves the sequential addition of several
nucleic acid segments to a vector in order to construct a desired
clone. One example of this type of cloning is in the construction
of gene targeting vectors. Gene targeting vectors typically include
two nucleic acid segments, each identical to a portion of the
target gene, flanking a selectable marker. In order to construct
such a vector, it may be necessary to clone each segment
sequentially, i.e., first one gene fragment is inserted into the
vector, then the selectable marker and then the second fragment of
the target gene. This may require a number of digestion,
purification, ligation and isolation steps for each fragment
cloned. Subcloning nucleic acid fragments is thus often viewed as a
chore to be done as few times as possible.
[0017] Several methods for facilitating the cloning of nucleic acid
segments have been described, e.g., as in the following
references.
[0018] Ferguson, J., et al., Gene 16:191 (1981), disclose a family
of vectors for subcloning fragments of yeast nucleic acids. The
vectors encode kanamycin resistance. Clones of longer yeast nucleic
acid segments can be partially digested and ligated into the
subcloning vectors. If the original cloning vector conveys
resistance to ampicillin, no purification is necessary prior to
transformation, since the selection will be for kanamycin.
[0019] Hashimoto-Gotoh, T., et al., Gene 41:125 (1986), disclose a
subcloning vector with unique cloning sites within a streptomycin
sensitivity gene; in a streptomycin-resistant host, only plasmids
with inserts or deletions in the dominant sensitivity gene will
survive streptomycin selection.
[0020] Notwithstanding the improvements provided by these methods,
traditional subclonings using restriction and ligase enzymes are
time consuming and relatively unreliable. Considerable labor is
expended, and if two or more days later the desired subclone can
not be found among the candidate plasmids, the entire process must
then be repeated with alternative conditions attempted.
[0021] Recombinational Cloning
[0022] Cloning systems that utilize recombination at defined
recombination sites have been previously described in U.S. Pat.
Nos. 5,888,732, 6,143,557, 6,171,861, 6,270,969, and 6,277,608
which are specifically incorporated herein by reference. In brief,
the Gateway.TM. Cloning System, described in this application and
the applications referred to in the related applications section,
utilizes vectors that contain at least one and preferably at least
two different site-specific recombination sites based on the
bacteriophage lambda system (e.g., attl and att2) that are mutated
from the wild type (attO) sites. Each mutated site has a unique
specificity for its cognate partner att site of the same type (for
example attBl with attPl, or attL1 with attRl) and will not
cross-react with recombination sites of the other mutant type or
with the wild-type attO site. Nucleic acid fragments flanked by
recombination sites are cloned and subcloned using the Gateway.TM.
system by replacing a selectable marker (for example, ccdB) flanked
by att sites on the recipient plasmid molecule, sometimes termed
the Destination Vector. Desired clones are then selected by
transformation of a ccdB sensitive host strain and positive
selection for a marker on the recipient molecule. Similar
strategies for negative selection (e.g., use of toxic genes) can be
used in other organisms such as thymidine kinase (TK) in mammals
and insects.
[0023] Mutating specific residues in the core region of the att
site can generate a large number of different att sites. As with
the attl and att2 sites utilized in Gateway.TM., each additional
mutation potentially creates a novel alt site with unique
specificity that will recombine only with its cognate partner aut
site bearing the same mutation and will not cross-react with any
other mutant or wild-type att site. Novel mutated alt sites (e.g.,
attB 1-10, attP 1-10, attR 1-10 and attL 1-10) are described in
commonly owned U.S. application Ser. No. 09/517,466, filed Mar. 2,
2000, which is specifically incorporated herein by reference. Other
recombination sites having unique specificity (i.e., a first site
will recombine with its corresponding site and will not recombine
or not substantially recombine with a second site having a
different specificity) may be used to practice the present
invention. Examples of suitable recombination sites include, but
are not limited to, loxP sites and derivatives such as loxP511 (see
U.S. Pat. No. 5,851,808), frt sites and derivatives, dif sites and
derivatives, psi sites and derivatives and cer sites and
derivatives. The present invention provides novel methods using
such recombination sites to join or link multiple nucleic acid
molecules or segments and more specifically to clone such multiple
segments into one or more vectors containing one or more
recombination sites (such as any Gateway.TM. Vector including
Destination Vectors).
SUMMARY OF THE INVENTION
[0024] The invention relates; in part, to nucleic acid molecules
which comprise one or more (e.g., one, two, three, four, five,
etc.) recombination sites (e.g., one or more alt sites, one or more
lox sites, etc.) and/or one or more (e.g., one, two, three, four,
five, etc.) topoisomerase recognition sites (e.g., one or more
recognition sites for a type IA topoisomerase, a type IB
topoisomerase, a type II topoisomerase, etc.), as well as nucleic
acid molecules which have undergone cleavage with a topoisomerase
(e.g., a site specific topoisomerase). The invention also relates
to nucleic acid molecules which comprise one or more recombination
sites and/or one or more topoisomerases. The invention more
specifically relates to combining or joining at least a first
nucleic acid molecule which comprises at least a first nucleic acid
molecule which comprises at least one recombination site and at
least a second nucleic acid molecule which comprises at least one
topoisomerase recognition site and/or at least one topoisomerase.
Upon joining these at least first and second molecules, at least a
third (or chimeric) molecule may be produced which comprises (1) at
least one recombination site and (2) at least one topoisomerase
recognition site and/or at least one topoisomerase. These nucleic
acid molecules may be linear or closed circular (e.g., relaxed,
supercoiled, etc.). Such recombination sites, topoisomerase
recognition sites and topoisomerase can be located at any position
on any number of nucleic acid molecules of the invention, including
at or near the termini of the nucleic acid molecules and/or within
the nucleic acid molecules. Moreover, any combination of the same
or different recombination sites, topoisomerase recognition sites
and/or topoisomerases may be used in accordance with the
invention.
[0025] The invention includes, in part, nucleic acid molecules and
compositions comprising nucleic acid molecules (e.g., reaction
mixtures), wherein the nucleic acid molecules comprise (1) at least
one (e.g., one, two, three, four, five, six, seven eight, etc.)
recombination site and (2) at least one (e.g., one, two, three,
four, five, six, seven eight, etc.) topoisomerase (e.g., a
covalently linked topoisomerase) or at least one (e.g., one, two,
three, four, five, six, seven eight, etc.) toposiomerase
recognition site. In particular embodiments, the topoisomerases or
toposiomerase recognition sites, as well as the recombination
sites, of the nucleic acid molecules referred to above can be
either internal or at or near one or both termini. For example, one
or more (e.g., one, two, three, four, five, six, seven eight, etc.)
of the at least one topoisomerase or the at least one topoisomerase
recognition site, as well as one or more of the at least one
recombination site, can be located at or near a 5' terminus, at or
near a 3' terminus, at or near both 5' termini, at or near both 3'
termini, at or near a 5' terminus and a 3' terminus, at or near a
5' terminus and both 3' termini, or at or near a 3' terminus and
both 5' termini. The invention further provides methods for
preparing and using nucleic acid molecules and compositions of the
invention.
[0026] In specific aspects, the invention provides nucleic acid
molecules (1) to which topoisomerases of various types (e.g., a
type IA toposiomerase, a type IB toposiomerase, a type II
topoisomerase, etc.) are attached (e.g., covalently bound) and/or
(2) which contain two or more topoisomerase recognition sites which
are recognized by various types of topoisomerases, as well as
methods for preparing and using compositions comprising such
nucleic acid molecules. In many embodiments, these nucleic acid
molecules will further comprise one or more (e.g., one, two, three,
four, five, six, seven eight, etc.) recombination site.
[0027] The invention further provides methods for joining two or
more nucleic acid segments, wherein at least one of the nucleic
acid segments contains at least one toposiomerase or topoisomerase
recognition site and/or one or more recombination sites. Further,
when nucleic acid segments used in methods of the invention contain
more than one (e.g., two, three, four, five, six, seven eight,
etc.) toposiomerase, either on the same or different nucleic acid
segments, these toposiomerase may be of the same type or of
different types. Similarly, when nucleic acid segments used in
methods of the invention contain more than one toposiomerase
recognition site, either on the same or different nucleic acid
segments, these toposiomerase recognition sites may be recognized
by topoisomerases of the same type or of different types.
Additionally, when nucleic acid segments used in methods of the
invention contain one or more recombination sites, these
recombination sites may be able to recombine with one or more
recombination sites on the same or different nucleic acid segments.
Thus, the invention provides methods for joining nucleic acid
segments using methods employing any one toposiomerase or
topoisomerase recognition site. The invention provides further
methods for joining nucleic acid segments using methods employing
(1) any combination of topoisomerases or topoisomerase recognition
sites and/or (2) any combination of recombination sites. The
invention also provides nucleic acid molecules produced by the
methods described above, as well as uses of these molecules and
compositions comprising these molecules.
[0028] In general, the invention provides, in part, methods for
joining any number of nucleic acid segments (e.g., two, three,
four, five, six, seven, eight, nine, ten, etc.) which contain
different functional or structural elements. The invention thus
provides, in part, methods for bringing together any number of
nucleic acid segments (e.g., two, three, four, five, six, seven,
eight, nine, ten, etc.) which confer different properties upon a
nucleic acid molecule product. In many instances, methods of the
invention will result in the formation of nucleic acid molecules
wherein there is operable interaction between properties and/or
elements of individual nucleic acid segments which are joined
(e.g., operable interaction/linkage between an expression control
sequence and an open reading frame). Examples of (1) functional and
structural elements and (2) properties which may be conferred upon
product molecules include, but are not limited to, multiple cloning
sites (e.g., nucleic acid regions which contain at least two
restriction endonuclease cleavage sites), packaging signals (e.g.,
adenoviral packaging signals, alphaviral packaging signals, etc.),
restriction endonuclease cleavage sites, open reading frames (e.g.,
intein coding sequence, affinity purification tag coding sequences,
etc.), expression control sequences (e.g., promoters, operators,
etc.), etc. Additional elements and properties which can be
conferred by nucleic acid segments upon a product nucleic acid
molecule are described elsewhere herein. The invention also
provides nucleic acid molecules produced by the methods described
above, as well as uses of these molecules and compositions
comprising these molecules.
[0029] The invention further includes, in part, methods for joining
two or more (e.g., 2, 3, 4, 5, 6, 7, 8, etc.) nucleic acid
segments, wherein at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, etc.)
of the nucleic acid segments comprises one or more (e.g., 1, 2, 3,
4, 5, 6, 7, 8, etc.) topoisomerases and/or one or more
topoisomerase recognition sites and at least one of the nucleic
acid segments comprises one or more recombination sites. In
particular embodiments, the invention provides methods for joining
at least two (e.g., 2, 3, 4, 5, 6, 7, 8, etc.) nucleic acid
molecules (e.g., methods employing recombination and/or mediated by
one or more topoisomerases), wherein one of the nucleic acid
segments comprises one or more topoisomerases or topoisomerase
recognition sites but does not contain a recombination site and the
other nucleic acid segments comprises one or more recombination
site but does not contain a topoisomerase or topoisomerase
recognition site. Thus, methods of the invention can be used to
prepare joined or chimeric nucleic acid molecules by the joining of
nucleic acid segments, wherein the product nucleic acid molecules
comprise (1) one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, etc.)
topoisomerases and/or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8,
etc.) topoisomerase recognition sites and (2) one or more (e.g., 1,
2, 3, 4, 5, 6, 7, 8, etc.) recombination sites. The invention
further provides nucleic acid molecules prepared by such methods,
compositions comprising such nucleic acid molecules, and methods
for using such nucleic acid molecules.
[0030] The invention also provides compositions comprising one or
more nucleic acid segments and/or nucleic acid molecules described
herein. Such compositions may comprise one or a number of other
components selected from the group consisting of one or more other
nucleic acid molecules (which may comprise recombination sites,
topoisomerase recognition sites, topoisomerases, etc.), one or more
nucleotides, one or more polymerases, one or more reverse
transcriptases, one or more recombination proteins, one or more
topoisomerases, one or more buffers and/or salts, one or more solid
supports, one or more polyamines, one or more vectors, one or more
restriction enzymes and the like. For example, compositions of the
invention include, but are not limited to, mixtures (e.g., reaction
mixtures) comprising a nucleic acid segment which comprises at
least one topoisomerase recognition site and at least one
topoisomerase which recognizes at least one of the at least one
topoisomerase recognition sites of the nucleic acid segment.
Compositions of the invention further include at least one nucleic
acid segment comprising (1) at least one topoisomerase recognition
site or at least one nucleic acid segment to which at least one
topoisomerase is attached (e.g., covalently bound) and (2) one or
more additional components. Examples of such additional components
include, but are not limited to, topoisomerases; additional nucleic
acid segments, which may or may not comprise one or more
topoisomerases or topoisomerase recognition sites; buffers; salts;
polyamines (e.g., spermine, spermidine, etc.); water; etc. Nucleic
acid segments present in compositions of the invention may further
comprise one or more recombination sites and/or one or more
recombinase.
[0031] Nucleic acid molecules or segments produced by or used in
conjunction with the methods of the invention, as well as nucleic
acid molecules or segments thereof of the invention, include those
molecules or segments specifically described herein as well as
those molecules or segments that have substantial sequence identity
to those molecules or segments specifically described herein. By a
molecule or segment having "substantial sequence identity" to a
given molecule or segment is meant that the molecule or segment is
at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99%, identical to the given (or "reference") molecule or segment.
By a nucleic acid molecule or segment having a nucleotide sequence
at least, for example, 65% "identical" to a reference nucleic acid
molecule or segment is intended that the nucleotide sequence of the
nucleic acid molecule or segment is identical to that of the
reference sequence except that the nucleic acid molecule or segment
may include up to 35 point mutations per each 100 nucleotides of
the reference nucleotide sequence. In other words, to obtain a
polynucleotide having a nucleotide sequence at least 65% identical
to a reference nucleotide sequence, up to 5% of the nucleotides in
the reference sequence may be deleted or substituted with another
nucleotide, or a number of nucleotides up to 35% of the total
nucleotides in the reference sequence may be inserted into the
reference sequence. These mutations of the reference sequence may
occur at the 5' or 3' terminal positions (or both) of the reference
nucleotide sequence, or anywhere between those terminal positions,
interspersed either individually among nucleotides in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0032] As a practical matter, whether any particular nucleic acid
molecule or segment is at least about 65%, 70%, 75%, 80%, 85%, 90%,
95%, 96%, 97%, 98% or 99% identical to a given reference molecule
or segment can be determined conventionally using known computer
programs such as FASTA (Heidelberg, Germany), BLAST (Washington,
D.C.) or BESTFIT (Wisconsin Sequence Analysis Package, Version 8
for Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711), which employs a local homology
algorithm (Smith and Waterman, Advances in Applied Mathematics 2:
482-489 (1981)) to find the best segment of homology between two
sequences. When using FASTA, BLAST, BESTFIT or any other sequence
alignment program to determine whether a particular sequence is,
for instance, 65% identical to a reference sequence according to
the present invention, the parameters are set such that the
percentage of identity is calculated over the full length of the
reference nucleotide sequence and that gaps in homology of up to
35% of the total number of nucleotides in the reference sequence
are allowed.
[0033] Often, nucleic acid molecules which have undergone cleavage
with a topoisomerase (e.g., a site specific topoisomerase) will
further have a topoisomerase molecule covalently bound to a
phosphate group of the nucleic acid molecules. The invention
further includes methods for preparing nucleic acid molecules
described above and elsewhere herein, as well as recombinant
methods for using such molecules.
[0034] In particular embodiments, nucleic acid molecules of the
invention will be vectors. In additional embodiments, the invention
includes host cells which contain nucleic acid molecules of the
invention, as well as methods for making and using such host cells,
for example, to produce expression products (e.g., proteins,
polypeptides, antigens, antigenic determinants, epitopes, and the
like, or fragments thereof).
[0035] In specific embodiments, nucleic acid molecules of the
invention comprise two or more recombination sites with one or more
(e.g., one, two, three, four, five, etc.) topoisomerase recognition
site located between the recombination sites. In additional
specific embodiments, nucleic acid molecules of the invention may
comprise two or more topoisomerase recognition sites with one or
more (e.g., one, two, three, four, five, etc.) recombination sites
located between the two or more topoisomerase recognition
sites.
[0036] In additional specific embodiments, nucleic acid molecules
of the invention comprise two recombination sites with two
topoisomerase recognition sites located between the two
recombination sites. Thus, if such molecules are linearized by
cleavage between the topoisomerase recognition sites, the
topoisomerase recognition sites in the resulting linear molecule
will be located distal (i.e., closer to the two ends of the linear
molecule) to the recombination sites. The invention thus provides
linear nucleic acid molecules which contain one or more
recombination sites and one or more topoisomerase recognition
sites. In particular embodiments, the one or more topoisomerase
recognition sites are located distal to the one or more
recombination sites. Examples of such molecules are set out below
in Example 8.
[0037] The positioning of recombination sites and topoisomerase
recognition sites of a first nucleic acid molecule can be such that
topoisomerase mediated linkage of this molecule to a second nucleic
acid molecule results in the second nucleic acid molecule being
positioned between the two or more recombination sites. As an
example, a linear first nucleic acid molecule may contain one
recombination site at or near each end and may further comprise a
topoisomerase recognition site located distal to one of the two
recombination sites. In such a case, incubation of the linear first
nucleic acid molecule with a topoisomerase can be designed to
result in the covalent linkage of the topoisomerase to the first
nucleic acid molecule, wherein the topoisomerase is positioned at
or near the end of the first nucleic acid molecule and distal to
the adjacent/nearest recombination site. This end of the first
nucleic acid molecule may be blunt or may have either a 5' or 3'
overhang. When incubated with a suitable second nucleic acid
molecule (e.g., a molecule with sequence complementarity to at
least one strand of the topoisomerase modified end of the first
nucleic acid molecule), one or both strands of one end of the
second nucleic acid molecule can be covalently joined to one or
both strands of one end of the first nucleic acid molecule.
Further, if a circular nucleic acid molecule is desired, then the
second end of the second nucleic acid molecule can be joined to the
second end of the first nucleic acid molecule by a topoisomerase, a
ligase or other method. The result of the process described above
is the generation of a nucleic acid molecule which contains a
nucleic acid insert positioned between two recombination sites.
Specific examples of related processes are set out below in Example
8. Methods for covalently linking nucleic acid molecules using
topoisomerase are described in more detail elsewhere herein.
[0038] Once a nucleic acid insert has been positioned between one
or more recombination sites, this insert, as well as adjacent
nucleic acid, may be transferred to other nucleic acid molecules by
recombinational cloning. The invention thus also provides methods
for generating the nucleic acid molecules described above and
elsewhere herein.
[0039] The distance, in terms of the number of nucleotides, between
recombination sites and topoisomerase recognition sites which
reside in a nucleic acid molecule of the invention will vary with
the particular application for which the molecule is to be used,
but can be zero, one, two, three, four, five, six, seven, eight,
nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,
twenty, twenty-five, thirty, forty, fifty, sixty, eighty, one
hundred, one hundred fifty, two hundred, three hundred, five
hundred, seven hundred, nine hundred, one thousand, etc., or more,
nucleotides. Further, the distance, in terms of the number of
nucleotides, between recombination sites and topoisomerase
recognition sites which reside in a nucleic acid molecule of the
invention may fall within the following ranges: 0-10 nucleotides,
10-30 nucleotides, 20-50 nucleotides, 40-80 nucleotides, 70-100
nucleotides, 90-200 nucleotides, 120-400 nucleotides, 200-400
nucleotides, 200-1000 nucleotides, 200-2,000 nucleotides, etc.
[0040] The present invention also generally provides materials and
methods for joining or combining two or more (e.g., three or more,
four or more, five or more etc.) segments or molecules of nucleic
acid of the invention. In one aspect, for such molecules to be
combined, at least one of the segments or molecules may comprise at
least one recombination site and at least one of the segments or
molecules may comprise at least one topoisomerase recognition site.
Such methods for joining multiple nucleic acid molecules according
to the invention may be conducted in vivo or in vitro. Accordingly,
the invention relates to methods to create novel or unique
combinations of sequences and to the sequences created by such
methods. The nucleic acid molecules created by the methods of the
invention may be used for any purpose known to those skilled in the
art. In one aspect, at least one (and often two or more) of the
nucleic acid molecules or segments to be joined by the methods of
the invention comprise at least one, and preferably at least two,
recombination sites, although each molecule may comprise multiple
recombination sites (e.g., three or more, four or more, five or
more, etc.). In another aspect, the nucleic acid molecules may
comprise at least one topoisomerase recognition site and/or at
least one topoisomerase. In yet another aspect, the molecules may
comprise (1) at least one recombination site and (2) at least one
topoisomerase recognition site and/or at least one topoisomerase.
Such recombination sites and topoisomerase recognition sites (which
may be the same or different) may be located at various positions
in each nucleic acid molecule or segment and the nucleic acid used
in the invention may have various sizes and be in different forms
including circular, supercoiled, linear, and the like. The nucleic
acid molecules used in the invention may also comprise one or more
vectors or one or more sequences allowing the molecule to function
as a vector in a host cell (such as an origin of replication). In
one aspect, nucleic acid molecules or segments for use in the
invention are linear molecules having at least one recombination
site at or near at least one termini of the molecule and preferably
comprise at least one recombination site at or near both termini of
the molecule. In another aspect, when multiple recombination sites
are located on a nucleic acid molecule of interest, such sites do
not substantially recombine or do not recombine with each other on
that molecule. In this embodiment, the corresponding binding
partner recombination sites preferably are located on one or more
other nucleic acid molecules to be linked or joined by the methods
of the invention. For instance, a first nucleic acid molecule used
in the invention may comprise at least a first and second
recombination site and a second nucleic acid molecule may comprise
at least a third and fourth recombination site, wherein the first
and second sites do not recombine with each other and the third and
fourth sites do not recombine with each other, although the first
and third and/or the second and fourth sites may recombine.
[0041] The nucleic acid molecules to be joined by the methods of
the invention (e.g., the "starting molecules") may be used to
produce one or more hybrid molecules containing all or a portion of
the starting molecules (e.g., the "product nucleic acid
molecules"). The starting molecules can be any nucleic acid
molecule derived from any source or produced by any method. Such
molecules may be derived from natural sources (such as cells,
tissue, and organs from any animal or non-animal source) or may be
non-natural (e.g., derivative nucleic acids) or synthetically
derived. The segments or molecules for use in the invention may be
produced by any means known to those skilled in the art including,
but not limited to, amplification such as by PCR, isolation from
natural sources, chemical synthesis, shearing or restriction digest
of larger nucleic acid molecules (such as genomic or cDNA),
transcription, reverse transcription and the like, and
recombination sites and/or topoisomerase recognition sites and/or
topoisomerases may be added to such molecules by any means known to
those skilled in the art including ligation of adapters containing
recombination sites and/or topoisomerase recognition sites and/or
topoisomerases, amplification or nucleic acid synthesis using
primers containing recombination sites and/or topoisomerase
recognition sites and/or topoisomerases, insertion or integration
of nucleic acid molecules (e.g., transponsons or integration
sequences) containing recombination sites and/or topoisomerase
recognition sites and/or topoisomerases, etc. In one aspect, the
nucleic acid molecules used in the invention are populations of
molecules such as nucleic acid libraries or cDNA libraries.
[0042] Once nucleic acid molecules are joined by recombination
using methods such as those described herein, these nucleic acid
molecules may then be joined to other nucleic acid molecules using
topoisomerase-mediated joining methods and/or
recombination-mediated joining methods also described herein.
[0043] Recombination sites for use in the invention may be any
recognition sequence on a nucleic acid molecule which participates
in a recombination reaction catalyzed or facilitated by
recombination proteins. In those embodiments of the present
invention utilizing more than one recombination site, such
recombination sites may be the same or different and may recombine
with each other or may not recombine or not substantially recombine
with each other. Recombination sites contemplated by the invention
also include mutants, derivatives or variants of wild-type or
naturally occurring recombination sites. Preferred recombination
site modifications include those that enhance recombination, such
enhancement selected from the group consisting of substantially (i)
favoring integrative recombination; (ii) favoring excisive
recombination; (iii) relieving the requirement for host factors;
(iv) increasing the efficiency of co-integrate or product
formation; and (v) increasing the specificity of co-integrate or
product formation. Preferred modifications include those that
enhance recombination specificity, remove one or more stop codons,
and/or avoid hair-pin formation. Desired modifications can also be
made to the recombination sites to include desired amino acid
changes to the transcription or translation product (e.g., mRNA or
protein) when translation or transcription occurs across the
modified recombination site. Recombination sites that may be used
in accordance with the invention include att sites, frt sites, dif
sites, psi sites, cer sites, and lox sites or mutants, derivatives
and variants thereof (or combinations thereof). Recombination sites
contemplated by the invention also include portions of such
recombination sites.
[0044] Each starting nucleic acid molecule may comprise, in
addition to one or more recombination sites and/or one or more
topoisomerase recognition sites and/or one or more topoisomerases,
a variety of sequences (or combinations thereof) including, but not
limited to sequences suitable for use as primer sites (e.g.,
sequences which a primer such as a sequencing primer or
amplification primer may hybridize to initiate nucleic acid
synthesis, amplification or sequencing), transcription or
translation signals or regulatory sequences such as promoters
and/or operators, ribosomal binding sites, topoisomerase
recognition sequences (or sites), Kozak sequences, and start
codons, transcription and/or translation termination signals such
as stop codons (which may be optimally suppressed by one or more
suppressor tRNA molecules), tRNAs (e.g., suppressor tRNAs), origins
of replication, selectable markers, and genes or portions of genes
which may be used to create protein fusion (e.g., N-terminal or
carboxy terminal) such as GST, GUS, GFP, open reading frame (orf)
sequences, and any other sequence of interest which may be desired
or used in various molecular biology techniques including sequences
for use in homologous recombination (e.g., gene targeting).
[0045] The present invention also relates to methods of generating
a covalently linked recombinant nucleic acid molecule by contacting
two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc) nucleic acid
molecules (which may be alternatively and equivalently referred to
herein as "nucleotide sequences"), e.g., double-stranded ("ds") or
single-stranded ("ss") nucleic acid molecules, with at least one
(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) topoisomerase. As will
be understood by the ordinarily skilled artisan, any and all of the
nucleic acid molecules or nucleotide sequences referred to herein,
for example those used in or generated by the methods, compositions
and kits disclosed herein, may be ss or ds nucleic acid molecules
or nucleotide sequences, whether or not the molecules or sequences
are specifically referred to herein as being ss and/or ds.
[0046] In one such aspect, the methods of the invention allow
joining of such nucleic acid sequences in a desired orientation
and/or order, which, if desired, can be further manipulated or used
in a variety of assays or procedures, including, for example, for a
transcription or transfection procedure, which can be performed in
vitro or in vivo, a translation reaction or other protein
expression procedure, recombination reactions, and the like. In
another aspect, three or more, four or more, five or more, etc., or
a population or library of the same or different nucleic acid
sequences can be linked according to a method of the invention. In
still another aspect, the methods of the invention can be used to
link each end of a single nucleic acid molecule to form a
covalently closed circular or supercoiled molecule.
[0047] The nucleic acid sequences to be linked can be derived from
any source, and can be naturally occurring and chemically or
recombinantly synthesized nucleic acid molecules such as cDNA,
genomic DNA, vectors, oligonucleotides, and the like. Furthermore,
the nucleic acid sequences can, but need not, contain one or more
functional sequences such as gene regulatory elements, origins of
replication, splice sites, polyadenylation sites, open reading
frames, which can encode, for example, tag sequences, detectable or
selectable markers, cell localization domains, or other peptide or
polypeptide, and the like. As such, the invention allows any number
of nucleic acid sequences, which can be the same or different, to
be linked, including, if desired, in a predetermined order or
orientation or both.
[0048] The nucleic acid molecules (e.g., ds or ss nucleic acid
molecules) to be linked can be in any form, for example,
single-stranded or double-stranded, linear, circular, or
supercoiled, and are characterized, in part, in that each nucleic
acid molecule to be linked is a substrate for a topoisomerase or
can be modified to be such a substrate. The topoisomerase can be
any topoisomerase that can covalently link at least one strand of a
nucleic acid molecule to at least one strand of another nucleic
acid molecule, preferably through a phosphodiester bond. The
topoisomerase can be a site specific topoisomerase or can have
relaxed specificity, and preferably forms a stable complex (e.g., a
covalent complex) with one strand of the nucleic acid molecule at
or near the site at which cleavage is effected.
[0049] A method of the invention generally is performed by
contacting topoisomerase and the nucleic acid molecules (e.g., ds
or ss nucleic acid molecules) to be joined under conditions such
that both strands of an end of one nucleic acid molecule are
ligated to both strands of an end of at least one (e.g., 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, etc.) other nucleic acid molecule. As such, a
method of the invention generates a covalently linked recombinant
nucleic acid molecule (which may be either single-stranded or
double-stranded), which does not contain a nick at the site or
sites at which the substrate nucleic acid molecules are ligated.
The present invention also provides recombinant nucleic acid
molecules prepared by such a method. In certain such aspects of the
invention, such recombinant nucleic acid molecules will further
comprise one or more recombination sites.
[0050] A method of the invention can be performed using various
combinations of components. For example, the method can be
performed by contacting two or more substrate nucleic acid
molecules (e.g., ss nucleic acid molecules or ds nucleic acid
molecules) to be covalently linked and at least one topoisomerase,
wherein the topoisomerase cleaves one or both strands of the
nucleic acid molecules and forms a stable complex with a nucleotide
at a terminus of the cleavage site. The topoisomerase-charged ends
or topoisomerase-charged nucleic acid molecules are then contacted
with each other such that each strand of the substrate nucleic acid
molecules are linked, thereby generating one or more covalently
linked recombinant nucleic molecules. Preferably, the topoisomerase
mediates the formation of phosphodiester bond at each linkage site.
The method also can be performed by contacting two or more
topoisomerase-charged nucleic acid molecules, either alone, or in
the presence of excess topoisomerase, or by contacting one or more
topoisomerase-charged nucleic acid molecules (which may be ss or
ds) with one or more nucleic acid molecules (which may also be ss
or ds) that contain a topoisomerase cleavage site, and a
topoisomerase. The present invention also provides recombinant
nucleic acid molecules prepared by such a method. In certain such
aspects of the invention, such recombinant nucleic acid molecules
will further comprise one or more recombination sites. In various
embodiments, the topoisomerase can have a relatively relaxed
specificity such that it can bind to and cleave a variety of
different nucleotide sequences, or the topoisomerase can be a site
specific topoisomerase, which binds to and cleaves a specific
nucleotide sequence. The topoisomerase also can be a type I
topoisomerase, which cleaves one strand of a ds nucleic acid
molecule, or can be a type II topoisomerase, which cleaves both
strands of a ds nucleic acid molecule. Where the topoisomerase is a
type II topoisomerase, cleavage is effected such that a linear ds
nucleic acid molecule is produced, and is topoisomerase-charged at
one or both ends. In certain such aspects, the strand of the ds
nucleic acid molecule that is complementary to the strand
containing the bound topoisomerase will form an overhanging
sequence.
[0051] An advantage of performing a method of the invention is that
the ligation reaction performed by a topoisomerase occurs very
quickly and over a wide range of temperatures. An additional
advantage is that recombinant nucleic acid molecules generated
according to the methods of the invention do not contain nicks at
the sites where two nucleic acid molecules are joined together. As
such, the covalently linked recombinant nucleic acid molecules can
be used directly in a subsequent procedure, for example, as a
substrate for an amplification reaction such as a polymerase chain
reaction (PCR).
[0052] By way of example, a method of the invention can be
performed by contacting 1) a first nucleic acid molecule (which may
be ss or ds) having a first end and a second end, wherein, at the
first end or second end or both, the first nucleic acid molecule
has a topoisomerase recognition site at or near the 3' terminus; 2)
at least a second nucleic acid molecule (which may also be ss or
ds) having a first end and a second end, wherein, at the first end
or second end or both, the at least second double stranded
nucleotide sequence has a topoisomerase recognition site at or near
the 3' terminus; and 3) a site specific topoisomerase, under
conditions such that all components are in contact and the
topoisomerase can effect its activity. The strand complementary to
that containing the topoisomerase recognition sequence may comprise
a 5' hydroxyl group and, upon cleavage by the topoisomerase, may
further comprise a 5' overhanging sequence.
[0053] A method of the invention also can be performed by
contacting 1) a nucleic acid molecule (which may be ss or ds)
having a first end and a second end, wherein each of the first end
and second end contains a topoisomerase recognition site at or near
the 3' terminus, and 2) a site specific topoisomerase, under
conditions such that the components are in contact and the
topoisomerase can effect its activity. For example, the
topoisomerase can be a type IB topoisomerase such as a Vaccinia
topoisomerase or an S. cerevisiae topoisomerase. Such a method
provides a means to prepare a covalently closed circular or
supercoiled ds nucleic acid molecule.
[0054] A method of the invention also can be performed by
contacting 1) a first nucleic acid molecule (which may be ss or ds)
having a first end and a second end, wherein the first nucleic acid
molecule has a topoisomerase recognition site at or near the 5'
terminus of the first end or the second end or both; 2) at least a
second nucleic acid molecule (which may also be ss or ds) having a
first end and a second end, wherein the at least second double
stranded nucleotide sequence has a topoisomerase recognition site
at or near the 5' terminus of the first end or the second end or
both; and 3) at least one site specific topoisomerase, under
conditions such that all components are in contact and the at least
one topoisomerase can effect its activity. For example, the
topoisomerase can be a type IA topoisomerase such as an E. coli
topoisomerase I or topoisomerase III, or eukaryotic topoisomerase
III. Upon cleavage of a nucleic acid molecule, the topoisomerase
preferably is stably bound to the 5' terminus. The 3' terminus of
the end containing the topoisomerase recognition site, or bound
topoisomerase, can comprise a 3' hydroxyl group, or can be modified
to comprise a 3' hydroxyl group. Upon cleavage by the
topoisomerase, the cleaved nucleic acid molecule may comprise a 3'
overhanging sequence.
[0055] The methods as exemplified herein can be performed using two
or more site specific topoisomerases, wherein the first, second or
other nucleic acid substrates correspondingly have, at or near a 3'
terminus or 5' terminus of an end, a topoisomerase recognition site
for one of the two or more topoisomerases. The use of two or more
topoisomerases, and corresponding topoisomerase recognition sites,
can facilitate the joining of the nucleic acid molecules (which may
be ss or ds) in a predetermined order, orientation, or combination
thereof. Thus, it will be recognized that, where a method of the
invention is exemplified using a topoisomerase, the method
similarly can be performed using two or more topoisomerases. In
some cases, reference is made to the use of at least one
topoisomerase, although, unless indicated otherwise, the methods
can be performed using one, two, three or more topoisomerases,
provided the substrate nucleic acid molecules contain the
appropriate topoisomerase recognition sites. Similar considerations
are relevant to topoisomerase-charged nucleic acid substrates, in
that the topoisomerases can be the same or different.
[0056] In another embodiment, a method of the invention can be
performed by contacting 1) a first nucleic acid molecule (which may
be ss or ds) having a first end and a second end, wherein the first
nucleic acid molecule has a topoisomerase recognition site at or
near the 3' terminus and a topoisomerase recognition site at or
near the 5' terminus of the first end or of the second end or of
both ends; 2) at least a second nucleic acid molecule (which may
also be ss or ds) having a first end and a second end; and 3) at
least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) site specific
topoisomerases, under conditions such that all components are in
contact and each of the topoisomerases can effect its activity.
Upon cleavage of the termini of the substrate first nucleic acid
molecule by the topoisomerases, the 5' terminus or the 3' terminus
of one or both ends can comprise an overhanging sequence, or can be
blunt ended, or one end can contain an overhang and the second end
can be blunt ended. Where present, an overhanging sequence
generally has sufficient complementarity to an overhanging sequence
of the second (or other) nucleic acid molecule to allow for
specific hybridization of the two molecules to each other.
[0057] Once nucleic acid molecules are joined by topoisomerase
mediated joining methods of the invention, the resulting nucleic
acid molcules may then be used in recombination reactions, such as
those described elsewhere herein.
[0058] The number of different topoisomerases useful in such an
embodiment will depend, in part, on whether the first nucleic acid
molecule contains topoisomerase recognition sites at only the first
end or the second end, or contains topoisomerase recognition sites
at both ends, and further, where the nucleic acid molecule contains
topoisomerase recognition sites on both ends, whether at least the
3' recognition sites or the 5' recognition sites are different. In
addition, the method can be performed such that one or more of the
at least second nucleic acid molecule also can contain a
topoisomerase recognition site at or near the 3' terminus and/or a
topoisomerase recognition site at or near the 5' terminus of the
first end or of the second end or of both ends, wherein the
topoisomerase recognition sites at or near the 3' terminus or the
5' terminus or both of the other nucleic acid molecule can the same
as or different from the topoisomerase recognition sites in the
first nucleic acid molecule. As such, the number of different
topoisomerase further will depend on the number of different
substrate nucleic acid molecules being linked according to a method
of the invention.
[0059] An advantage of performing a method of the invention using a
site specific topoisomerase is that the first nucleic acid
molecule, the second nucleic acid molecule, and one or more
additional nucleic acid molecules (which may be ss or ds) can be
covalently linked in a predetermined directional orientation. An
additional advantage is that a functional product can be selected
in vitro by performing an amplification reaction using primers
specific for the termini of the desired covalently linked
recombinant nucleic acid molecule. As such, a covalently linked
recombinant nucleic acid molecule (which may be ss or ds) generated
according to a method of the invention can be used directly in
further procedures, for example, for transfecting a cell, or as a
template for performing amplification (e.g., PCR), a recombination
reaction (e.g., a recombination reaction such as those described
herein), an in vitro transcription reaction, or a coupled
transcription/translation reaction. Accordingly, the covalently
linked recombinant nucleic acid molecule is useful, without further
manipulation, for various purposes.
[0060] In an aspect of the invention, the first nucleic acid
molecules, as well as other nucleic acids used in methods of the
invention, may be derived from at least a first population of
nucleic acid molecules, for example, from a cDNA library or a
combinatorial library such as a combinatorial library of synthetic
oligonucleotides, and the second nucleic acid molecules, as well as
other nucleic acids used in methods of the invention, may be
derived from at least a second population of source nucleic acid
molecules. According to such a method, linking of first nucleic
acid molecules with second nucleic acid molecules provides a means
to generate combinatorial populations of covalently linked
recombinant nucleic acid molecules (which may be ss or ds). In
accordance with such a method, one or more target nucleic acid
molecules also can be linked with the recombinant nucleic acid
molecules of the population to produce additional populations. Such
populations of combinatorial molecules can be further manipulated
or analyzed, for example, by protein expression and screening for
fusion proteins having desirable characteristics.
[0061] In one embodiment, a method of the invention is performed
such that the first nucleic acid molecule (which may be ss or ds),
as well as other nucleic acids used in methods of the invention,
comprises an open reading flame, for example, an isolated cDNA or
coding sequence of a gene, and a second nucleic acid molecule
(which may be ss or ds) comprises a regulatory element such as a
promoter, which can be operably covalently linked to the 5' end of
the coding sequence such that the coding sequence can be
transcribed therefrom. A second nucleic acid molecule, as well as
other nucleic acids used in methods of the invention, also can
comprise two or more regulatory elements, for example, a promoter
(e.g., a GAL4 promoter), an operator (e.g., a tet operator, a
galactose operon operator, a lac operon operator, and the like), an
internal ribosome entry site and an ATG initiator methionine codon,
in operative linkage with each other, which can be operably
covalently linked to the 5' end of a first nucleic acid molecule
comprising a coding sequence according to a method of the
invention. Such a method can further include contacting a third
nucleic acid molecule (which may be ss or ds) comprising, for
example, a polyadenylation signal, which can be operably covalently
linked to the 3' end of the coding sequence. Such a method can be
useful for generating an expressible nucleic acid molecule, which
can be transcribed, translated, or both as a functional unit. In
addition, or alternatively, a nucleic acid molecule encoding a
detectable marker, for example, an epitope tag, can be operably
linked to a first or second (or other) nucleic acid molecule(s)
according to a method of the invention. The generation of a
recombinant nucleic acid molecule (which may be ss or ds) having a
desired directional orientation of the nucleotide sequences in such
a construct may be facilitated, for example, by including
complementary 5' overhanging sequences at the termini of the
nucleic acid molecules to be covalently linked together by the
topoisomerase.
[0062] In another embodiment, a method of the invention is
performed such that at least the first nucleic acid molecule or the
at least second nucleic acid molecule, as well as other nucleic
acids used in methods of the invention, is one of a plurality of
nucleotide sequences, for example, a cDNA library, a combinatorial
library of nucleotide sequences, or a variegated population of
nucleotide sequences. In another embodiment, a method of the
invention includes further contacting a generated covalently linked
ds recombinant nucleic acid molecule (e.g., a recombinant nucleic
acid molecule which is covalently linked in one or both strands)
with a PCR primer pair, and amplifying all or a portion of the
covalently linked recombinant nucleic acid molecule. In addition to
generating a large amount of product, the amplification reaction
can be selective for constructs comprising a desired covalently
linked ds recombinant nucleic acid molecule, particularly where the
nucleic acid molecules to be covalently linked comprise
complementary overhanging sequences. As such, a method of the
invention provides an in vitro selection means that is suitable for
high throughput analysis.
[0063] A method of the invention is also exemplified by contacting
1) a first nucleic acid molecule (which may be ss or ds) having a
first end and a second end, wherein, at the first end or second end
or both, the first nucleic acid molecule has a topoisomerase
covalently bound to the 3' terminus ("topoisomerase-charged"); and
2) at least a second topoisomerase-charged nucleic acid molecule
(which may be ss or ds). Preferably, the topoisomerase-charged
nucleic acid molecules contain a 5' hydroxyl group at the ends
containing the bound topoisomerase, although 5' hydroxy groups also
can be generated using a phosphatase. The methods of the invention
can be performed using only a first nucleic acid molecule and a
second nucleic acid molecule, or can include a third, fourth or
more nucleic acid molecules (which may be ss or ds) as desired,
wherein each nucleotide sequence is as defined. A first or second
(or other) nucleic acid molecule independently can have a
topoisomerase covalently bound to a 3' terminus of one end or at
both ends of the nucleotide sequence, and, unless indicated
otherwise, the first and second (or other) nucleic acid molecules
can be the same or can be different. In certain such aspects, at
least one of the nucleic acid molecules used in the methods
described herein will comprise at least one recombination site.
Further, nucleic acid molecules generated by methods described
above may be used in recombination reactions, such as those
described elsewhere herein.
[0064] Methods of the invention are further exemplified by
contacting 1) a first nucleic acid molecule (which may be ss or ds)
having a first end and a second end, wherein, at the first end or
second end or both, the first nucleic acid molecule has a
topoisomerase covalently bound to a 5' terminus (i.e., a
topoisomerase-charged 5' terminus); and 2) at least a second
topoisomerase-charged nucleic acid molecule (which may be ss or ds)
comprising at least one topoisomerase-charged 5' terminus. The
topoisomerase-charged nucleic acid molecules can contain a 3'
hydroxyl group at the ends containing the bound topoisomerase, or a
3' hydroxyl group can be generated using a phosphatase. As
disclosed herein, such a method can be performed using only a first
nucleic acid molecule and a second nucleic acid molecule, or can
include a third, fourth or more nucleic acid molecules (which may
be ss or ds) as desired, wherein each nucleotide sequence is as
defined, including comprising at least one topoisomerase-charged 5'
terminus. A first or second (or other) nucleic acid molecule
independently can have a topoisomerase covalently bound to a 5'
terminus of one end or at both ends of the nucleic acid molecule,
and, unless indicated otherwise, the first and second (or other)
nucleic acid molecules can be the same or can be different. In
certain such aspects, at least one of the nucleic acid molecules
used in the methods described herein will comprise at least one
recombination site. Further, nucleic acid molecules generated by
methods described above and elsewhere herein may also be used in
recombination reactions, such as those described elsewhere
herein.
[0065] A method of the invention is additionally exemplified by
contacting 1) a first nucleic acid molecule having a first end and
a second end, wherein, at the first end or second end or both, the
first nucleic acid molecule has a first topoisomerase covalently
bound to the 5' terminus and a second topoisomerase covalently
bound to the 3' terminus of the first end or the second end or both
(i.e., one or both ends contain a topoisomerase charged 5' terminus
and a topoisomerase-charged 3' terminus); and 2) at least a second
nucleic acid molecule, which, preferably, has or can be made to
have hydroxyl groups at the 5' terminus and 3' terminus of an end
to be covalently linked to an end of the first nucleic acid
molecule containing the topoisomerases. The method also can be
performed wherein either the 5' terminus or 3' terminus of the end
containing a topoisomerase-charged 3' terminus or
topoisomerase-charged 5' terminus, respectively, contains a
topoisomerase recognition site, wherein the method further includes
contacting the components with a topoisomerase that can effect its
activity with respect to the topoisomerase recognition site. In
certain such aspects, at least one of the nucleic acid molecules
used in the methods described herein will comprise at least one
recombination site. Further, nucleic acid molecules generated by
methods described above and elsewhere herein may also be used in
recombination reactions, such as those described elsewhere
herein.
[0066] Such a method of the invention can be performed using only a
first nucleic acid molecule and a second nucleic acid molecule, or
can include a third, fourth or more nucleic acid molecule as
desired, wherein the nucleic acid molecules are as defined for the
first nucleic acid molecule, the second nucleic acid molecule, or a
combination thereof. A first or second (or other) nucleic acid
molecule independently can, but need not, have one or more
topoisomerases covalently bound to a 5' terminus, 3' terminus, or
both 5' and 3' termini of the second end (i.e., the undefined end).
Further, one or more of these nucleic acid molecules may
additionally comprise one or more recombination sites. Unless
indicated otherwise, the first and second (or other) nucleic acid
molecules can be the same or can be different.
[0067] The present invention further relates to a method of
generating a covalently linked ds recombinant nucleic acid molecule
by 1) amplifying a portion of a first nucleic acid molecule using a
PCR primer pair, wherein at least one primer of the primer pair
encodes a complement of a topoisomerase recognition site, and,
optionally, of one or more recombination sites, thereby producing
an amplified first nucleic acid molecule having a first end and a
second end, wherein the first end or second end or both has a
topoisomerase recognition site at or near the 3' terminus; and 2)
contacting a) the amplified first nucleic acid molecule; b) at
least a second nucleic acid molecule having a first end and a
second end, wherein the first end or second end or both has a
topoisomerase recognition-site, or cleavage product thereof, at or
near the 3' terminus and has, or can be made to have, a hydroxyl
group at the 5' terminus of the same end; and c) a site specific
topoisomerase, under conditions such that the topoisomerase can
cleave the end of the amplified first nucleic acid molecule having
a topoisomerase recognition site and the end (or ends) of the at
least second nucleic acid molecule having a topoisomerase
recognition site, and can effect its ligating activity. The PCR
primer that encodes a complement of topoisomerase recognition site
can have a hydroxyl group at its 5' terminus, or the amplified
first nucleic acid molecule generated using the primer can be
contacted with a phosphatase to generate a hydroxyl group at its 5'
terminus. The PCR primer encoding the complement of a topoisomerase
recognition site also can comprise a nucleotide sequence at its 5'
terminus such that, upon cleavage by a site specific topoisomerase
of a first nucleic acid molecule amplified using the primer, the
nucleic acid molecule contains a 5' overhanging sequence, which is
complementary to a 5' overhanging sequence of a second (or other)
nucleic acid molecule to which the first nucleic acid molecule is
to be covalently linked according to a method of the invention. In
certain such aspects, at least one of the nucleic acid molecules
used in the methods described herein will comprise at least one
recombination site. Further, nucleic acid molecules generated by
methods described above and elsewhere herein may also be used in
recombination reactions, such as those described elsewhere
herein.
[0068] The present invention also relates to a method of generating
a covalently linked ds recombinant nucleic acid molecule by 1)
amplifying a portion of a first nucleic acid molecule using a PCR
primer pair, wherein at least one primer of the primer pair encodes
a topoisomerase recognition site, and, optionally, one or more
recombination sites, thereby producing an amplified first nucleic
acid molecule having a first end and a second end, wherein the
first end or second end or both has a topoisomerase recognition
site at or near the 5' terminus; and 2) contacting a) the amplified
first nucleic acid molecule; b) at least a second nucleic acid
molecule having a first end and a second end, wherein the first end
or second end or both has a topoisomerase recognition site at or
near the 5' terminus and has, or can be made to have, a hydroxyl
group at the 3' terminus of the same end; and c) at least one site
specific topoisomerase, under conditions such that the at least one
topoisomerase can cleave the end of the amplified first nucleic
acid molecule having a topoisomerase recognition site and the end
(or ends) of the at least second nucleic acid molecule having a
topoisomerase recognition site, and can effect its ligating
activity. The amplified first nucleic acid molecule generally has a
hydroxyl group at the 3' terminus of the end containing the
topoisomerase recognition site, or can be modified to contain such
a 3' hydroxyl group. The PCR primer encoding the topoisomerase
recognition site can further comprise a nucleotide sequence at its
5' terminus, i.e., 5' to the topoisomerase recognition site, such
that, upon cleavage of the amplified first nucleic acid molecule by
a site specific topoisomerase, the nucleic acid molecule contains a
3' overhanging sequence, which is complementary to a 3' overhanging
sequence of a second (or other) nucleic acid molecule to which the
first nucleic acid molecule is to be covalently linked according to
a method of the invention. In certain such aspects, at least one of
the nucleic acid molecules used in the methods described herein
will comprise at least one recombination site. Further, nucleic
acid molecules generated by methods described above and elsewhere
herein may also be used in recombination reactions, such as those
described elsewhere herein.
[0069] The present invention further relates to a method of
generating a covalently linked ds recombinant nucleic acid molecule
by 1) amplifying a portion of a first nucleic acid molecule using a
PCR primer pair, wherein at least one primer of the primer pair
includes a topoisomerase recognition site, a nucleotide sequence
complementary to a topoisomerase recognition site, such that PCR
introduces a functional recognition site in the opposite strand
(see primer sequences in FIG. 9D), and, optionally, a recombination
site, thereby producing an amplified first nucleic acid molecule
having a first end and a second end, wherein the amplified first
nucleic acid molecule has a topoisomerase recognition site at or
near the 5' terminus and a topoisomerase recognition site at or
near the 3' terminus of the first end or of the second end or of
both ends; and 2) contacting a) the amplified first nucleic acid
molecule; b) at least a second nucleic acid molecule having a first
end and a second end, wherein the second nucleic acid molecule has,
or can be made to have, a 5' hydroxyl group and a 3' hydroxyl group
at the first end or at second end or at both ends; and c) at least
two site specific topoisomerases, under conditions such that i) at
least one topoisomerase can cleave the topoisomerase recognition
site at or near the 5' terminus of the end of the amplified first
nucleic acid molecule, and can effect its ligating activity, and
ii) at least one topoisomerase can cleave the topoisomerase
recognition site at or near the 3' terminus of the end of the
amplified first nucleic acid molecule, and can effect its ligating
activity. Accordingly, the present invention provides a nucleic
acid molecule containing, at one or both ends, a topoisomerase
recognition site at or near the 5' terminus and a topoisomerase
recognition site at or near the 3' terminus. In addition, the
invention provides such a nucleic acid molecule, which is
topoisomerase charged at the 5' terminus or the 3' terminus or
both. In certain such aspects, at least one of the nucleic acid
molecules used in the methods described herein will comprise at
least one recombination site. Further, nucleic acid molecules
generated by methods described above and elsewhere herein may also
be used in recombination reactions, such as those described
elsewhere herein.
[0070] The present invention further relates to an oligonucleotide
containing at least one recognition site of one or more type IA
site specific topoisomerases, at least one nucleotide sequence
complementary to a recognition site of one or more type IB site
specific topoisomerases and, optionally, at least one recombination
site. Such an oligonucleotide is useful, for example, as a primer
for a primer extension reaction or as one of a primer pair for
performing an amplification reaction such as PCR. Such an
oligonucleotide, referred to herein as an oligonucleotide primer,
can be one of a primer pair, which can be useful for generating a
ds nucleic acid amplification product that contains, at one end, a
type IA topoisomerase recognition site at or near the 5' terminus
and, at the same end, a type IB topoisomerase recognition site at
or near the 3' terminus. The oligonucleotide primer can further
contain a nucleotide sequence encoding (or complementary to) any
other nucleotide sequence or peptide of interest, for example, a
restriction endonuclease recognition site, a peptide tag, and, if
desired, one or more additional type IA or type IB topoisomerase
recognition sites, thereby allowing selection of one or more
convenient or readily available topoisomerases for practicing a
method of the invention. The oligonucleotide primer can further
comprise a nucleotide sequence at its 5' terminus, i.e., 5' to the
type IA topoisomerase recognition site or to the nucleotide
sequence complementary to the type IB topoisomerase recognition
site, such that, upon cleavage of the amplified first nucleic acid
molecule by a site specific topoisomerase, the nucleic acid
molecule contains a 3' or 5' overhanging sequence, respectively,
which is complementary to a 3' or 5' overhanging sequence,
respectively, of a second (or other) nucleic acid molecule to which
the first nucleic acid molecule is to be covalently linked
according to a method of the invention, or the oligonucleotide
primer can be designed such that, upon cleavage of an amplified
nucleic acid molecule generated therefrom, a blunt end
topoisomerase charged nucleic acid molecule is generated.
[0071] The invention further relates to an oligonucleotide which
contains at least one topoisomerase recognition site, or a
nucleotide sequence complementary thereto, and at least one
recombination site. Such an oligonucleotide may be used as
described above, for example as one member of a primer pair.
[0072] Oligonucleotides of the invention will often be between
15-20, 15-30, 15-50, 20-30, 20-50, 30-40, 30-50, 30-80, 30-100,
40-50, 40-70, 40-80, 40-100, 50-60, 50-80, 50-100, 15-80, 15-100,
or 20-100 (or the like) nucleotides in length.
[0073] The present invention also provides a primer pair, which
includes at least-one oligonucleotide primer as defined above,
wherein one of the primers is useful as a forward primer and the
primer is useful as a reverse primer in an amplification reaction.
The second primer in such a primer pair can, but need not, include
a type IA topoisomerase recognition site, a nucleotide sequence
complementary to a type IB topoisomerase recognition site, or both,
and can include any other nucleotide sequence of interest and/or at
least one recombination site. In one embodiment, the primer pair
includes two oligonucleotide primers of the invention, wherein one
oligonucleotide primer is useful as a forward primer and the second
oligonucleotide primer is useful as a reverse primer, such a primer
pair being useful, for example, for generating a nucleic acid
molecule amplification product having topoisomerase recognition
sites at both termini of both ends and/or one or more recombination
sites, wherein the type IA or type IB or both topoisomerase
recognition sites at the termini are the same or different.
[0074] Accordingly, the present invention further relates to a
nucleic acid molecule, which has a first end and a second end, and
which contains a type IA topoisomerase recognition site at or near
the 5' terminus and a type IB topoisomerase recognition site at or
near the 3' terminus of the first end or of the second end or of
both ends. In addition, the present invention provides a nucleic
acid molecule as defined above, except wherein the nucleic acid
molecule is a topoisomerase charged molecule, comprising a stably
bound type IA topoisomerase or a type IB topoisomerase or both, at
one or both ends, as desired. These nucleic acid molecules may
further comprise one or more recombination sites.
[0075] In one embodiment, the first nucleic acid molecule, as well
as other nucleic acids used in methods of the invention, comprises
an expressible nucleotide sequence which encodes molecules such as
a polypeptide (which may be, e.g., a polypeptide with an intein),
an antisense nucleotide sequence, interference RNA (i.e., "RNAi")
molecule(s), a ribozyme, a transfer RNA (i.e., a tRNA, including
but not limited to a supressor tRNA), a triplexing nucleotide
sequence, and the like, and the second (or other) nucleic acid
molecule comprises a transcription regulatory element such as a
promoter (e.g., a GAL4 operator), an operator (e.g., a tet
operator, a galactose operon operator, a lac operon operator, and
the like), an enhancer, a silencer, a translation start site, or a
polyadenylation signal, or encodes a translation regulatory element
such as an initiator methionine, a STOP codon, a cell
compartmentalization domain, a homology domain, or the like, or a
combination thereof in operative linkage. A second (or other)
nucleic acid molecule, as well as other nucleic acids used in
methods of the invention, which can be an amplified second (or
other) nucleic acid molecule prepared as for the amplified first
nucleic acid molecule, also can comprise one or more multiple
cloning sites ("MCS"), a detectable label, for example, an enzyme,
a substrate for an enzyme, a fluorescent compound, a luminescent
compound, a chemiluminescent compound, a radionuclide, a
paramagnetic compound, and biotin; or can include a tag, which can
be an oligonucleotide tag or can be a peptide tag, for example, a
polyhistidine tag, a V5 epitope, or a myc epitope.
[0076] In another embodiment, a method of the invention is
performed using a first nucleic acid molecule that encodes a
polypeptide (e.g., a polypeptide which contains an intein), or a
domain thereof, and a second (or other) nucleic acid molecule that
encodes a transcription activation domain or a DNA binding domain.
Such a method can be used to generate covalently linked ds
recombinant nucleic acid molecules that encode chimeric
polypeptides useful for performing a two hybrid assay system,
particularly a high throughput two hybrid assay. In still another
embodiment, the first nucleic acid molecules comprises a plurality
of nucleotide sequences, which can be a cDNA library, a
combinatorial library of nucleotide sequences, a variegated
population of nucleotide sequences, or the like.
[0077] A method of the invention provides a means to generate a
covalently linked ds recombinant nucleic acid molecule useful for
site specific insertion into a target genomic DNA sequence. The
target genomic DNA sequence can be any genomic sequence,
particularly a gene, and preferably a gene for which some or all of
the nucleotide sequence is known. The method can be performed
utilizing two sets of PCR primer pairs and a nucleic acid molecule.
The nucleic acid molecule has a first end and a second end and
encodes a polypeptide, for example, a selectable marker, wherein
the nucleic acid molecule comprises a topoisomerase recognition
site or cleavage product thereof at the 3' terminus of each end
and, optionally, a hydroxyl group at the 5' terminus of each end,
and wherein, preferably, the 5' termini comprise overhanging
sequences, which are different from each other. Similarly, the
nucleic acid molecule can comprise a topoisomerase recognition site
or cleavage product thereof at or near the 5' terminus of one or
both ends and, optionally, a hydroxyl group at the 3' terminus of
one or both end, and wherein one or both the 3' termini can
comprise overhanging sequences, which can be the same as or,
preferably, different from each other; or the 5' terminus and 3'
terminus of one or both ends of the nucleic acid molecule each can
comprise a topoisomerase recognition site or cleavage product
thereof (see FIG. 11). In certain such aspects, at least one of the
nucleic acid molecules used in the methods described herein will
comprise at least one recombination site. Further, nucleic acid
molecules generated by methods described above and elsewhere herein
may also be used in recombination reactions, such as those
described elsewhere herein.
[0078] The two sets of PCR primer pairs will generally be selected
such that, in the presence of an appropriate DNA polymerase such as
Taq polymerase and a template comprising the sequences to be
amplified, the primers amplify portions of a genomic DNA sequence
that are upstream (and adjacent to) and downstream (and adjacent
to) of the target site for insertion of the polypeptide (e.g.,
selectable marker). The sets of PCR primer pairs also are designed
such that the amplification products contain a topoisomerase
recognition site at least at the end to be covalently linked to the
selectable marker, including at or near the 5' terminus, or the 3'
terminus, or both, as appropriate for the particular method of the
invention being practiced. As such, the first PCR primer pair can
include, for example, 1) a first primer, which comprises, in an
orientation from 5' to 3', a nucleotide sequence complementary to a
5' overhanging sequence of the end of the selectable marker to
which the amplification product is to be covalently linked, a
nucleotide sequence complementary to a topoisomerase recognition
site, such that PCR introduces a functional recognition site in the
opposite strand (see primer sequences in FIG. 9D), and a nucleotide
sequence complementary to a 3' sequence of a target genomic DNA
sequence; and 2) a second primer, which comprises a nucleotide
sequence of the target genomic DNA upstream of the 3' sequence to
which the first primer is complementary. The second PCR primer pair
includes 1) a first primer, which comprises, from 5' to 3', a
nucleotide sequence complementary to the 5' overhanging sequence of
the end of the selectable marker to which it is to be covalently
linked, a nucleotide sequence complementary to a topoisomerase
recognition site, such that PCR introduces a functional recognition
site in the opposite strand (see primer sequences in FIG. 9D), and
a nucleotide sequence of a 5' sequence of a target genomic DNA
sequence, wherein the 5' sequence of the target genomic DNA is
downstream of the 3' sequence of the target genomic DNA to which
the first primer of the first PCR primer pair is complementary; and
2) a second primer, which comprises a nucleotide sequence
complementary to a 3' sequence of the target genomic DNA that is
downstream of the 5' sequence of the target genomic DNA contained
in the first primer.
[0079] Upon contact of the nucleic acid molecule comprising the
selectable marker, the PCR amplification products, and at least one
topoisomerase, a covalently linked ds recombinant nucleic acid
molecule is generated according to a method of the invention. The
generated ds recombinant nucleic acid molecule is useful for
performing homologous recombination in a genome, for example, to
knock-out the function of a gene in a cell, or to confer a novel
phenotype on the cell containing the generated ds recombinant
nucleic acid molecule. The method can further be used to produce a
transgenic non-human organism having the generated recombinant
nucleic acid molecule stably maintained in its genome.
[0080] The present invention also relates to compositions prepared
according to the methods of the invention, and to compositions
useful for practicing the methods. Such compositions can include
one or more reactants used in the methods of the invention and/or
one or more ds recombinant nucleic acid molecules produced
according to a method of the invention. Such compositions can
include, for example, one or more nucleic acid molecules with one
or more topoisomerase recognition sites; one or more
topoisomerase-charge nucleic acid molecules; one or more nucleic
acid molecules comprising one or more recombination sites; one or
more primers useful for preparing a nucleic acid molecule
containing a topoisomerase recognition site at one or both termini
of one or both ends of an amplification product prepared using the
primer; one or more topoisomerases; one or more substrate nucleic
acid molecules, including, for example, nucleotide sequences
encoding tags, markers, regulatory elements, or the like; one or
more covalently linked ds recombinant nucleic acid molecules
produced according to a method of the invention; one or more cells
containing or useful for containing a nucleic acid molecule,
primer, or recombinant nucleic acid molecule as disclosed herein;
one or more polymerases for performing a primer extension or
amplification reaction; one or more reaction buffers; and the like.
In one embodiment, a composition of the invention comprises two or
more different topoisomerase-charged nucleic acid molecules and/or
two or more different recombination sites. The composition can
further comprise at least one topoisomerase. A composition of the
invention also can comprise a site specific topoisomerase and a
covalently linked ds recombinant nucleic acid molecule, wherein the
recombinant nucleic acid molecule contains at least one
topoisomerase recognition site for the site specific topoisomerase
in each strand, and wherein a topoisomerase recognition site in one
strand is within about 100 nucleotides of a topoisomerase
recognition site in the complementary strand, generally within
about five, ten, twenty or thirty nucleotides.
[0081] Product molecules produced by methods of the invention may
comprise any combination of starting molecules (or portions
thereof) and can be any size and be in any form (e.g., circular,
linear, supercoiled, etc.), depending on the starting nucleic acid
molecule or segment, the location of the recombination sites on the
molecule, and the order of recombination of the sites.
[0082] Any of the product molecules of the invention may be further
manipulated, analyzed or used in any number of standard molecular
biology techniques or combinations of such techniques (in vitro or
in vivo). These techniques include sequencing, amplification,
nucleic acid synthesis, protein or peptide expression (for example,
fusion protein expression, antibody expression, hormone expression
etc.), protein-protein interactions (2-hybrid or reverse 2-hybrid
analysis), homologous recombination or gene targeting, and
combinatorial library analysis and manipulation. The invention also
relates to cloning the nucleic acid molecules of the invention
(preferably by recombination) into one or more vectors or
converting the nucleic acid molecules of the invention into a
vector by the addition of certain functional vector sequences
(e.g., origins of replication). In one aspect, recombination and/or
topoisomerase-mediated joining is accomplished in vitro and further
manipulation or analysis is performed directly in vitro. Thus,
further analysis and manipulation will not be constrained by the
ability to introduce the molecules of the invention into a host
cell and/or maintained in a host cell. Thus, less time and higher
throughput may be accomplished by further manipulating or analyzing
the molecules of the invention directly in in vitro, although in
vitro analysis or manipulation can be done after passage through
host cells or can be done directly in vivo (while in the host
cells).
[0083] Nucleic acid synthesis steps, according to the invention,
may comprise:
[0084] (a) mixing a nucleic acid molecule of interest or template
with one or more primers and one or more nucleotides to form a
mixture; and
[0085] (b) incubating said mixture under conditions sufficient to
synthesize a nucleic acid molecule complementary to all or a
portion of said molecule or template.
[0086] The synthesized molecule may then be used as a template for
further synthesis of a nucleic acid molecule complementary to all
or a portion of the first synthesized molecule. Accordingly, a
double stranded nucleic acid molecule (e.g., DNA) may be prepared.
Preferably, such second synthesis step is preformed in the presence
of one or more primers and one or more nucleotides under conditions
sufficient to synthesize the second nucleic acid molecule
complementary to all or a portion of the first nucleic acid
molecule. Typically, synthesis of one or more nucleic acid
molecules is performed in the presence of one or more polymerases
(preferably DNA polymerases which may be thermostable or
mesophilic), although reverse transcriptases may also be used in
such synthesis reactions. Accordingly, the nucleic acid molecules
used as templates for the synthesis of additional nucleic acid
molecules may be RNA, mRNA, DNA or non-natural or derivative
nucleic acid molecules. Nucleic acid synthesis, according to the
invention, may be facilitated by incorporating one or more primer
sites into the product molecules through the use of starting
nucleic acid molecules containing such primer sites. Thus, by the
methods of the invention, primer sites may be added at one or a
number of desired locations in the product molecules, depending on
the location of the primer site within the starting molecule and
the order of addition of the starting molecule in the product
molecule.
[0087] Sequencing steps, according to the invention, may
comprise:
[0088] (a) mixing a nucleic acid molecule to be sequenced with one
or more primers, one or more nucleotides and one or more
termination agents to form a mixture;
[0089] (b) incubating said mixture under conditions sufficient to
synthesize a population of molecules complementary to all or a
portion of said molecules to be sequenced; and
[0090] (c) separating said population to determine the nucleotide
sequence of all or a portion of said molecule to be sequenced.
[0091] Such sequencing steps are preferably performed in the
presence of one or more polymerases (e.g., DNA polymerases and/or
reverse transcriptases) and one or more primers. Preferred
terminating agents for sequencing include derivative nucleotides
such as dideoxynucleotides (ddATP, ddTTP, ddGTP, ddCTP and
derivatives thereof). Nucleic acid sequencing, according to the
invention, may be facilitated by incorporating one or more
sequencing primer sites into the product molecules through the use
of starting nucleic acid molecules containing such primer sites.
Thus, by the methods of the invention, sequencing primer sites may
be added at one or a number of desired locations in the product
molecules, depending on the location of the primer site within the
starting molecule and the order of addition of the starting
molecule in the product molecule.
[0092] Protein expression steps, according to the invention, may
comprise:
[0093] (a) obtaining a nucleic acid molecule to be expressed which
comprises one or more expression signals; and
[0094] (b) expressing all or a portion of the nucleic acid molecule
under control of said expression signal thereby producing a peptide
or protein encoded by said molecule or portion thereof.
[0095] In this context, the expression signal may be said to be
operably linked to the sequence to be expressed. The protein or
peptide expressed is preferably expressed in a host cell (in vivo),
although expression may be conducted in vitro using techniques well
known in the art. Upon expression of the protein or peptide, the
protein or peptide product may optionally be isolated or purified.
Moreover, the expressed protein or peptide may be used in various
protein analysis techniques including 2-hybrid interaction, protein
functional analysis and agonist/antagonist-protein interactions
(e.g., stimulation or inhibition of protein function through drugs,
compounds or other peptides). The novel and unique hybrid proteins
or peptides (e.g., fusion proteins) produced by the invention and
particularly from expression of the combinatorial molecules of the
invention may generally be useful for therapeutics. Protein
expression, according to the invention, may be facilitated by
incorporating one or more transcription or translation signals or
regulatory sequences, start codons, termination signals, splice
donor/acceptor sequences (e.g., intronic sequences) and the like
into the product molecules through the use of starting nucleic acid
molecules containing such sequences. Thus, by the methods of the
invention, expression sequences may be added at one or a number of
desired locations in the product molecules, depending on the
location of such sequences within the starting molecule and the
order of addition of the starting molecule in the product
molecule.
[0096] Homologous recombination, according to the invention, may
comprise:
[0097] (a) mixing at least a first nucleic acid molecule of the
invention (which is preferably a product molecule) comprising one
or more recombination sites and/or one or more toposiomerase
recognition sites with at least one target nucleic molecule,
wherein Said first and target molecules have one or more homologous
sequences; and
[0098] (b) causing said first and target nucleic acid molecules to
recombine by homologous recombination. One example of a nucleic
acid construct that can be used for homologous recombination is
depicted in FIG. 37. The invention further includes methods for
preparing nucleic acid molecules which can be used for homologous
recombination, and nucleic acid molecules prepared by such methods,
as well as cells which have undergone homologous recombination
according to methods of the invention.
[0099] Such homologous recombination may occur in vitro, but
preferably is accomplished in vivo (e.g., in a host cell).
Preferably, homologous recombination causes transfer of all or a
portion of a nucleic acid molecule of the invention containing
recombination sites (the first nucleic acid molecule) into one or
more positions of the target nucleic acid molecule containing
homologous sequences. Selection of such homologous recombination
may be facilitated by positive or negative selection (e.g., using
selectable markers) to select for a desired product and/or against
an undesired product. In a preferred aspect, the nucleic acid
molecule of the invention comprises at least one selectable marker
and at least two sequences which are homologous to the target
molecule. Preferably, the first molecule comprises at least two
homologous sequences flanking at least one selectable marker.
[0100] The present invention thus facilitates construction of gene
targeting nucleic acid molecules or vectors which may be used to
knock-out or mutate a sequence or gene of interest (or alter
existing sequences, for example to convert a mutant sequence to a
wild type sequence), particularly genes or sequences within a host
or host cells such as animal, plant, human, insect, bacteria, and
the like or sequences of adventitious agents such as viruses within
such host or host cells. Such gene targeting may preferably
comprise targeting a sequence on the genome of such host cells.
Such gene targeting may be conducted in vitro or in vivo. Thus, in
a preferred aspect, the invention relates to a method of targeting
or mutating a sequence or a gene comprising:
[0101] (a) obtaining at least one nucleic acid molecule of the
invention comprising one or more recombination sites and/or one or
more topoisomerase recognition sites (and preferably one or more
selectable markers), wherein said molecule comprises one or more
sequences homologous to the target gene or sequence of interest
(said one or more homologous sequences preferably flank one or more
selectable markers on the molecule of the invention); and
[0102] (b) contacting said molecule with one or more target genes
or sequences of interest under conditions sufficient to cause
homologous recombination at one or more sites between said target
sequence or gene of interest and said molecule of the invention,
thereby causing insertion of all or a portion of the molecule of
the invention within the target sequence or gene.
[0103] Such targeting method may cause deletion, inactivation or
partial inactivation of the sequence or target gene such that an
expression product (typically a protein or peptide) normally
expressed by such sequence is not produced or produced at a higher
or lower level or to the extent produced is has an altered protein
sequence which may result in more or less activity or in an
inactive or partially inactive expression product. The selectable
marker preferably present on the molecule of the invention
facilitates selection of candidates (for example host cells) in
which the homologous recombination event was successful. Thus, the
present invention provides a method to produce host cells, tissues,
organs, and animals (e.g., transgenic animals) containing the
modified gene or sequence produced by the targeting methods of the
invention. The modified sequence or gene preferably comprises at
least one recombination site and/or at least one selectable marker
provided by the molecule of the invention.
[0104] Thus, the present invention more specifically relates to a
method of targeting or mutating a sequence or a gene
comprising:
[0105] (a) obtaining at least one nucleic acid molecule of the
invention comprising one or more recombination sites, at least one
selectable marker flanked by one or more sequences homologous to
the target gene or sequence of interest and, optionally, one or
more topoisomerase recognition sites;
[0106] (b) contacting said molecule with one or more target genes
or sequences of interest under conditions sufficient to cause
homologous recombination at one or more sites between said target
sequence or gene of interest and said molecule, thereby causing
insertion of all or a portion of the molecule of the invention (and
preferably causing insertion of at least one selectable marker
and/or at least one recombination site) within the target sequence
or gene; and
[0107] (c) optionally selecting for said sequence or gene
comprising all or a portion of the molecule of the invention or for
a host cell containing said gene or sequence containing all or a
portion of said molecule of the invention.
[0108] In another aspect of the invention, recombination sites
introduced into targeted sequences according to the invention may
be used to excise or remove all or a portion of the molecule
inserted into the target sequence. Thus, the invention allows for
in vitro or in vivo removal of such sequences and thus may allow
for reactivation of the target gene or sequence. In some
embodiments, after identification and isolation of a sequence
containing the alterations introduced as above, a selectable marker
present on the molecule of the present invention may be
removed.
[0109] The present invention also provides methods for cloning the
starting or product nucleic acid molecules of the invention into
one or more vectors or converting the product molecules of the
invention into one or more vectors. In one aspect, the starting
molecules are recombined to make one or more product molecules and
such product molecules are cloned, (preferably by recombination)
into one or more vectors. In another aspect, the starting molecules
are cloned directly into one or more vectors such that a number of
starting molecules are joined within the vector, thus creating a
vector containing the product molecules of the invention. In
another aspect, the starting molecules are cloned directly into one
or more vectors such that the starting molecules are not joined
within the vector (i.e., the starting molecules are separated by
vector sequences). In yet another aspect, a combination of product
molecules and starting molecules may be cloned in any order into
one or more vectors, thus creating a vector comprising a new
product molecule resulting from a combination of the original
starting and product molecules.
[0110] Thus, the invention relates to a method of cloning
comprising:
[0111] (a) obtaining at least one nucleic acid molecule of the
invention comprising one or more recombination sites and/or one or
more topoisomerase recognition sites; and
[0112] (b) transferring all or a portion of said molecule into one
or more vectors. The invention further includes vectors prepared by
such methods, compositions comprising these vectors, and methods
using these vectors.
[0113] Such vectors will often comprise one or more recombination
sites and/or one or more topoisomerase recognition sites, and the
transfer of the molecules into such vectors is preferably
accomplished by recombination between one or more sites on the
vectors and one or more sites on the molecules of the invention. In
another aspect, the product molecules of the invention may be
converted to molecules which function as vectors by including the
necessary vector sequences (e.g., origins of replication). Thus,
according to the invention, such vectors sequences may be
incorporated into the product molecules through the use of starting
molecules containing such sequences. Such vector sequences may be
added at one or a number of desired locations in the product
molecules, depending on the location of the sequence within the
starting molecule and the order of addition of the starting
molecules in the product molecule. The product molecule containing
the vector sequences may be in linear form or may be converted to a
circular or supercoiled form by causing recombination of
recombination sites within the product molecule or by a
topoisomerase-mediated joining reaction. Often, circularization of
such product molecule is accomplished by recombining recombination
sites at or near both termini of the product molecule.
[0114] The vector sequences used in the invention may comprise one
or a number of elements and/or functional sequences and/or sites
(or combinations thereof) including one or more sequencing or
amplification primer sites, one or more multiple cloning sites, one
or more selectable markers (e.g., toxic genes, antibiotic
resistance genes, selectable markers etc.), one or more
transcription or translation sites or signals, one or more
transcription or translation termination sites, one or more
topoisomerase recognition sites, one or more topoisomerases, one or
more origins of replication, one or more recombination sites (or
portions thereof), etc. The vector sequences used in the invention
may also comprise stop codons which may be suppressed to allow
expression of desired fusion proteins as described herein. Thus,
according to the invention, vector sequences may be used to
introduce one or more of such elements, functional sequences and/or
sites into any of the nucleic acid molecule of the invention, and
such sequences may be used to further manipulate or analyze any
such nucleic acid molecule cloned into such vectors. For example,
primer sites provided by a vector (preferably located on both sides
of the insert cloned in such vector) allow sequencing or
amplification of all or a portion of a product molecule cloned into
the vector. Additionally, transcriptional or regulatory sequences
contained by the vector allows expression of peptides, polypeptides
or proteins encoded by all or a portion of the product molecules
cloned to the vector. Likewise, genes, portion of genes or sequence
tags (such as GUS, GST, GFP, His tags, epitope tags and the like)
provided by the vectors allow creation of populations of gene
fusions with the product molecules cloned in the vector or allows
production of a number of peptide, polypeptide or protein fusions
encoded by the sequence tags provided by the vector in combination
with the product sequences cloned in such vector. Such genes,
portions of genes or sequence tags may be used in combination with
optionally suppressed stop codons to allow controlled expression of
fusion proteins encoded by the sequence of interest being cloned
into the vector and the vector supplied gene or tag sequence. In a
construct, the vector may comprise one or more recombination sites,
one or more stop codons and one or more tag sequences. In some
embodiments, the tag sequences may be adjacent to a recombination
site. Optionally, a stop codon may be incorporated into the
sequence of the tag or in the sequence of the recombination site in
order to allow controlled addition of the tag sequence to the gene
of interest. In embodiments of this type, the gene of interest may
be inserted into the vector by recombinational cloning such that
the tag and the coding sequence of the gene of interest are in the
same reading frame. The gene of interest may be provided with
translation initiation signals, e.g., Shine-Delgarno sequences,
Kozak sequences and/or IRES sequences, in order to permit the
expression of the gene with a native N-terminal when the stop codon
is not suppressed. The gene of interest may also be provided with a
stop codon at the 3'-end of the coding sequence. In some
embodiments, a tag sequence may be provided at both the N- and
C-terminals of the gene of interest. Optionally, the tag sequence
at the N-terminal may be provided with a stop codon and the gene of
interest may be provided with a stop codon and the tag at the
C-terminal may be provided with a stop codon. The stop codons may
be the same or different. In some embodiments, the stop codon of
the N-terminal tag is different from the stop codon of the gene of
interest. In embodiments of this type, suppressor tRNAs
corresponding to one or both of the stop codons may be provided.
When both are provided, each of the suppressor tRNAs may
independently be provided on the same vector, a different vector or
in the host cell genome. The suppressor tRNAs need not both be
provided in the same way, for example, one may be provided on the
vector containing the gene of interest while the other may be
provided in the host cell genome. In this way, the nucleic acid
molecules of one such aspect of the invention may comprise a
suppressible stop codon that separates two coding regions.
Depending on the location of the expression signals (e.g.,
promoters), expression of the suppressor tRNA results in
suppression of the stop codon(s), thereby allowing the production
of a fusion peptide, for example a fusion peptide having an
affinity tag sequence at the N- and/or C-terminus of the expressed
protein. By not suppressing the stop codon(s), expression of the
sequence of interest without the N- and/or C-terminal tag sequence
may be accomplished. Thus, the invention allows through
recombination efficient construction of vectors containing a gene
or sequence of interest (e.g., one or more open reading frames or
"orfs") for controlled expression of fusion proteins depending on
the need. Preferably, the starting nucleic acid molecules or
product molecules of the invention which are cloned into one or
more vectors comprise at least one open reading frame (orf). Such
starting or product molecules may also comprise functional
sequences (e.g., primer sites, transcriptional or translation sites
or signals, termination sites (e.g., stop codons which may be
optionally suppressed), origins of replication, and the like) and
preferably comprises sequences that regulate gene expression
including transcriptional regulatory sequences and sequences that
function as internal ribosome entry sites (IRES). Preferably, at
least one of the starting or product molecules and/or vectors
comprise sequences that function as a promoter. Such starting or
product molecules and/or vectors may also comprise transcription
termination sequences, selectable markers, restriction enzyme
recognition sites, and the like.
[0115] In some embodiments, the vector comprises two copies of the
same selectable marker, each copy flanked by recombination sites
and/or topoisomerase recognition sites. In other embodiments, the
vector comprises two different selectable markers each flanked by
two recombination sites. In some embodiments, one or more of the
selectable markers may be a negative selectable marker.
[0116] In a specific aspect, the invention provides a method of
cloning comprising providing at least a first nucleic acid molecule
comprising at least a first and a second recombination site and at
least a second nucleic acid molecule comprising at least a third
and a fourth recombination site, wherein either the first or the
second recombination site is capable of recombining with either the
third or the fourth recombination site and conducting a
recombination reaction such that the two nucleic acid molecules are
recombined into one or more product nucleic acid molecules and
cloning the product nucleic acid molecules into one or more
vectors. In certain such embodiments, the recombination sites flank
the first and/or second nucleic acid molecules. Moreover, the
cloning step is often accomplished by the recombination reaction of
the product molecule into a vector comprising one or more
recombination sites. In one aspect, the cloning step comprises
conducting a recombination reaction between the sites in the
product nucleic acid molecule that did not react in the first
recombination reaction with a vector having recombination sites
capable of recombining with the unreacted sites.
[0117] In some embodiments, a recombination site and/or a
topoisomerase recognition site may be attached to a molecule of
interest using conventional conjugation technology. For example,
oligonucleotides comprising the recombination site and/or
topoisomerase recognition site can be synthesized so as to include
one or more reactive functional moieties which may be the same or
different. Suitable reactive functional moieties include, but are
not limited to, amine groups, epoxy groups, vinyl groups, thiol
groups and the like. The synthesis of oligonucleotides comprising
one or more reactive functional moieties is routine in the art.
Once synthesized, oligonucleotides comprising one or more reactive
functional moieties may be attached to one or more reactive groups
present on the molecule or compound of interest. The
oligonucleotides may be attached directly by reacting one or more
of the reactive functional moieties with one or more of the
reactive functional groups. In some embodiments, the attachment may
be effected using a suitable linking group capable of reacting with
one or more of the reactive functional moieties present on the
oligonucleotide and with one or more of the reactive groups present
on the molecule of interest. In other embodiments, both direct
attachment and attachment through a linking group may be used.
Those skilled in the art will appreciate that the reactive
functional moieties on the oligonucleotide may be the same or
different as the reactive functional moieties on the molecules
and/or compounds of interest. Suitable reagents and techniques for
conjugation of the oligonucleotide to the molecule of interest may
be found in Hermanson, Bioconjugate Techniques, Academic Press
Inc., San Diego, Calif., 1996.
[0118] The invention also relates to compositions for carrying out
the methods of the invention, and kits comprising such
compositions, and to compositions created while carrying out the
methods of the invention.
[0119] Compositions, methods and kits of the invention may be
prepared and carried out using a phage-lambda site-specific
recombination system. Further, such compositions, methods and kits
may be prepared and carried out using the GATEWAYT.TM.
Recombinational Cloning System and/or the TOPO.RTM. Cloning System
and/or the pENTR Directional TOPO.RTM. Cloning System, which are
available from Invitrogen Corporation (Carlsbad, Calif.).
[0120] In other aspects, the invention provides isolated nucleic
acid molecules comprising one or more (e.g., one, two, three, four,
five, etc.) recombination sites and/or one or more (e.g., one, two,
three, four, five, etc.) topoisomerase recognition sites. One such
molecule of the invention will contain two or more recombination
sites flanking one topoisomerase recognition site. Another such
molecule of the invention will contain two or more recombination
sites and two or more topoisomerase recognition sites, wherein each
recombination site may flank a topoisomerase recognition site.
Nucleic acid molecules according to this aspect of the invention
may be linear, circular, or have any of a variety of geometries and
structures, such as coiled, supercoiled, etc. Recombination sites
advantageously used in nucleic acid molecules according to this
aspect of the invention include, but are not limited to, att sites
(including, but not limited to, attB sites, attP sites, attL sites,
attR sites, and the like), lox sites (including, but not limited
to, loxP sites, loxP511 sites, and the like), psi sites, dif sites,
cer sites, frt sites, and mutants, variants, and derivatives of
these recombination sites that retain the ability to undergo
recombination. Topoisomerase recognition sites advantageously used
in the nucleic acid molecules of this aspect of the invention are
preferably recognized and bound by a type I topoisomerase (such as
type IA topoisomerases (including but not limited to E. coli
topoisomerase I, E. coli topoisomerase III, eukaryotic
topoisomerase II, archeal reverse gyrase, yeast topoisomerase III,
Drosophila topoisomerase III, human topoisomerase III,
Streptococcus pneumoniae topoisomerase III, and the traE protein of
plasmid RP4) and type IB topoisomerases (including but not limited
to eukaryotic nuclear type I topoisomerase and a poxvirus (such as
that isolated from or produced by vaccinia virus, Shope fibroma
virus, ORF virus, fowlpox virus, molluscum contagiosum virus and
Amsacta moorei entomopoxvirus)), and type II topoisomerase
(including, but not limited to, bacterial gyrase, bacterial DNA
topoisomerase IV, eukaryotic DNA topoisomerase II (such as calf
thymus type II topoisomerase), and T-even phage-encoded DNA
topoisomerase).
[0121] The invention also provides vectors (which may be expression
vectors) comprising such isolated nucleic acid molecules. Exemplary
vectors according to this aspect of the invention include, but are
not limited to, pcDNAGW-DT(sc), pENTR-DT(sc), pcDNA-DEST41,
pENTR/D-TOPO, pENTR/SD/D-TOPO, pcDNA3.2N5/GWD-TOPO and
pcDNA6.2N5/GWD-TOPO. The invention also provides host cells
comprising such the isolated nucleic acid molecules or vectors of
the invention.
[0122] In related aspects, the invention provides in vitro methods
of cloning a nucleic acid molecule. Methods according to this
aspect of the invention may comprise one or more steps,
including:
[0123] (a) obtaining a nucleic acid molecule to be cloned (which in
certain embodiments may be a linear molecule (and which may be
blunt-ended or not) such as a PCR product, and which may optionally
comprise one or more genes or open reading frames);
[0124] (b) mixing the nucleic acid molecule to be cloned in vitro
with a vector (which may be an expression vector) comprising at
least a first topoisomerase recognition site flanked by at least a
first recombination site and at least a second recombation site,
wherein the first and second recombination sites do not recombine
with each other, and with at least one topoisomerase; and
[0125] (c) incubating the mixture under conditions such that the
nucleic acid molecule to be cloned is inserted into the vector
between the first and second topoisomerase recognition sites,
thereby producing a first product molecule comprising the nucleic
acid molecule localized between the first and second recombination
sites. The invention further includes nucleic acid molecules
prepared by the above methods.
[0126] Methods according to this aspect of the invention may
comprise one or more additional steps, including, for example,
contacting the first product molecule with at least one vector
comprising at least a third and fourth recombination sites that do
not recombine with each other, under conditions favoring
recombination between the first and third and between the second
and fourth recombination sites, thereby producing at least one
second product molecule. According to the invention, the first
and/or second product molecules produced by these methods may be
inserted into a host cell. The vectors used in this aspect of the
invention may comprise at least one additional nucleic acid
sequence selected from the group consisting of a selectable marker,
a cloning site, a restriction site, a promoter, an operon, an
origin of replication, and a gene or partial gene (i.e., a gene
fragment or element).
[0127] Recombination sites and topoisomerase recognition sites used
in the methods of this aspect of the invention include, but are not
limited to, those described elsewhere herein. In particular
methods, the second product nucleic acid molecule and the vector
are combined in the presence of at least one recombination protein,
which may be but is not limited to Cre, Int, IHF, X is, F is, Hin,
Gin, Cin, Tn3 resolvase, TndX, XerC, or XerD. In certain such
embodiments, the recombination protein is Cre, Int, X is, IHF or
Fis.
[0128] The invention also provides kits comprising these isolated
nucleic acid molecules of the invention, which may optionally
comprise one or more additional components selected from the group
consisting of one or more topoisomerases, one or more recombination
proteins, one or more vectors, one or more polypeptides having
polymerase activity, and one or more host cells.
[0129] Other preferred embodiments of the invention will be
apparent to one or ordinary skill in the art in light of what is
known in the art, in light of the following drawings and
description of the invention, and in light of the claims.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0130] FIG. 1 is a schematic representation of a basic
recombinational cloning reaction.
[0131] FIG. 2 is a schematic representation of the use of the
present invention to clone two nucleic acid segments by performing
an LR recombination reaction.
[0132] FIG. 3 is a schematic representation of the use of the
present invention to clone two nucleic acid segments by joining the
segments using an LR reaction and then inserting the joined
fragments into a Destination Vector using a BP recombination
reaction.
[0133] FIG. 4 is a schematic representation of the use of the
present invention to clone two nucleic acid segments by performing
a BP reaction followed by an LR reaction.
[0134] FIG. 5 is a schematic representation of two nucleic acid
segments having attB sites being cloned by performing a first BP
reaction to generate an attL site on one segment and an attR on the
other followed by an LR reaction to combine the segments. In
variations of this process, P1, P2, and/or P3 can be
oligonucleotides or linear stretches of nucleotides.
[0135] FIG. 6 is a schematic representation of the cloning of two
nucleic acid segments into two separate sites in a Destination
Vector using an LR reaction.
[0136] FIG. 7 is a schematic representation of the cloning of two
nucleic acid segments into two separate sites in a Destination
Vector using a BP reaction.
[0137] FIGS. 8A and 8B depict generating a covalently linked double
stranded nucleotide sequence containing an element on each end
according to a method of the invention. "PCR" indicates polymerase
chain reaction; "TOPO" indicates topoisomerase; topoisomerase shown
as circle attached to sequence; "P1" and "P2" indicate PCR primers.
Topoisomerase recognition site is indicated in bold print.
(5'-CGGAACAAGGG (SEQ ID NO: 63); 3'-GGGAACCGGAT (SEQ ID NO: 64);
5'-CCCTTCGGAACAAGGG (SEQ ID NO: 65); 5'-CCCTTGGCCATAAGGG (SEQ ID
NO: 66); 5'-GGCCATAAGGG (SEQ ID NO: 135); 3'-GGGAAGCCTTG (SEQ ID
NO: 136))
[0138] FIGS. 9A-9C show the ends of PCR products representing a
cytomegalovirus promoter element ("CMV"), a green fluorescent
protein element ("GFP"), and a bovine growth hormone
polyadenylation signal ("BGH") element. Primers used to construct
the PCR products of FIGS. 9A, 9B and 9C are indicated by an "F"
number (see FIG. 9D). The portion of one or both ends including the
topoisomerase recognition site (CCCTT) is shown. Bold print
indicates overhanging sequences. In FIGS. 9A and 9B, one (FIG. 9B)
or both (FIG. 9A) of the overhang sequences are palindromic in
nature. Sequences are shown in conventional orientation, with the
top strand in a 5' to 3' orientation from left to right, and the
bottom strand in a 3' to 5' orientation from left to right. Number
in parentheses above or below sequences indicates SEQ ID NOs.
[0139] FIGS. 10A and 10B show constructs (FIG. 10A) and results
(FIG. 10B) of studies examining the ability to use covalently
linked ds recombinant nucleic acid molecules that encode
polypeptides for performing a two hybrid assay. FIG. 10A shows the
amount of each construct used for transfection. A "p" preceding an
amount or volume of reactant indicates plasmid form, "1" indicates
linear form, and "PCR" indicates PCR amplification reaction
mixture. FIG. 10B shows the level of .beta.-galactosidase activity
("LacZ activity") associated with each transfected sample.
Increased LacZ activity is indicative of a positive
interaction.
[0140] FIGS. 11A to 11F represent various embodiments of the
composition and methods for generating a ds recombinant nucleic
acid molecule covalently linked in one strand. Note nicks in one or
both strands of the molecules shown in FIGS. 11B-11F.
[0141] FIGS. 12A to 12D illustrate various embodiments of
compositions and methods of the invention for generating a
covalently linked ds recombinant nucleic acid molecule.
Topoisomerase is shown as a solid circle, and is either attached to
a terminus of a substrate nucleic acid molecule or is released
following a linking reaction. As illustrated, the substrate nucleic
acid molecules have 5' overhangs, although they similarly can have
3' overhangs or can be blunt ended. In addition, while the
illustrated nucleic acid molecules are shown having the
topoisomerases bound thereto (topoisomerase-charged), one or more
of the termini shown as having a topoisomerase bound thereto also
can be represented as having a topoisomerase recognition site, in
which case the joining reaction would further require addition of
one or more site specific topoisomerases, as appropriate.
[0142] FIG. 12A shows a first nucleic acid molecule having a
topoisomerase linked to each of the 5' terminus and 3' terminus of
one end, and further shows linkage of the first nucleic acid
molecule to a second nucleic acid molecule.
[0143] FIG. 12B shows a first nucleic acid molecule having a
topoisomerase bound to the 3' terminus of one end, and a second
nucleic acid molecule having a topoisomerase bound to the 3'
terminus of one end, and further shows a covalently linked ds
recombinant nucleic acid molecule generated due to contacting the
ends containing the topoisomerase-charged substrate nucleic acid
molecules.
[0144] FIG. 12C shows a first nucleic acid molecule having a
topoisomerase bound to the 5' terminus of one end, and a second
nucleic acid molecule having a topoisomerase bound to the 5'
terminus of one end, and further shows a covalently linked ds
recombinant nucleic acid molecule generated due to contacting the
ends containing the topoisomerase-charged substrate nucleic acid
molecules.
[0145] FIG. 12D shows a nucleic acid molecule having a
topoisomerase linked to each of the 5' terminus and 3' terminus of
both ends, and further shows linkage of the topoisomerase-charged
nucleic acid molecule to two nucleic acid molecules, one at each
end. The topoisomerases at each of the 5' termini and/or at each of
the 3' termini can be the same or different.
[0146] FIG. 13 illustrates the generation of an expressible ds
recombinant nucleic acid molecule and amplification of the
expressible ds recombinant nucleic acid molecule. The expressible
ds recombinant nucleic acid molecule is generated from three
nucleic acid molecules, including a nucleotide sequence comprising
a promoter, a nucleotide sequence comprising a coding sequence, and
a nucleotide sequence comprising a polyadenylation signal.
Generation of the nucleic acid molecule can be facilitated by the
incorporation of complementary 5' and/or 3' overhanging sequences
at the ends of the ds nucleotides sequences to be joined. The
expressible ds recombinant nucleic acid molecule is generated by
contacting a first nucleic acid molecule having a type IA
topoisomerase at a 5' terminus of a first end and a type IB
topoisomerase at a 3' terminus of a second end, with a second
nucleic acid molecule and a third double stranded nucleotide
sequence. The expressible ds recombinant nucleic acid molecule is
amplified using a first primer that hybridizes to the second ds
recombinant nucleic acid molecule upstream of the promoter, and a
second primer that hybridizes to the third ds recombinant nucleic
acid molecule downstream of the polyadenylation signal.
[0147] FIG. 14 shows one example of a process for preparing a
double stranded nucleic acid molecule which contains a
topoisomerase (e.g., a type IA topoisomerase) bound to the 5'
terminus of one end of the molecule, wherein the same end of the
molecule further comprise a 3' overhang (see (4) in this
figure).
[0148] FIG. 15 shows two embodiments of the invention in which a
single stranded or double stranded DNA nucleotide sequence is
joined with single stranded RNA nucleotide sequence.
[0149] FIG. 16 is a schematic demonstrating the flexibility in
entry point for PCR cloning using the TOPO-Gateway.TM. or standard
Gateway.TM. cloning methodologies.
[0150] FIG. 17 is a schematic diagram of the production of
expression clones using the Gateway.TM. system and a directional
TOPO-Gateway.TM. expression vector.
[0151] FIG. 18 is a map of the multiple cloning site in plasmids
pcDNAGW-DT(sc) and pENTR-DT(sc). (SEQ ID NO: 67; amino acid
sequences SEQ ID NO: 68 and SEQ ID NO: 69)
[0152] FIG. 19 is a physical map of plasmid pcDNAGW-DT.
[0153] FIG. 20 is a physical map of plasmid pcDNA-DEST41.
[0154] FIG. 21 is a physical map of plasmid pENTR-DT.
[0155] FIG. 22 is a depiction of the physical map (FIG. 22A)
showing the TOPO cloning site in, and the nucleotide sequence (FIG.
22B (SEQ ID NO. 70)) of, plasmid pENTR/D-TOPO. The physical map
depicts the adapted, supercoiled form of the vector, while the
nucleotide sequence depicts the vector containing a start codon and
an open reading frame (atgnnnnnn . . . ). Restriction sites are
labeled to indicate the actual cleavage site. The boxed region
indicates attL sequences in the entry clone that will be
transferred into the destination vector following
recombination.
[0156] FIG. 23 is a depiction of the physical map (FIG. 23A)
showing the TOPO cloning site in, and the nucleotide sequence (FIG.
23B (SEQ ID NO. 71)) of, plasmid pENTR/SD/D-TOPO. The physical map
depicts the adapted, supercoiled form of the vector, while the
nucleotide sequence depicts the vector containing a start codon and
an open reading frame (atgnnnnnn . . . ). Restriction sites are
labeled to indicate the actual cleavage site. The boxed region
indicates attL sequences in the entry clone that will be
transferred into the destination vector following
recombination.
[0157] FIG. 24 is a depiction of the physical map (FIG. 24A) and
the nucleotide sequence (FIG. 24B-C) (SEQ ID NO: 72) of plasmid
pcDNA3.2N5/GWD-TOP07. The physical map depicts the adapted,
supercoiled form of the vector, while the nucleotide sequence
depicts the vector containing a start codon and an open reading
frame (atgnnnnnn . . . ).
[0158] FIG. 25 is a depiction of the physical map (FIG. 25A) and
the nucleotide sequence (FIG. 25B-C) (SEQ ID NO: 73) of plasmid
pcDNA6.2N5/GWD-TOP07. The physical map depicts the adapted,
supercoiled form of the vector, while the nucleotide sequence
depicts the vector containing a start codon and an open reading
frame (atgnnnnnn . . . ).
[0159] FIG. 26 is a depiction of an exemplary adaptation strategy
for pENTR/SD-dTopo (SEQ ID NO: 74: SEQ ID NO: 75: SEQ ID NO: 138:
SEQ ID NO 139), pENTR-dTopo (SEQ ID NO: 76: SEQ ID NO: 78: SEQ ID
NO: 140: SEQ ID NO: 141), and pcDNAGW-dTopo (SEQ ID NO: 77: SEQ ID
NO: 79: SEQ ID NO: 142).
[0160] FIG. 27 is a photograph of a Western blot analysis of HLA
and CAT expressed in COS cells. The genes encoding CAT (26 kDa) and
HLA (41 kDa) were amplified by PCR and either Topo-cloned into
pENTR-dTopo and transferred into pcDNA-DEST40 (lanes 2 and 5,
respectively), or cloned directly into pcDNAGW-dTopo (lanes 3 and
6, respectively). These constructs were used to transfect COS cells
and the lysates probed for recombinant V5tagged protein by Western
blot, using V5-HRP antibody conjugate. Lanes 1 and 4 represent
cells only controls.
[0161] FIG. 28 is a photograph of a gel depicting HLA and CAT
expression in E. coli. The genes encoding HLA (41 kDa) and CAT (26
kDa) were amplified by PCR and either topo cloned into
pENTR/SD-dTopo and transferred into pET-DEST42 (lanes 3 and 6,
respectively) or cloned directly into pET101-dTopo (lanes 4 and 7,
respectively). These constructs were used to transform BL21(DE3)
cells and induced to express by addition of IPTG to 1 mM for 3
hours at 37 C. Cell lysates were run on a NuPage and stained with
SafeStain.TM.. Lanes 2 and 5 represent cells uninduced cell lysates
from the respective pET-DEST42 cultures.
[0162] FIG. 29 is a schematic depiction of the binding of a
topoisomerase to a recognition site near the 3' terminus of a
target nucleic acid molecule. Upon binding of the topoisomerase,
the downstream sequence (3' to the cleavage site) can dissociate,
leaving a nucleic acid molecule having the topoisomerase covalently
bound to the newly generated 3' end. (SEQ ID NO: 80)
[0163] FIG. 30 depicts protein expression results (Western blot)
for mammalian expression cassettes that were constructed by PCR
amplification of expression elements and a gene of interest (CAT or
V5) followed by a TOPO joining reaction performed with or without
secondary PCR. Protein expression data from the expression cassette
transfected into suspension TRex-CHO cells (FIG. 30A), adherent
TRex-CHO cells (FIG. 30B), and adherent TRex-293 cells (FIG. 30C).
For the Western blot, anti-V5 or anti-CAT antibodies were used for
detection. Arrows indicate the position of the bands corresponding
to the V5 or CAT proteins.
[0164] FIG. 31 is a photograph of an ethidium bromide-stained
agarose gel containing PCR products showing that the
Gateway-compatible cassette contained inserts of the expected size.
The Gateway-compatible cassette was constructed by first generating
a CAT insert by PCR and then using a TOPO joining reaction to
introduce attBl and attB2 adaptors. The purified DNA product was
inserted into pDONR 222 using a BP reaction. Following
transformation into E. coli, PCR was performed on the colonies and
the PCR product was checked on an ethidium bromide-stained agarose
gel.
[0165] FIG. 32 is a schematic diagram depicting the preparation of
topoisomerase-charged pENTR vectors, by charging pDONR vectors with
topoisomerase and carrying out a BxP GATEWAY cloning reaction
according to methods of the invention.
[0166] FIG. 33 is a schematic diagram depicting the preparation of
topoisomerase-charged pEXP vectors, by charging pDEST vectors with
topoisomerase and carrying out an LxR GATEWAY cloning reaction,
then adding TOPO adaptors to the cut ends of the pEXP vector,
according to methods of the invention.
[0167] FIG. 34 shows a schematic outline of methods of the
invention. In the first step, nucleic acid molecules to be
assembled are generated using, for example, PCR. In the second
step, nucleic acid molecules of the first step are assembled using
methods of the invention (e.g., methods involving the use of
topoisomerase to covalently linking at least one strand of one
nucleic acid segments to another nucleic acid segment). In the
third step, assembled nucleic acid molecules generated in the
second step either may be used directly or may be amplified and
then used. Examples of uses of the assembled molecules are
described elsewhere herein.
[0168] FIG. 35 shows a schematic representation of a process for
using topoisomerase to link two nucleic acid segments, followed by
single site recombination to recombine the linked nucleic acid
segment with another nucleic acid segment. In the first step, a
topoisomerase adapted nucleic acid segment which contains an attL1
recombination site is linked to another nucleic acid segment,
referred to here as an insert (labeled "I"), using any of the
topoisomerase mediated methods described herein for connecting
nucleic acid molecules. The topoisomerase assembled nucleic acid
segments are then contacted with another nucleic acid segment which
contains a promoter, labeled "P", and an attR1 recombination site
in the presence of LR CLONASE.TM. under conditions which allow for
recombination between the two recombination sites. Recombination
results in the formation of a nucleic acid molecule which contains
the insert nucleic acid segment in operable linkage with the
promoter. Further, an attB1 recombination site is located between
the promoter and the insert in the end product. The recombination
sites shown in this figure are attL and attB sites, but any
suitable recombination sites could be used.
[0169] FIG. 36 shows a schematic representation of a process for
using topoisomerase and recombination to recombine and/or link five
separate nucleic acid segments and circularize the resulting
product. In the first step, a topoisomerase adapted nucleic acid
segment which contains attL1 and attL2 recombination sites and a
negative selection marker (labeled "NM") is linked to another
nucleic acid segment, referred to here as an insert (labeled "I"),
using any of the topoisomerase mediated methods described herein
for connecting nucleic acid molecules. The topoisomerase assembled
nucleic acid segments are then contacted with two additional
nucleic acid segments, each of which contains at least one attR
recombination site, in the presence of LR CLONASE.TM. (Invitrogen
Corporation, Carlsbad, Calif.) under conditions which allow for
recombination between the various recombination sites. In certain
such methods, for example, TOPO-adapted vectors are incubated with
one or more nucleic acid segments (e.g., one or more PCR products)
at room temperature (e.g., about 20-20.degree. C.) for about 5-30
(and preferably about 10) minutes; the reaction is then
heat-treated by incubation at about 80.degree. C. for about 20
minutes, and the reaction mixture then used in a standard LR
reaction according to manufacturer's instructions (Invitrogen
Corporation, Carlsbad Calif.), except the incubation time for the
LR reaction is increased to about 3 hours. The recombination
reactions result in the formation of a product molecule in which
the promoter is linked to (1) the insert molecule and (2) an origin
of replication (labeled "ori"). This product molecule is then
connected to a nucleic acid segment which is topoisomerase adapted
at both termini and contains a positive selection marker (labeled
"PM"). Further, the final topoisomerase linkage step results in the
formation of a circular nucleic acid molecule. The recombination
sites shown in this figure are attL and attB sites, but any
suitable recombination sites could be used.
[0170] FIG. 37 shows a schematic representation of a process for
the preparation of nucleic acid molecules for performing homologous
recombination. In this instance, three nucleic acid segments are
connected to each other using methods which involve topoisomerase
mediated covalent linkage of nucleic acid strands of the individual
segments. Two of these nucleic acid segments each contain a
positive selection marker and two attL sites which flank a negative
selection marker. Thus, the nucleic acid molecule which results
from the first step contains a nucleic acid segment, referred to
here as an insert. On each side of the insert is (1) a positive
selection marker and (2) two recombination sites which flank a
negative selection marker. LR CLONASE.TM. catalyzed recombination
in the presence of two nucleic acid segments which contain regions
that share homology to a chromosomal locus where the nucleic acid
end product is designed to integrate (labeled "HR1" and HR2")
results in the formation of the end product nucleic acid molecule
shown. As one skilled in the art would recognize, any suitable
recombination sites could be used in the process set out in this
figure.
[0171] FIG. 38 shows a schematic representation of the linking of
four nucleic acid segments using toposiomerase to generate a linear
nucleic acid molecule with recombination sites (labeled "L1" and
"L2") located near the termini. Upon toposiomerase mediated linkage
of the nucleic acid strands, no nicks are present at the junction
points. In a second step, the topoisomerase assembled nucleic acid
segments are contacted with another nucleic acid segment which
contains an origin of replication (labeled "ori"), a positive
selection marker (labeled "PM"), an attR1 recombination site, and
an attR2 recombination site in the presence of LR CLONASE.TM. under
conditions which allow for recombination between the recombination
sites. Recombination results in the formation of a circular nucleic
acid molecule as shown. The recombination sites shown in this
figure are attL and attB sites, but any suitable recombination
sites could be used.
[0172] FIG. 39 shows a schematic representation of the linking of
two nucleic acid segments in a single step process using
toposiomerase and recombination sites to generate a circular
nucleic acid molecule. One of the nucleic acid segments contains an
attL1 recombination site (labeled "L1"), a promoter (labeled "P"),
and toposiomerase molecule covalently linked to one terminus. The
other nucleic acid segment contains an attR1 recombination site
(labeled "R1"), an open reading frame (labeled "ORF"), an origin of
replication (labeled "ORI"), a positive selection marker (labeled
"PM"), and topoisomerase molecule covalently linked to one
terminus. Thus, when these two nucleic acid segments are contacted
with each other in the presence of LR CLONASE.TM. under conditions
which allow for recombination between the attL and attR
recombination sites and topoisomerase mediated linkage of nucleic
acid strands, a circular molecule is formed having the structure
indicated. The recombination sites shown in this figure are attL
and attB sites, but any suitable recombination sites could be
used.
[0173] FIG. 40 shows a schematic representation of the linking of
two nucleic acid segments using toposiomerase mediated methods to
generate a circular nucleic acid molecule. This circular molecule
contains an open reading frame (labeled "ORF") positioned between
attL1 and attL2 recombination site (labeled "L1" and "L2"). The
topoisomerase assembled product then undergoes recombination with
another circular molecule which contains attR1 and attR2
recombination sites to generate a third circular nucleic acid
molecule which contains the open reading frame positioned between
attB1 and attB2 recombination sites. Further, the open reading
frame is operably linked to a promoter. The recombination sites
shown in this figure are attL and attB sites, but any suitable
recombination sites could be used.
[0174] FIG. 41 shows an example of a process by which two nucleic
acid segments may be covalently linked to each other in both
strands at the junction where the two nucleic acid segments are
connected. As in other figures presented herein, the "lollipop"
type symbol represents a topoisomerase molecule. Further, the
arrows within the boxes represent the functional directionality of
the particular nucleic acid segment. For example, if the PCR
product is an open reading frame, then the 3' end of the coding
region (i.e., the end of the coding region which encodes the
C-terminal end of the polypeptide) would be at the point of the
arrow and the 5' end would be at the other end.
[0175] FIG. 42. T7 TOPO-linker. (A) Diagram of TOPO-activated T7
linker Three oligonucleotides were annealed, incubated with TOPO,
and the diagrammed TOPO-DNA covalent complex was purified as
described in Materials and Methods. The T7 promoter is shown in
bold, and the TOPO recognition site is underlined. (B) Use of
TOPO-linker to add a T7 RNA polymerase promoter to a PCR product
generated by Taq polymerase (A-tailed).
[0176] FIG. 43. FPLC purification. (A) Chromatogram of FPLC
purification of TOPO-activated T7 linker. A.sub.254 trace is shown
in blue, conductivity (salt concentration) in brown, and the
relative concentration of buffer B in the mixing chamber in green.
The boundaries of eleven 1 ml flow-through fractions and 39-0.2 ml
elution fractions are designated with broken red lines. The
A.sub.254 trace is actually shifted approximately 3 elution
fractions to the left as determined by gel analysis (see FIG. 44A).
(B) Magnified A.sub.254 peak showing small right shoulder
corresponding to the position of free topoisomerase (see also FIG.
44B).
[0177] FIG. 44. Gel analysis of fractions. (A) EtBr-stained 10% TBE
polyacrylamide gels. Except the undigested load, all fractions were
incubated with proteinase K prior to loading. 10 bp DNA ladder,
annealed oligos, the load, flow-through (F-T) fractions 2-5, and
elution fractions 29-40 are shown. (B) Coomassie-stained 4-12%
Bis-Tris NuPAGE gels. The sizes of some of the marker bands in kDa
are shown. "TOPO" designates the free topoisomerase control lane
(see Materials & Methods). Load, flow-through, and elution
fractions are labeled as in (A).
[0178] FIG. 45. PCR, linking reactions, and transcription. (A-D).
EtBr-stained 1.2% agarose-TAE gels. (A,C) The 1.2 kb ladder band
corresponds to 25 ng DNA. (A) Primary PCR reactions loaded to
estimate product yields. "T7-" indicates that the reverse primer
contained a 5' T7 promoter sequence. (B) The products of linking
reactions with ("+") or without ("-") T7 TOPO linker and with actin
or GFP primary PCR products from (A). (C) Products from the
secondary amplification of the linking reactions in (B). The volume
loaded from each PCR reaction is indicated. "F+T" indicates that
gene-specific forward primers and T7amp1 primer were used in the
amplification. "T" indicates that the T7amp1 primer alone was used.
Negative control ("neg ctrl") reactions used the mock linking
reactions in (B) as templates. (D) Products of transcription. "neg
ctrl" transcriptions used the negative control "F+T" secondary
amplification products shown in (C). The "T7-" transcriptions were
performed with the "T7-" primary PCR products in (A) as
templates.
[0179] FIG. 46. pUC19/actin positive control plasmid. (A) Vector
map of the positive control plasmid. The actin template sequence
was cloned into BamHI and HindIII sites in the pUC19 polylinker as
described in Materials & Methods. (B)-(D) Photographs of
ethidium bromide stained gels. (B) 6% polyacrylamide TBE gel of
linking reactions of a primary PCR product amplified from
pUC19/actin with actinF and actinR primers in the presence (right)
or absence (left) of T7 TOPO linker. (C) 1.2% agarose-TAE gel of
secondary amplification product of linked actin from (B) and the
primary product of pUC19/actin amplified with actinF and T7-actinR
primers. (D) 1.2% agarose-TAE gel DNase I digested transcription
reactions using the PCR products in (C) as templates.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0180] In the description that follows, a number of terms used in
recombinant nucleic acid technology are utilized extensively. In
order to provide a clear and more consistent understanding of the
specification and claims, including the scope to be given such
terms, the following definitions are provided.
[0181] Gene: As used herein, a gene is a nucleic acid sequence that
contains information necessary for expression of a polypeptide,
protein or functional RNA (e.g., a ribozyme, tRNA, rRNA, mRNA,
etc.). It includes the promoter and the structural gene open
reading flame sequence (orf) as well as other sequences involved in
expression of the protein.
[0182] Structural gene: As used herein, a structural gene refers to
a nucleic acid sequence that is transcribed into messenger RNA that
is then translated into a sequence of amino acids characteristic of
a specific polypeptide.
[0183] Host: As used herein, a host is any prokaryotic or
eukaryotic organism that is a recipient of a replicable expression
vector, cloning vector or any nucleic acid molecule. The nucleic
acid molecule may contain, but is not limited to, a structural
gene, a transcriptional regulatory sequence (such as a promoter,
enhancer, repressor, and the like) and/or an origin of replication.
As used herein, the terms "host," "host cell," "recombinant host"
and "recombinant host cell" may be used interchangeably. For
examples of such hosts, see Maniatis et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y. (1982).
[0184] Derivative: As used herein, a "derivative" of a specified
host is a progeny of the specified host, a modified or mutated host
obtained or derived from the specified host or its progeny, or
other recipient host that contains genetic material obtained
directly or indirectly from the specified host. Such a derivative
host may, for example, be formed by removing genetic material from
a specified host and subsequently introducing it into another host
(i.e., the progeny or other recipient host) by any conventional
methodology including, but not limited to, transformation,
conjugation, electroporation, transduction and the like. A
derivative may be formed by introducing one or more mutations or
modifications into the genome or other genetic material (e.g.
vectors, plasmids, extrachromosomal elements, etc.) of a host. Such
mutations or modifications may include one or more insertion
mutations, deletion mutations and/or substitutions or various
combinations thereof. The mutations or modifications may be
insertions into the genome or other genetic material (e.g. vectors,
plasmids, extrachromosomal elements, etc.) of the host.
Alternatively, the mutations may be deletions of one or more bases
and/or nucleic acid sequences from the genome or other genetic
material (e.g. vectors, plasmids, extrachromosomal elements, etc.)
of the host. In some instances, the mutations may be the alteration
of one or more bases in the genome of the host. Such modifications
or mutations may also comprise substituting one or more nucleic
acid bases and/or nucleic acid molecules for other nucleic acid
molecules and/or bases. In addition, one host is a derivative of a
parent host if it contains the genome of the parent host but does
not contain some or all of the same extrachromosomal nucleic acid
molecules. Thus, a strain produced by curing some or all of the
endogenous vectors from a parent strain is a derivative of the
parent strain. Derivatives of a host of the invention may also
include those hosts obtained by the addition of one or more nucleic
acid molecules into the host of interest. Nucleic acid molecules
which may be introduced into a host will be recognized by one
skilled in the art and may include, but is not limited to, vectors,
plasmids, transposons, oligonucleotides, RNA, DNA, RNA/DNA hybrids,
phage sequences, virus sequences, regardless of the form or
conformation (e.g. linear, circular, supercoiled, single stranded,
double stranded, single/double stranded hybrids and the like).
Examples of mutations or other genetic alterations which may be
incorporated into the hosts of the present invention include, but
are not limited to, mutations or alterations that create: a recK
genotype such as recA1/recA13 or recA deletions, a lacZ-genotype
that allows alpha complementation such as lacX74, lacZ.DELTA.M15 or
other lacZ deletion, a protease deficient genotype such as
.DELTA.lon and/or ompT.sup.-, an endonuclease minus genotype such
as endA1, a genotype suitable for M13 phage infection by including
the F' episome, a restriction negative, modification positive
genotype such as hsdR17(r.sub.K.sup.-, m.sub.B.sup.-), a
restriction negative, modification negative genotype such as
hsdS20(r.sub.B.sup.-, m.sub.B.sup.-), a methylase deficient
genotype such as mcrA and/or mcrB and/or mrr, a genotype suitable
for taking up large plasmids such as deoR, a genotype containing
suppressor mutations such as supE and/or supF. Other suitable
modifications are known to those skilled in the art and such
modifications are considered to be within the scope of the present
invention.
[0185] Transcriptional Regulatory Sequence: As used herein,
transcriptional regulatory sequence is a functional stretch of
nucleotides contained on a nucleic acid molecule, in any
configuration or geometry, that acts to regulate the transcription
of one or more structural genes into messenger RNA. Examples of
transcriptional regulatory sequences include, but are not limited
to, promoters, operators, enhancers, repressors, and the like.
Transcriptional regulatory sequences may also regulate the
transcription of nucleic acid molecules which encode functional
RNAs (e.g., ribozymes, tRNAs, rRNAs, mRNAs, etc.).
[0186] Promoter: As used herein, a promoter is an example of a
transcriptional regulatory sequence, and is specifically a nucleic
acid sequence generally described as the 5'-region of a gene
located proximal to the start codon. The transcription of an
adjacent nucleic acid segment is initiated at the promoter region.
A repressible promoter's rate of transcription decreases in
response to a repressing agent. An inducible promoter's rate of
transcription increases in response to an inducing agent. A
constitutive promoter's rate of transcription is not specifically
regulated, though it can vary under the influence of general
metabolic conditions.
[0187] Insert: As used herein, an insert is a desire nucleic acid
segment that is a part of a larger nucleic acid molecule.
[0188] Target Nucleic Acid Molecule: As used herein, target nucleic
acid molecule is a nucleic acid segment of interest preferably
nucleic acid which is to be acted upon using the compounds and
methods of the present invention. Such target nucleic acid
molecules preferably contain one or more genes or portions of
genes.
[0189] Insert Donor: As used herein, an insert donor is one of the
two parental nucleic acid molecules (e.g. RNA or DNA) of the
present invention which carries the Insert. The Insert Donor
molecule comprises the Insert flanked on both sides with
recombination sites. The Insert Donor can be linear or circular. In
one embodiment of the invention, the Insert Donor is a circular
nucleic acid molecule, optionally supercoiled, and further
comprises a cloning vector sequence outside of the recombination
signals (see FIG. 1). When a population of Inserts or population of
nucleic acid segments are used to make the Insert Donor, a
population of Insert Donors result and may be used in accordance
with the invention.
[0190] Product: As used herein, a product is one the desired
daughter molecules comprising the A and D sequences which is
produced after the second recombination event during the
recombinational cloning process (see FIG. 1). The Product contains
the nucleic acid which was to be cloned or subcloned. In accordance
with the invention, when a population of Insert Donors are used,
the resulting population of Product molecules will contain all or a
portion of the population of Inserts of the Insert Donors and
preferably will contain a representative population of the original
molecules of the Insert Donors.
[0191] Recognition sequence: As used herein, a recognition sequence
(alternatively and equivalently referred to herein as a
"recognition site") is a particular sequence to which a protein,
chemical compound, DNA, or RNA molecule (e.g., restriction
endonuclease, a topoisomerase, a modification methylase, or a
recombinase) recognizes and binds. In the present invention, a
recognition sequence will usually refer to a recombination site
(which may alternatively be referred to as a recombinase
recognition site) or a topoisomerase recognition site. For example,
the recognition sequence for Cre recombinase is loxP which is a 34
base pair sequence comprised of two 13 base pair inverted repeats
(serving as the recombinase binding sites) flanking an 8 base pair
core sequence. See FIG. 1 of Sauer, B., Current Opinion in
Biotechnology 5:521-527 (1994). Other examples of such recognition
sequences are the attB, attP, attL, and attR sequences which are
recognized by the recombinase enzyme (Integrase. attB is an
approximately 25 base pair sequence containing two 9 base pair
core-type Int binding sites and a 7 base pair overlap region. attP
is an approximately 240 base pair sequence containing core-type Int
binding sites and arm-type Int binding sites as well as sites for
auxiliary proteins integration host factor (IHF), FIS and
excisionase (Xis). See Landy, Current Opinion in Biotechnology
3:699-707 (1993). Such sites may also be engineered according to
the present invention to enhance production of products in the
methods of the invention. When such engineered sites lack the P1 or
H1 domains to make the recombination reactions irreversible (e.g.,
attR or attP), such sites may be designated attR' or attP' to show
that the domains of these sites have been modified in some way.
Examples of topoisomerase recognitions sites include, but are not
limited to, the sequence 5'-GCAACTT-3' that is recognized by E.
coli topoisomerase III (a type I topoisomerase); the sequence
5'-(C/T)CCTT-3' which is a topoisomerase recognition site that is
bound specifically by most poxvirus topoisomerases, including
vaccinia virus DNA topoisomerase I; and others that are known in
the art as discussed elsewhere herein.
[0192] Recombination proteins: As used herein, recombination
proteins include excisive or integrative proteins, enzymes,
co-factors or associated proteins that are involved in
recombination reactions involving one or more recombination sites,
which may be wild-type proteins (See Landy, Current Opinion in
Biotechnology 3:699-707 (1993)), or mutants, derivatives (e.g.,
fusion proteins containing the recombination protein sequences or
fragments thereof), fragments, and variants thereof.
[0193] Recombination site: A used herein, a recombination site is a
recognition sequence on a nucleic acid molecule participating in an
integration/recombination reaction by recombination proteins.
Recombination sites are discrete sections or segments of nucleic
acid on the participating nucleic acid molecules that are
recognized and bound by a site-specific recombination protein
during the initial stages of integration or recombination. For
example, the recombination site for Cre recombinase is loxP which
is a 34 base pair sequence comprised of two 13 base pair inverted
repeats (serving as the recombinase binding sites) flanking an 8
base pair core sequence. See FIG. 1 of Sauer, B., Curr. Opin.
Biotech. 5:521-527 (1994). Other examples of recognition sequences
include the attB, attP, attL, and attR sequences described herein,
and mutants, fragments, variants and derivatives thereof, which are
recognized by the recombination protein (Int and by the auxiliary
proteins integration host factor (IHF), FIS and excisionase (Xis).
See Landy, Curr. Opin. Biotech. 3:699-707 (1993).
[0194] Recombinational Cloning: As used herein, recombinational
cloning is a method, such as that described in U.S. Pat. Nos.
5,888,732, 6,143,557, 6,171,861, 6,270,969, and 6,277,608 (the
contents of which are fully incorporated herein by reference), and
as also described herein, whereby segments of nucleic acid
molecules or populations of such molecules are exchanged, inserted,
replaced, substituted or modified, in vitro or in vivo. Preferably,
such cloning method is an in vitro method.
[0195] Repression cassette: As used herein, repression cassette is
a nucleic acid segment that contains a repressor or a Selectable
marker present in the subcloning vector.
[0196] Selectable marker: As used herein, selectable marker is a
nucleic acid segment that allows one to select for or against a
molecule (e.g., a replicon) or a cell that contains it, often under
particular conditions. These markers can encode an activity, such
as, but not limited to, production of RNA, peptide, or protein, or
can provide a binding site for RNA, peptides, proteins, inorganic
and organic compounds or compositions and the like. Examples of
selectable markers include but are not limited to: (1) nucleic acid
segments that encode products which provide resistance against
otherwise toxic compounds (e.g., antibiotics); (2) nucleic acid
segments that encode products which are otherwise lacking in the
recipient cell (e.g., tRNA genes, auxotrophic markers); (3) nucleic
acid segments that encode products which suppress the activity of a
gene product; (4) nucleic acid segments that encode products which
can be readily identified (e.g., phenotypic markers such as
(-galactosidase, green fluorescent protein (GFP), and cell surface
proteins); (5) nucleic acid segments that bind products which are
otherwise detrimental to cell survival and/or function; (6) nucleic
acid segments that otherwise inhibit the activity of any of the
nucleic acid segments described in Nos. 1-5 above (e.g., antisense
oligonucleotides); (7) nucleic acid segments that bind products
that modify a substrate (e.g. restriction endonucleases); (8)
nucleic acid segments that can be used to isolate or identify a
desired molecule (e.g. specific protein binding sites); (9) nucleic
acid segments that encode a specific nucleotide sequence which can
be otherwise non-functional (e.g., for PCR amplification of
subpopulations of molecules); (10) nucleic acid segments, which
when absent, directly or indirectly confer resistance or
sensitivity to particular compounds; and/or (11) nucleic acid
segments that encode products which are toxic in recipient
cells.
[0197] Selection scheme: As used herein, selection scheme is any
method which allows selection, enrichment, or identification of a
desired Product or Product(s) from a mixture containing an Entry
Clone or Vector, a Destination Vector, a Donor Vector, an
Expression Clone or Vector, any intermediates (e.g. a Cointegrate
or a replicon), and/or Byproducts. The selection schemes of one
preferred embodiment have at least two components that are either
linked or unlinked during recombinational cloning. One component is
a Selectable marker. The other component controls the expression in
vitro or in vivo of the Selectable marker, or survival of the cell
(or the nucleic acid molecule, e.g., a replicon) harboring the
plasmid carrying the Selectable marker. Generally, this controlling
element will be a repressor or inducer of the Selectable marker,
but other means for controlling expression or activity of the
Selectable marker can be used. Whether a repressor or activator is
used will depend on whether the marker is for a positive or
negative selection, and the exact arrangement of the various
nucleic acid segments, as will be readily apparent to those skilled
in the art. In some preferred embodiments, the selection scheme
results in selection of or enrichment for only one or more desired
Products. As defined herein, selecting for a nucleic acid molecule
includes (a) selecting or enriching for the presence of the desired
nucleic acid molecule, and (b) selecting or enriching against the
presence of nucleic acid molecules that are not the desired nucleic
acid molecule.
[0198] In one embodiment, the selection schemes (which can be
carried out in reverse) will take one of three forms, which will be
discussed in terms of FIG. 1. The first, exemplified herein with a
Selectable marker and a repressor therefore, selects for molecules
having segment D and lacking segment C. The second selects against
molecules having segment C and for molecules having segment D.
Possible embodiments of the second form would have a nucleic acid
segment carrying a gene toxic to cells into which the in vitro
reaction products are to be introduced. A toxic gene can be a
nucleic acid that is expressed as a toxic gene product (a toxic
protein or RNA), or can be toxic in and of itself (In the latter
case, the toxic gene is understood to carry its classical
definition of "heritable trait".)
[0199] Examples of such toxic gene products are well known in the
art, and include, but are not limited to, restriction endonucleases
(e.g., DpnI), apoptosis-related genes (e.g. ASK1 or members of the
bc1-2/ced-9 family), retroviral genes including those of the human
immunodeficiency virus (HIV), defensins such as NP-1, inverted
repeats or paired palindromic nucleic acid sequences, bacteriophage
lytic genes such as those from (X174 or bacteriophage T4;
antibiotic sensitivity genes such as rpsL, antimicrobial
sensitivity genes such as pheS, plasmid killer genes, eukaryotic
transcriptional vector genes that produce a gene product toxic to
bacteria, such as GATA-1, and genes that kill hosts in the absence
of a suppressing function, e.g., kicB, ccdb, (X174 E (Liu, Q. et
al., Curr. Biol. 8:1300-1309 (1998)), and other genes that
negatively affect replicon stability and/or replication. A toxic
gene can alternatively be selectable in vitro, e.g., a restriction
site.
[0200] Many genes coding for restriction endonucleases operably
linked to inducible promoters are known, and may be used in the
present invention. See, e.g. U.S. Pat. Nos. 4,960,707 (DpnI and
DpnII); 5,000,333, 5,082,784 and 5,192,675 (KpnI); 5,147,800
(NgoARIII and NgoAI); 5,179,015 (FspI and HaeIII): 5,200,333 (HaeII
and TaqI); 5,248,605 (HpaII); 5,312,746.sup.o ClaI); 5,231,021 and
5,304,480 (XhoI and XhoII); 5,334,526 (Alul); 5,470,740 (NsiI);
5,534,428 (SstI/SacI); 5,202,248 (NcoI); 5,139,942 (NdeI); and
5,098,839 (Pad). See also Wilson, G. G., Nucl. Acids Res.
19:2539-2566 (1991); and Lunnen, K. D., et al., Gene 74:25-32
(1988).
[0201] In the second form, segment D carries a Selectable marker.
The toxic gene would eliminate transformants harboring the Vector
Donor, Cointegrate, and Byproduct molecules, while the Selectable
marker can be used to select for cells containing the Product and
against cells harboring only the Insert Donor.
[0202] The third form selects for cells that have both segments A
and D in cis on the same molecule, but not for cells that have both
segments in trans on different molecules. This could be embodied by
a Selectable marker that is split into two inactive fragments, one
each on segments A and D.
[0203] The fragments are so arranged relative to the recombination
sites that when the segments are brought together by the
recombination event, they reconstitute a functional Selectable
marker. For example, the recombinational event can link a promoter
with a structural nucleic acid molecule (e.g., a gene), can link
two fragments of a structural nucleic acid molecule, or can link
nucleic acid molecules that encode a heterodimeric gene product
needed for survival, or can link portions of a replicon.
[0204] Site-specific recombinase: As used herein, a site specific
recombinase is a type of recombinase which typically has at least
the following four activities (or combinations thereof): (1)
recognition of one or two specific nucleic acid sequences; (2)
cleavage of said sequence or sequences; (3) topoisomerase activity
involved in strand exchange; and (4) ligase activity to reseal the
cleaved strands of nucleic acid. See Sauer, B., Current Opinions in
Biotechnology 5:521-527 (1994). Conservative site-specific
recombination is distinguished from homologous recombination and
transposition by a high degree of specificity for both partners.
The strand exchange mechanism involves the cleavage and rejoining
of specific nucleic acid sequences in the absence of DNA synthesis
(Landy, A. (1989) Ann. Rev. Biochem. 58:913-949).
[0205] Vector: As used herein, a vector is a nucleic acid molecule
(preferably DNA) that provides a useful biological or biochemical
property to an Insert. Examples include plasmids, phages,
autonomously replicating sequences (ARS), centromeres, and other
sequences which are able to replicate or be replicated in vitro or
in a host cell, or to convey a desired nucleic acid segment to a
desired location within a host cell. A Vector can have one or more
restriction endonuclease recognition sites at which the sequences
can be cut in a determinable fashion without loss of an essential
biological function of the vector, and into which a nucleic acid
fragment can be spliced in order to bring about its replication and
cloning. Vectors can further provide primer sites, e.g., for PCR,
transcriptional and/or translational initiation and/or regulation
sites, recombinational signals, replicons, Selectable markers, etc.
Clearly, methods of inserting a desired nucleic acid fragment which
do not require the use of recombination, transpositions or
restriction enzymes (such as, but not limited to, UDG cloning of
PCR fragments (U.S. Pat. No. 5,334,575, entirely incorporated
herein by reference), TA Cloning.RTM. brand PCR cloning (Invitrogen
Corporation, Carlsbad, Calif.) (also known as direct ligation
cloning), and the like) can also be applied to clone a fragment
into a cloning vector to be used according to the present
invention. The cloning vector can further contain one or more
selectable markers suitable for use in the identification of cells
transformed with the cloning vector.
[0206] Subcloning vector: As used herein, a subcloning vector is a
cloning vector comprising a circular or linear nucleic acid
molecule which includes preferably an appropriate replicon. In the
present invention, the subcloning vector (segment D in FIG. 1) can
also contain functional and/or regulatory elements that are desired
to be incorporated into the final product to act upon or with the
cloned nucleic acid Insert (segment A in FIG. 1). The subcloning
vector can also contain a Selectable marker (preferably DNA).
[0207] Vector Donor: As used herein, a Vector Donor is one of the
two parental nucleic acid molecules (e.g. RNA or DNA) of the
present invention which carries the nucleic acid segments
comprising the nucleic acid vector which is to become part of the
desired Product. The Vector Donor comprises a subcloning vector D
(or it can be called the cloning vector if the Insert Donor does
not already contain a cloning vector) and a segment C flanked by
recombination sites (see FIG. 1). Segments C and/or D can contain
elements that contribute to selection for the desired Product
daughter molecule, as described above for selection schemes. The
recombination signals can be the same or different, and can be
acted upon by the same or different recombinases. In addition, the
Vector Donor can be linear or circular.
[0208] Primer: As used herein, a primer is a single stranded or
double stranded oligonucleotide that is extended by covalent
bonding of nucleotide monomers during amplification or
polymerization of a nucleic acid molecule (e.g. a DNA molecule). In
one aspect, the primer may be a sequencing primer (for example, a
universal sequencing primer). In another aspect, the primer may
comprise a recombination site or portion thereof.
[0209] Template: As used herein, a template is a double stranded or
single stranded nucleic acid molecule which is to be amplified,
synthesized or sequenced. In the case of a double-stranded DNA
molecule, denaturation of its strands to form a first and a second
strand is preferably performed before these molecules may be
amplified, synthesized or sequenced, or the double stranded
molecule may be used directly as a template. For single stranded
templates, a primer complementary to at least a portion of the
template is hybridized under appropriate conditions and one or more
polypeptides having polymerase activity (e.g. DNA polymerases
and/or reverse transcriptases) may then synthesize a molecule
complementary to all or a portion of the template. Alternatively,
for double stranded templates, one or more transcriptional
regulatory sequences (e.g., one or more promoters) may be used in
combination with one or more polymerases to make nucleic acid
molecules complementary to all or a portion of the template. The
newly synthesized molecule, according to the invention, may be of
equal or shorter length compared to the original template. Mismatch
incorporation or strand slippage during the synthesis or extension
of the newly synthesized molecule may result in one or a number of
mismatched base pairs. Thus, the synthesized molecule need not be
exactly complementary to the template. Additionally, a population
of nucleic acid templates may be used during synthesis or
amplification to produce a population of nucleic acid molecules
typically representative of the original template population.
[0210] Incorporating: As used herein, incorporating means becoming
a part of a nucleic acid (e.g., DNA) molecule or primer.
[0211] Library: As used herein, a library is a collection of
nucleic acid molecules (circular or linear). In one embodiment, a
library may comprise a plurality (i.e., two or more) of nucleic
acid molecules, which may or may not be from a common source
organism, organ, tissue, or cell. In another embodiment, a library
is representative of all or a portion or a significant portion of
the nucleic acid content of an organism (a "genomic" library), or a
set of nucleic acid molecules representative of all or a portion or
a significant portion of the expressed nucleic acid molecules (a
cDNA library or segments derived therefrom) in a cell, tissue,
organ or organism. A library may also comprise random sequences
made by de novo synthesis, mutagenesis of one or more sequences and
the like. Such libraries may or may not be contained in one or more
vectors.
[0212] Amplification: As used herein, amplification is any in vitro
method for increasing a number of copies of a nucleotide sequence
with the use of one or more polypeptides having polymerase activity
(e.g., one or more nucleic acid polymerases or one or more reverse
transcriptases). Nucleic acid amplification results in the
incorporation of nucleotides into a DNA and/or RNA molecule or
primer thereby forming a new nucleic acid molecule complementary to
a template. The formed nucleic acid molecule and its template can
be used as templates to synthesize additional nucleic acid
molecules. As used herein, one amplification reaction may consist
of many rounds of nucleic acid replication. DNA amplification
reactions include, for example, polymerase chain reaction (PCR).
One PCR reaction may consist of 5 to 100 cycles of denaturation and
synthesis of a DNA molecule.
[0213] Nucleotide: As used herein, a nucleotide is a
base-sugar-phosphate combination. Nucleotides are monomeric units
of a nucleic acid molecule (DNA and RNA). The term nucleotide
includes ribonucleoside triphosphates ATP, UTP, CTG, GTP and
deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP,
dGTP, dTTP, or derivatives thereof. Such derivatives include, for
example, [S]dATP, 7-deaza-dGTP and 7-deaza-dATP. The term
nucleotide as used herein also refers to dideoxyribonucleoside
triphosphates (ddNTPs) and their derivatives. Illustrated examples
of dideoxyribonucleoside triphosphates include, but are not limited
to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. According to the present
invention, a "nucleotide" may be unlabeled or detectably labeled by
well known techniques. Detectable labels include, for example,
radioactive isotopes, fluorescent labels, chemiluminescent labels,
bioluminescent labels and enzyme labels.
[0214] Nucleic acid molecule: As used herein, a nucleic acid
molecule is a sequence of contiguous nucleotides (riboNTPs, dNTPs
or ddNTPs, or combinations thereof) of any length, which may encode
a full-length polypeptide or a fragment of any length thereof, or
which may be non-coding. As used herein, the terms "nucleic acid
molecule" and "polynucleotide" may be used interchangeably.
[0215] Oligonucleotide: As used herein, an oligonucleotide is a
synthetic or natural molecule comprising a covalently linked
sequence of nucleotides which are joined by a phosphodiester bond
between the 3' position of the pentose of one nucleotide and the 5'
position of the pentose of the adjacent nucleotide.
[0216] Polypeptide: As used herein, a polypeptide is a sequence of
contiguous amino acids, of any length. As used herein, the terms
"peptide," "oligopeptide," or "protein" may be used interchangeably
with the term "polypeptide."
[0217] Hybridization: As used herein, the terms hybridization and
hybridizing refer to base pairing of two complementary
single-stranded nucleic acid molecules (RNA and/or DNA) to give a
double stranded molecule. As used herein, two nucleic acid
molecules may be hybridized, although the base pairing is not
completely complementary. Accordingly, mismatched bases do not
prevent hybridization of two nucleic acid molecules provided that
appropriate conditions, well known in the art, are used. In some
aspects, hybridization is said to be under "stringent conditions."
By "stringent conditions" as used herein is meant overnight
incubation at 42.degree. C. in a solution comprising: 50%
formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times.Denhardt's solution, 10%
dextran sulfate, and 20 g/ml denatured, sheared salmon sperm DNA,
followed by washing the filters in 0.1.times.SSC at about
65.degree. C.
[0218] Other terms used in the fields of recombinant nucleic acid
technology and molecular and cell biology as used herein will be
generally understood by one of ordinary skill in the applicable
arts.
Overview
[0219] The present invention relates to methods, compositions and
kits for the recombinational and/or topoisomerase-mediated joining
of two or more segments or molecules of nucleic acid or other
molecules and/or compounds (or combinations thereof). The invention
also relates to attaching such linked nucleic acids or other
molecules and/or compounds to one or more supports or structures
preferably through recombination sites (which may include
recombination protein recognition sequences, topoisomerase
recognition sequences, etc.) or portions thereof. Thus, the
invention generally relates to linking any number of nucleic acids
or other molecules and/or compounds via nucleic acid linkers
comprising one or more topoisomerase recognition sites and/or one
or more recombination sites or portions thereof. The linked
products produced by the invention may comprise any number of the
same or different nucleic acids or other molecules and/or
compounds, depending on the starting materials. Such starting
materials inlcude, but are not limited to, any nucleic acids (or
derivatives thereof such as peptide nucleic acids (PNAs)), chemical
compounds, detectably labeled molecules (such as fluorescent
molecules and chemiluminescent molecules), drugs, peptides or
protesins, lipids, carbohydrates and other molecules and/or
compounds comprising one or more recombination sites or portions
thereof. Through recombination of such recombination sites and/or
topoisomerase-mediated joining reactions according to the
invention, any number or combination of such starting molecules
and/or compounds can be linked to make linked products of the
invention. In addition, deletion or replacement of certain portions
or components of the linked products of the invention can be
accomplished by recombination.
[0220] In some embodiments, the joined segments may be inserted
into a different nucleic acid molecule such as vectors, such as by
recombinational cloning methods and/or topoisomerase-mediated
joining methods of the invention. Thus, in some embodiments, the
present invention relates to the construction of nucleic acid
molecules (RNA or DNA) by combining two or more segments of nucleic
acid by a recombination reaction and/or a topoisomerase-mediated
joining reaction and inserting the joined two or more segments into
a vector by recombinational cloning. In embodiments where the
joined nucleic acid molecules are to be further combined with an
additional nucleic acid molecule by a recombination reaction, the
timing of the two recombination events, i.e. the joining of the
segments and the insertion of the segments into a vector, is not
critical. That is to say, it is not critical to the present
invention whether the two or more nucleic acid segments are joined
together before insertion into the vector or whether, for example,
one recombination site on each segment first reacts with a
recombination site on the vector and subsequently the recombination
sites on the nucleic acid segments react with each other to join
the segments. Moreover, the nucleic acid segments can be cloned in
any one or a number of positions within the vector and do not need
to be inserted adjacent to each other, although, in some
embodiments, joining of two or more of such segments within the
vector is preferred. In accordance with the invention,
recombinational cloning allows efficient selection and
identification of molecules (particularly vectors) containing the
combined nucleic acid segments. Thus, two or more nucleic acid
segments of interest can be combined and, optionally, inserted into
a single vector suitable for further manipulation of the combined
nucleic acid molecule.
[0221] In additional embodiments, at least two (e.g., 2, 3, 4, 5,
6, 7, 8, etc.) nucleic acid segments, each comprising at least one
(e.g., 1, 2, 3, 4, 5, 6, 7, 8, etc.) recombination site and
optionally with at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, etc.)
topoisomerase recognition site, are contacted with suitable
recombination proteins and/or with topoisomerase to effect the
joining all or a portion of the two molecules, depending on the
position of the recombination sites in the molecules. In certain
such embodiments, such as in nucleic acid molecules comprising at
least two recombination sites, at least one of the two
recombination sites flanks each end of a topoisomerase recognitino
site in the molecule. By a recombination site (or a topoisomerase
recognition site) that "flanks" another recognition site (e.g.,
another recombination site or topoisomerase recognition site) is
meant that the two sites are within about 20 nucleotides of each
other, or within about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,
9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 nucleotides of each other. Each
individual nucleic acid segment may comprise a variety of sequences
including, but not limited to sequences suitable for use as primer
sites (e.g., sequences for which a primer such as a sequencing
primer or amplification primer may hybridize to initiate nucleic
acid synthesis, amplification or sequencing), transcription or
translation signals or regulatory sequences such as promoters,
ribosomal binding sites, Kozak sequences, and start codons,
termination signals such as stop codons, origins of replication,
recombination sites (or portions thereof), topoisomerase
recognition sites (or portions thereof), selectable markers, and
genes or portions of genes to create protein fusions (e.g.,
N-terminal or carboxy terminal) such as GST, GUS, GFP, 6
histidines, epitopes haptens and the like and combinations thereof.
The vectors used for cloning such segments may also comprise these
functional sequences (e.g., promoters, primer sites etc.). After
combination of the segments comprising such sequences and optimally
the cloning of the sequences into one or more vectors, the
molecules may be manipulated in a variety of ways including
sequencing or amplification of the target sequence (i.e., by using
at least one or the primer sites introduced by the integration
sequence), mutation of the target sequence (i.e., by insertion,
deletion or substitution in or on the target sequences), and
protein expression from the target sequence or portions thereof
(i.e., by expression of translation and/or transcription signals
contained by the segments and/or vectors).
[0222] The present invention also relates to the generation of
combinatorial libraries using the recombinational cloning methods
disclosed. Thus, one or more of the nucleic acid segments joined
may comprise a nucleic acid library. Such a library may comprise,
for example, nucleic acid sequences corresponding to permutations
of a sequence coding for a peptide, polypeptide or protein
sequence. The permutations can be joined to another nucleic acid
segment consisting of a single sequence or, alternatively, the
second nucleic acid segment may also be a library corresponding to
permutation of another peptide, polypeptide or protein sequence
such that joining of the two segments may produce a library
representing all possible combinations of all the permutations of
the two peptide, polypeptide or proteins sequences. Numerous
examples of the use of combinatorial libraries are known in the
art. See, for example, Waterhouse, et al., Nucleic Acids Research,
1993, Vol. 21, No. 9, 2265-2266, Tsurushita, et al., Gene, 1996,
Vol. 172 No. 1, 59-63, Persson, Int Rev Immunol 1993 10:2-3 153-63,
Chanock, et al., Infect Agents Dis 1993 Jun. 2:3 118-31, Burioni,
et al., Res Virol 1997 March-April 148:2 161-4, Leung, Thromb
Haemost 1995 July 74:1 373-6, Sandhu, Crit. Rev Biotechnol 1992
12:5-6 437-62 and U.S. Pat. Nos. 5,733,743, 5,871,907 and 5,858,657
all of which are specifically incorporated herein by reference.
Recombination Sites
[0223] Recombination sites for use in the invention may be any
nucleic acid sequence that can serve as a substrate in a
recombination reaction. Such recombination sites may be wild-type
or naturally occurring recombination sites or modified or mutant
recombination sites. Examples of recombination sites for use in the
invention include, but are not limited to, phage-lambda
recombination sites (such as attP, attB, attL, and attR and mutants
or derivatives thereof) and recombination sites from other
bacteriophage such as phi80, P22, P2, 186, P4 and P1 (including lox
sites such as loxp and loxP511). Novel mutated att sites (e.g.,
attB 1-10, attP 1-10, attR 1-10 and attL 1-10) are described in
previous patent application Ser. No. 60/136,744, filed May 28,
1999, which is specifically incorporated herein by reference. Other
recombination sites having unique specificity (i.e., a first site
will recombine with its corresponding site and will not recombine
with a second site having a different specificity) are known to
those skilled in the art and may be used to practice the present
invention. Other suitable recombination proteins and mutant,
modified, variant, or derivative recombination sites for use in the
invention include those described in U.S. Pat. Nos. 5,888,732,
6,143,557, 6,171,861, 6,270,969, and 6,277,608 and in U.S.
application Ser. No. 09/438,358 (filed Nov. 12, 1999), based upon
U.S. provisional application No. 60/108,324 (filed Nov. 13, 1998).
Mutated att sites (e.g., attB 1-10, attP 1-10, attR 1-10 and attL
1-10) are described in U.S. provisional patent application No.
60/122,389, filed Mar. 2, 1999, 60/126,049, filed Mar. 23, 1999,
60/169,983, filed Dec. 10, 1999, and 60/188,000, filed Mar. 9,
2000, and in U.S. application Ser. No. 09/517,466, filed Mar. 2,
2000, and Ser. No. 09/732,914, filed Dec. 11, 2000 (published as
20020007051-A1) the disclosures of which are specifically
incorporated herein by reference in their entirety. Other suitable
recombination sites and proteins are those associated with the
GATEWAY.TM. Cloning Technology available from Invitrogen
Corporation, Carlsbad, Calif., and described in the product
literature of the GATEWAY.TM. Cloning Technology, the entire
disclosures of all of which are specifically incorporated herein by
reference in their entireties.
[0224] Sites that may be used in the present invention include att
sites. The 15 bp core region of the wildtype att site (GCTTTTTTAT
ACTAA (SEQ ID NO:81)), which is identical in all wildtype att
sites, may be mutated in one or more positions. The inventors have
determined that att sites that specifically recombine with other
att sites can be constructed by altering nucleotides in and near
the 7 base pair overlap region, bases 6-12 of the core region.
Thus, recombination sites suitable for use in the methods,
compositions, and vectors of the invention include, but are not
limited to, those with insertions, deletions or substitutions of
one, two, three, four, or more nucleotide bases within the 15 base
pair core region (see U.S. application Ser. No. 08/663,002, filed
Jun. 7, 1996 (now U.S. Pat. No. 5,888,732) and 09/177,387, filed
Oct. 23, 1998, which describes the core region in further detail,
and the disclosures of which are incorporated herein by reference
in their entireties). Recombination sites suitable for use in the
methods, compositions, and vectors of the invention also include
those with insertions, deletions or substitutions of one, two,
three, four, or more nucleotide bases within the 15 base pair core
region that are at least 50% identical, at least 55% identical, at
least 60% identical, at least 65% identical, at least 70%
identical, at least 75% identical, at least 80% identical, at least
85% identical, at least 90% identical, or at least 95% identical to
this 15 base pair core region.
[0225] Analogously, the core regions in attB1, attP1, attL1 and
attR1 are identical to one another, as are the core regions in
attB2, attP2, attL2 and attR2. Nucleic acid molecules suitable for
use with the invention also include those comprising insertions,
deletions or substitutions of one, two, three, four, or more
nucleotides within the seven base pair overlap region (TTTATAC,
bases 6-12 in the core region). The overlap region is defined by
the cut sites for the integrase protein and is the region where
strand exchange takes place. Examples of such mutants, fragments,
variants and derivatives include, but are not limited to, nucleic
acid molecules in which (1) the thymine at position 1 of the seven
by overlap region has been deleted or substituted with a guanine,
cytosine, or adenine; (2) the thymine at position 2 of the seven by
overlap region has been deleted or substituted with a guanine,
cytosine, or adenine; (3) the thymine at position 3 of the seven by
overlap region has been deleted or substituted with a guanine,
cytosine, or adenine; (4) the adenine at position 4 of the seven by
overlap region has been deleted or substituted with a guanine,
cytosine, or thymine; (5) the thymine at position 5 of the seven by
overlap region has been deleted or substituted with a guanine,
cytosine, or adenine; (6) the adenine at position 6 of the seven by
overlap region has been deleted or substituted with a guanine,
cytosine, or thymine; and (7) the cytosine at position 7 of the
seven by overlap region has been deleted or substituted with a
guanine, thymine, or adenine; or any combination of one or more
such deletions and/or substitutions within this seven by overlap
region. The nucleotide sequences of the above described seven base
pair core regions are set out below in Table 1.
[0226] Altered att sites have been constructed that demonstrate
that (1) substitutions made within the first three positions of the
seven base pair overlap (TTTATAC) strongly affect the specificity
of recombination, (2) substitutions made in the last four positions
(TTTATAC) only partially alter recombination specificity, and (3)
nucleotide substitutions outside of the seven by overlap, but
elsewhere within the 15 base pair core region, do not affect
specificity of recombination but do influence the efficiency of
recombination. Thus, nucleic acid molecules and methods of the
invention include those comprising or employing one, two, three,
four, five, six, eight, ten, or more recombination sites which
affect recombination specificity, particularly one or more (e.g.,
one, two, three, four, five, six, eight, ten, twenty, thirty,
forty, fifty, etc.) different recombination sites that may
correspond substantially to the seven base pair overlap within the
15 base pair core region, having one or more mutations that affect
recombination specificity. Particularly preferred such molecules
may comprise a consensus sequence such as NNNATAC wherein "N"
refers to any nucleotide (i.e., may be A, G, T/U or C). Preferably,
if one of the first three nucleotides in the consensus sequence is
a T/U, then at least one of the other two of the first three
nucleotides is not a T/U.
[0227] The core sequence of each att site (attB, attP, attL and
attR) can be divided into functional units consisting of integrase
binding sites, integrase cleavage sites and sequences that
determine specificity. Specificity determinants are defined by the
first three positions following the integrase top strand cleavage
site. These three positions are shown with underlining in the
following reference sequence: CAACTTTTTTATAC AAAGTTG (SEQ ID
NO:82). Modification of these three positions (64 possible
combinations) can be used to generate att sites that recombine with
high specificity with other att sites having the same sequence for
the first three nucleotides of the seven base pair overlap region.
The possible combinations of first three nucleotides of the overlap
region are shown in Table 1.
TABLE-US-00001 TABLE 1 Modifications of the First Three Nucleotides
of the att Site Seven Base Pair Overlap Region that Alter
Recombination Specificity. AAA CAA GAA TAA AAC CAC GAC TAC CAG GAG
TAG AAG CAT GAT TAT AAT CCA GCA TCA ACA CCC GCC TCC ACC CCG GCG TCG
ACG CCT GCT TCT ACT CGA GGA TGA AGA CGC GGC TGC AGC CGG GGG TGG AGG
CGT GGT TGT AGT CTA GTA TTA ATA CTC GTC TTC ATC CTG GTG TTG ATG CTT
GTT TTT ATT
[0228] Representative examples of seven base pair att site overlap
regions suitable for in methods, compositions and vectors of the
invention are shown in Table 2. The invention further includes
nucleic acid molecules comprising one or more (e.g., one, two,
three, four, five, six, eight, ten, twenty, thirty, forty, fifty,
etc.) nucleotides sequences set out in Table 2. Thus, for example,
in one aspect, the invention provides nucleic acid molecules
comprising the nucleotide sequence GAAATAC, GATATAC, ACAATAC, or
TGCATAC.
TABLE-US-00002 TABLE 2 Representative Examples of Seven Base Pair
att Site Overlap Regions Suitable for use in the recombination
sites of the Invention. AAAATAC CAAATAC GAAATAC TAAATAC AACATAC
CACATAC GACATAC TACATAC AAGATAC CAGATAC GAGATAC TAGATAC AATATAC
CATATAC GATATAC TATATAC ACAATAC CCAATAC GCAATAC TCAATAC ACCATAC
CCCATAC GCCATAC TCCATAC ACGATAC CCGATAC GCGATAC TCGATAC ACTATAC
CCTATAC GGTATAC TCTATAC AGAATAC CGAATAC GGAATAC TGAATAC AGCATAC
CGCATAC GGCATAC TGCATAC AGGATAC CGGATAC GGGATAC TGGATAC AGTATAC
CGTATAC GGTATAC TGTATAC ATAATAC CTAATAC GTAATAC TTAATAC ATCATAC
CTCATAC GTCATAC TTCATAC ATGATAC CTGATAC GTGATAC TTGATAC ATTATAC
CTTATAC GTTATAC TTTATAC
[0229] As noted above, alterations of nucleotides located 3' to the
three base pair region discussed above can also affect
recombination specificity. For example, alterations within the last
four positions of the seven base pair overlap can also affect
recombination specificity.
[0230] For example, mutated att sites that may be used in the
practice of the present invention include attB1 (AGCCTGCTTT
TTTGTACAAA CTTGT (SEQ ID NO:83)), attP1 (TACAGGTCAC TAATACCATC
TAAGTAGTTG ATTCATAGTG ACTGGATATG TTGTGTTTTA CAGTATTATG TAGTCTGTTT
TTTATGCAAA ATCTAATTTA ATATATTGAT ATTTATATCA TTTTACGTTT CTCGTTCAGC
TTTTTTGTAC AAAGTTGGCA TTATAAAAAA GCATTGCTCA TCAATTTGTT GCAACGAACA
GGTCACTATC AGTCAAAATA AAATCATTAT TTG (SEQ ID NO:84)), attL1
(CAAATAATGA TTTTATTTTG ACTGATAGTG ACCTGTTCGT TGCAACAAAT TGATAAGCAA
TGCTTTTTTA TAATGCCAAC TTTGTACAAA AAAGCAGGCT (SEQ ID NO:85)), and
attR1 (ACAAGTTTGT ACAAAAAAGC TGAACGAGAA ACGTAAAATG ATATAAATAT
CAATATATTA AATTAGATTT TGCATAAAAA ACAGACTACA TAATACTGTA AAACACAACA
TATCCAGTCA CTATG (SEQ ID NO:86)). Table 3 provides the sequences of
the regions surrounding the core region for the wild type att sites
(attB0, P0, R0, and L0) as well as a variety of other suitable
recombination sites. Those skilled in the art will appreciated that
the remainder of the site is the same as the corresponding site (B,
P, L, or R) listed above.
TABLE-US-00003 TABLE 3 Nucleotide sequences of representative att
sites. attB0 AGCCTGCTTT TTTATACTAA (SEQ ID NO: 87) CTTGAGC attP0
GTTCAGCTTT TTTATACTAA (SEQ ID NO: 88) GTTGGCA attL0 AGCCTGCTTT
TTTATACTAA (SEQ ID NO: 89) GTTGGCA attR0 GTTCAGCTTT TTTATACTAA (SEQ
ID NO: 90) CTTGAGC attB1 AGCCTGCTTT TTTGTACAAA (SEQ ID NO: 83)
CTTGT attP1 GTTCAGCTTT TTTGTACAAA (SEQ ID NO: 91) GTTGGCA attL1
AGCCTGCTTT TTTGTACAAA (SEQ ID NO: 92) GTTGGCA attR1 GTTCAGCTTT
TTTGTACAAA (SEQ ID NO: 93) CTTGT attB2 ACCCAGCTTT CTTGTACAAA (SEQ
ID NO: 94) GTGGT attP2 GTTCAGCTTT CTTGTACAAA (SEQ ID NO: 95)
GTTGGCA attL2 ACCCAGCTTT CTTGTACAAA (SEQ ID NO: 96) GTTGGCA attR2
GTTCAGCTTT CTTGTACAAA (SEQ ID NO: 97) GTGGT attB5 CAACTTTATT
ATACAAAGTT (SEQ ID NO: 98) GT attP5 GTTCAACTTT ATTATACAAA (SEQ ID
NO: 99) GTTGGCA attL5 CAACTTTATT ATACAAAGTT (SEQ ID NO: 100) GGCA
attR5 GTTCAACTTT ATTATACAAA (SEQ ID NO: 101) GTTGT attB11
CAACTTTTCT ATACAAAGTT (SEQ ID NO: 102) GT attP11 GTTCAACTTT
TCTATACAAA (SEQ ID NO: 103) GTTGGCA attL11 CAACTTTTCT ATACAAAGTT
(SEQ ID NO: 104) GGCA attR11 GTTCAACTTT TCTATACAAA (SEQ ID NO: 105)
GTTGT attB17 CAACTTTTGT ATACAAAGTT (SEQ ID NO: 106) GT attP17
GTTCAACTTT TGTATACAAA (SEQ ID NO: 107) GTTGGCA attL17 CAACTTTTGT
ATACAAAGTT (SEQ ID NO: 108) GGCA attR17 GTTCAACTTT TGTATACAAA (SEQ
ID NO: 109) GTTGT attB19 CAACTTTTTC GTACAAAGTT (SEQ ID NO: 110) GT
attP19 GTTCAACTTT TTCGTACAAA (SEQ ID NO: 111) GTTGGCA attL19
CAACTTTTTC GTACAAAGTT (SEQ ID NO: 112) GGCA attR19 GTTCAACTTT
TTCGTACAAA (SEQ ID NO: 113) GTTGT attB20 CAACTTTTTG GTACAAAGTT (SEQ
ID NO: 114) GT attP20 GTTCAACTTT TTGGTACAAA (SEQ ID NO: 115)
GTTGGCA attL20 CAACTTTTTG GTACAAAGTT (SEQ ID NO: 116) GGCA attR20
GTTCAACTTT TTGGTACAAA (SEQ ID NO: 117) GTTGT attB21 CAACTTTTTA
ATACAAAGTT (SEQ ID NO: 118) GT attP21 GTTCAACTTT TTAATACAAA (SEQ ID
NO: 119) GTTGGCA attL21 CAACTTTTTA ATACAAAGTT (SEQ ID NO: 120) GGCA
attR21 GTTCAACTTT TTAATACAAA (SEQ ID NO: 121) GTTGT
[0231] Other recombination sites having unique specificity (i.e., a
first site will recombine with its corresponding site and will not
recombine with a second site having a different specificity) are
known to those skilled in the art and may be used to practice the
present invention. Corresponding recombination proteins for these
systems may be used in accordance with the invention with the
indicated recombination sites. Other systems providing
recombination sites and recombination proteins for use in the
invention include the FLP/FRT system from Saccharomyces cerevisiae,
the resolvase family (e.g., .gamma..delta., TndX, TnpX, Tn3
resolvase, Hin, Hjc, GIn, SpCCE1, ParA, and Cin), and IS231 and
other Bacillus thuringiensis transposable elements. Other suitable
recombination systems for use in the present invention include the
XerC and XerD recombinases and the psi, dif and cer recombination
sites in E. coli. Other suitable recombination sites may be found
in U.S. Pat. No. 5,851,808 issued to Elledge and Liu which is
specifically incorporated herein by reference.
[0232] Recombination sites used with the invention may also have
embedded functions or properties. An embedded functionality is a
function or property conferred by a nucleotide sequence in a
recombination site that is not directly associated with
recombination efficiency or specificity. For example, recombination
sites may contain protein coding sequences (e.g. intein coding
sequences), intron/exon splice sites, origins of replication,
and/or stop codons. Further, recombination sites that have more
than one (e.g., two, three, four, five, etc.) embedded functions or
properties may also be prepared.
[0233] In some instances it will be advantageous to remove either
RNA corresponding to recombination sites from RNA transcripts or
amino acid residues encoded by recombination sites from
polypeptides translated from such RNAs. Removal of such sequences
can be performed in several ways and can occur at either the RNA or
protein level. One instance where it may be advantageous to remove
RNA transcribed from a recombination site will be when constructing
a fusion polypeptide between a polypeptide of interest and a coding
sequence present on the vector. The presence of an intervening
recombination site between the ORF of the polypeptide of interest
and the vector coding sequences may result in the recombination
site (1) contributing codons to the mRNA that result in the
inclusion of additional amino acid residues in the expression
product, (2) contributing a stop codon to the mRNA that prevents
the production of the desired fusion protein, and/or (3) shifting
the reading frame of the mRNA such that the two protein are not
fused "in-frame."
[0234] In one aspect, the invention provides methods for removing
nucleotide sequences encoded by recombination sites from RNA
molecules. One example of such a method employs the use of
intron/exon splice sites to remove RNA encoded by recombination
sites from RNA transcripts. Nucleotide sequences that encode
intron/exon splice sites may be fully or partially embedded in the
recombination sites used in the present invention and/or may
encoded by adjacent nucleic acid sequence. Sequences to be excised
from RNA molecules may be flanked by splice sites that are
appropriately located in the sequence of interest and/or on the
vector. For example, one intron/exon splice site may be encoded by
a recombination site and another intron/exon splice site may be
encoded by other nucleotide sequences (e.g., nucleic acid sequences
of the vector or a nucleic acid of interest). Nucleic acid splicing
is well known to those skilled in the art and is discussed in the
following publications: R. Reed, Curr. Opin. Genet. Devel.
6:215-220 (1996); S. Mount, Nucl. Acids. Res. 10:459-472, (1982);
P. Sharp, Cell 77:805-815, (1994); K. Nelson and M. Green, Genes
and Devel. 23:319-329 (1988); and T. Cooper and W. Mattox, Am. J.
Hum. Genet. 61:259-266 (1997).
[0235] Splice sites can be suitably positioned in a number of
locations. For example, a Destination Vector designed to express an
inserted ORF with an N-terminal fusion--for example, with a
detectable marker--the first splice site could be encoded by vector
sequences located 3' to the detectable marker coding sequences and
the second splice site could be partially embedded in the
recombination site that separates the detectable marker coding
sequences from the coding sequences of the ORF. Further, the second
splice site either could abut the 3' end of the recombination site
or could be positioned a short distance (e.g., 2, 4, 8, 10, 20
nucleotides) 3' to the recombination site. In addition, depending
on the length of the recombination site, the second splice site
could be fully embedded in the recombination site.
[0236] A modification of the method described above involves the
connection of multiple nucleic acid segments that, upon expression,
results in the production of a fusion protein. In one specific
example, one nucleic acid segment encodes detectable marker--for
example, GFP--and another nucleic acid segment that encodes an ORF
of interest. Each of these segments is flanked by recombination
sites. In addition, the nucleic acid segments that encodes the
detectable marker contains an intron/exon splice site near its 3'
terminus and the nucleic acid segments that contains the ORF of
interest also contains an intron/exon splice site near its 5'
terminus. Upon recombination, the nucleic acid segment that encodes
the detectable marker is positioned 5' to the nucleic acid segment
that encodes the ORF of interest. Further, these two nucleic acid
segments are separated by a recombination site that is flanked by
intron/exon splice sites. Excision of the intervening recombination
site thus occurs after transcription of the fusion mRNA. Thus, in
one aspect, the invention is directed to methods for removing RNA
transcribed from recombination sites from transcripts generated
from nucleic acids described herein.
[0237] Splice sites may introduced into nucleic acid molecules to
be used in the present invention in a variety of ways. One method
that could be used to introduce intron/exon splice sites into
nucleic acid segments is by the use of PCR. For example, primers
could be used to generate nucleic acid segments corresponding to an
ORF of interest and containing both a recombination site and an
intron/exon splice site.
[0238] The above methods can also be used to remove RNA
corresponding to recombination sites when the nucleic acid segment
that is recombined with another nucleic acid segment encodes RNA
that is not produced in a translatable format. One example of such
an instance is where a nucleic acid segment is inserted into a
vector in a manner that results in the production of antisense RNA.
As discussed below, this antisense RNA may be fused, for example,
with RNA that encodes a ribozyme. Thus, the invention also provides
methods for removing RNA corresponding to recombination sites from
such molecules.
[0239] The invention further provides methods for removing amino
acid sequences encoded by recombination sites from protein
expression products by protein splicing. Nucleotide sequences that
encode protein splice sites may be fully or partially embedded in
the recombination sites that encode amino acid sequences excised
from proteins or protein splice sites may be encoded by adjacent
nucleotide sequences. Similarly, one protein splice site may be
encoded by a recombination site and another protein splice sites
may be encoded by other nucleotide sequences (e.g., nucleic acid
sequences of the vector or a nucleic acid of interest).
[0240] It has been shown that protein splicing can occur by
excision of an intein from a protein molecule and ligation of
flanking segments (see, e.g., Derbyshire et al., Proc. Natl. Acad.
Sci. (USA) 95:1356-1357 (1998)). In brief, inteins are amino acid
segments that are post-translationally excised from proteins by a
self-catalytic splicing process. A considerable number of intein
consensus sequences have been identified (see, e.g., Perler,
Nucleic Acids Res. 27:346-347 (1999)).
[0241] Similar to intron/exon splicing, N- and C-terminal intein
motifs have been shown to be involved in protein splicing. Thus,
the invention further provides compositions and methods for
removing amino acid residues encoded by recombination sites from
protein expression products by protein splicing. In particular,
this aspect of the invention is related to the positioning of
nucleic acid sequences that encode intein splice sites on both the
5' and 3' end of recombination sites positioned between two coding
regions. Thus, when the protein expression product is incubated
under suitable conditions, amino acid residues encoded these
recombination sites will be excised.
[0242] Protein splicing may be used to remove all or part of the
amino acid sequences encoded by recombination sites. Nucleic acid
sequence that encode inteins may be fully or partially embedded in
recombination sites or may adjacent to such sites. In certain
circumstances, it may be desirable to remove considerable numbers
of amino acid residues beyond the N- and/or C-terminal ends of
amino acid sequences encoded by recombination sites. In such
instances, intein coding sequence may be located a distance (e.g.,
30, 50, 75, 100, etc. nucleotides) 5' and/or 3' to the
recombination site.
[0243] While conditions suitable for intein excision will vary with
the particular intein, as well as the protein that contains this
intein, Chong et al., Gene 192:271-281 (1997), have demonstrated
that a modified Saccharomyces cerevisiae intein, referred to as Sce
VMA intein, can be induced to undergo self-cleavage by a number of
agents including 1,4-dithiothreitol (DTT), .beta.-mercaptoethanol,
and cysteine. For example, intein excision/splicing can be induced
by incubation in the presence of 30 mM DTT, at 4.degree. C. for 16
hours.
[0244] Corresponding recombination proteins for these systems may
be used in accordance with the invention with the indicated
recombination sites. Other systems providing recombination sites
and recombination proteins for use in the invention include the
FLP/FRT system from Saccharomyces cerevisiae, the resolvase family
(e.g., 4, Tn3 resolvase, Hin, GIn and Cin), and IS231 and other
Bacillus thuringiensis transposable elements. Other suitable
recombination systems for use in the present invention include the
XerC and XerD recombinases and the psi, dif and cer recombination
sites in E. coli. Other suitable recombination sites may be found
in U.S. Pat. No. 5,851,808 issued to Elledge and Liu which is
specifically incorporated herein by reference. Preferred
recombination proteins and mutant or modified recombination sites
for use in the invention include those described in U.S. Pat. Nos.
5,888,732, 6,171,861, 6,143,557, 6,270,969 and 6,277,608, and
commonly owned, co-pending U.S. application Ser. Nos. 09/438,358
(filed Nov. 12, 1999), 09/517,466 (filed Mar. 2, 2000), 09/695,065
(filed Oct. 25, 2000) and 09/732,914 (filed Dec. 11, 2000), the
disclosures of all of which are incorporated herein by reference in
their entireties, as well as those associated with the GATEWAY.TM.
Cloning Technology available from Invitrogen Corporation (Carlsbad,
Calif.).
Topoisomerase Cloning
[0245] The present invention also relates to methods of using one
or more topoisomerases to generate a recombinant nucleic acid
molecule from two or more nucleotide sequences. In a first aspect,
the invention provides a method for generating a ds recombinant
nucleic acid molecule that is covalently linked in one strand. Such
a method is directed to linking a first and at least a second
nucleotide sequence with at least one (e.g., 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, etc.) topoisomerase (e.g., a type IA, type IB, and/or
type II topoisomerase) such that one strand, but not both strands,
is covalently linked (see, for example, FIGS. 11A-F). In a second
aspect, the invention provides a method for generating a ds
recombinant nucleic acid molecule covalently linked in both
strands. Such a method is directed to linking a first and at least
a second nucleotide sequence with at least one topoisomerase, such
that ligated ends are covalently linked in both strands (i.e., the
ds recombinant nucleic acid molecule contain no nicks at the
positions where ends were ligated; see, for example, FIGS. 12A-D).
In a third aspect, the invention provides a method for generating a
recombinant nucleic acid molecule covalently linked in one strand,
wherein the substrate nucleotide sequences linked according to the
method include at least one single stranded nucleotide sequence,
which can be covalently linked to a second (or more) single
stranded nucleotide sequence or to a nucleic acid molecule (see,
for example, FIG. 15).
[0246] A method for generating a ds recombinant nucleic acid
molecule covalently linked in one strand can be performed by
contacting a first nucleic acid molecule which has a site-specific
topoisomerase recognition site (e.g., a type IA or a type II
topoisomerase recognition site), or a cleavage product thereof, at
a 5' or 3' terminus, with a second (or other) nucleic acid
molecule, and optionally, a topoisomerase (e.g., a type IA, type
IB, and/or type II topoisomerase), such that the second nucleotide
sequence can be covalently attached to the first nucleotide
sequence. As disclosed herein, the methods of the invention can be
performed using any number of nucleotide sequences, typically
nucleic acid molecules wherein at least one of the nucleotide
sequences has a site-specific topoisomerase recognition site (e.g.,
a type IA, or type II topoisomerase), or cleavage product thereof,
at one or both 5' termini (see, for example, FIGS. 11A-11F).
[0247] A method for generating a ds recombinant nucleic acid
molecule covalently linked in both strands can be performed, for
example, by contacting a first nucleic acid molecule having a first
end and a second end, wherein, at the first end or second end or
both, the first nucleic acid molecule has a topoisomerase
recognition site (or cleavage product thereof) at or near the 3'
terminus; at least a second nucleic acid molecule having a first
end and a second end, wherein, at the first end or second end or
both, the at least second double stranded nucleotide sequence has a
topoisomerase recognition site (or cleavage product thereof) at or
near a 3' terminus; and at least one site specific topoisomerase
(e.g., a type IA and/or a type IB topoisomerase), under conditions
such that all components are in contact and the topoisomerase can
effect its activity. A covalently linked ds recombinant nucleic
acid generated according to a method of this aspect of the
invention is characterized, in part, in that it does not contain a
nick in either strand at the position where the nucleic acid
molecules are joined. In one embodiment, the method is performed by
contacting a first nucleic acid molecule and a second (or other)
nucleic acid molecule, each of which has a topoisomerase
recognition site, or a cleavage product thereof, at the 3' termini
or at the 5' termini of two ends to be covalently linked. In
another embodiment, the method is performed by contacting a first
nucleic acid molecule having a topoisomerase recognition site, or
cleavage product thereof, at the 5' terminus and the 3' terminus of
at least one end, and a second (or other) nucleic acid molecule
having a 3' hydroxyl group and a 5' hydroxyl group at the end to be
linked to the end of the first nucleic acid molecule containing the
recognition sites. As disclosed herein, the methods can be
performed using any number of nucleic acid molecules having various
combinations of termini and ends (see, for example, FIG.
12A-12D).
[0248] Topoisomerases are categorized as type I, including type IA
and type IB topoisomerases, which cleave a single strand of a
double stranded nucleic acid molecule, and type II topoisomerases
(gyrases), which cleave both strands of a nucleic acid molecule.
Type IA and IB topoisomerases cleave one strand of a nucleic acid
molecule. Cleavage of a nucleic acid molecule by type IA
topoisomerases generates a 5' phosphate and a 3' hydroxyl at the
cleavage site, with the type IA topoisomerase covalently binding to
the 5' terminus of a cleaved strand. In comparison, cleavage of a
nucleic acid molecule by type IB topoisomerases generates a 3'
phosphate and a 5' hydroxyl at the cleavage site, with the type IB
topoisomerase covalently binding to the 3' terminus of a cleaved
strand. As disclosed herein, type I and type II topoisomerases, as
well as catalytic domains and mutant forms thereof, are useful for
generating ds recombinant nucleic acid molecules covalently linked
in both strands according to a method of the invention.
[0249] Type IA topoisomerases include E. coli topoisomerase I, E.
coli topoisomerase III, eukaryotic topoisomerase II, archeal
reverse gyrase, yeast topoisomerase III, Drosophila topoisomerase
III, human topoisomerase m, Streptococcus pneumoniae topoisomerase
III, and the like, including other type IA topoisomerases (see
Berger, Biochim. Biophys. Acta 1400:3-18, 1998; DiGate and Marians,
J. Biol. Chem. 264:17924-17930, 1989; Kim and Wang, J. Biol. Chem.
267:17178-17185, 1992; Wilson et al., J. Biol. Chem. 275:1533-1540,
2000; Hanai et al., Proc. Natl. Acad. Sci. USA 93:3653-3657, 1996,
U.S. Pat. No. 6,277,620, each of which is incorporated herein by
reference). E. coli topoisomerase III, which is a type IA
topoisomerase that recognizes, binds to and cleaves the sequence
5'-GCAACTT-3', can be particularly useful in a method of the
invention (Zhang et al., J. Biol. Chem. 270:23700-23705, 1995,
which is incorporated herein by reference). A homolog, the traE
protein of plasmid RP4, has been described by Li et al., J. Biol.
Chem. 272:19582-19587 (1997) and can also be used in the practice
of the invention. A DNA-protein adduct is formed with the enzyme
covalently binding to the 5'-thymidine residue, with cleavage
occurring between the two thymidine residues.
[0250] Type IB topoisomerases include the nuclear type I
topoisomerases present in all eukaryotic cells and those encoded by
vaccinia and other cellular poxviruses (see Cheng et al., Cell
92:841-850, 1998, which is incorporated herein by reference). The
eukaryotic type IB topoisomerases are exemplified by those
expressed in yeast, Drosophila and mammalian cells, including human
cells (see Caron and Wang, Adv. Pharmacol. 29B: 271-297, 1994;
Gupta et al., Biochim. Biophys. Acta 1262:1-14, 1995, each of which
is incorporated herein by reference; see, also, Berger, supra,
1998). Viral type IB topoisomerases are exemplified by those
produced by the vertebrate poxviruses (vaccinia, Shope fibroma
virus, ORF virus, fowlpox virus, and molluscum contagiosum virus),
and the insect poxvirus (Amsacta moorei entomopoxvirus) (see
Shuman, Biochim. Biophys. Acta 1400:321-337, 1998; Petersen et al.,
Virology 230:197-206, 1997; Shuman and Prescott, Proc. Natl. Acad.
Sci. USA 84:7478-7482, 1987; Shuman, J. Biol. Chem.
269:32678-32684, 1994; U.S. Pat. No. 5,766,891; PCT/US95/16099;
PCT/US98/12372, each of which is incorporated herein by reference;
see, also, Cheng et al., supra, 1998).
[0251] Type II topoisomerases include, for example, bacterial
gyrase, bacterial DNA topoisomerase IV, eukaryotic DNA
topoisomerase II, and T-even phage encoded DNA topoisomerases (Roca
and Wang, Cell 71:833-840, 1992; Wang, J. Biol. Chem.
266:6659-6662, 1991, each of which is incorporated herein by
reference; Berger, supra, 1998). Like the type IB topoisomerases,
the type II topoisomerases have both cleaving and ligating
activities. In addition, like type IB topoisomerase, substrate
nucleic acid molecules can be prepared such that the type II
topoisomerase can form a covalent linkage to one strand at a
cleavage site. For example, calf thymus type II topoisomerase can
cleave a substrate nucleic acid molecule containing a 5' recessed
topoisomerase recognition site positioned three nucleotides from
the 5' end, resulting in dissociation of the three nucleotide
sequence 5' to the cleavage site and covalent binding the of the
topoisomerase to the 5' terminus of the nucleic acid molecule
(Andersen et al., supra, 1991). Furthermore, upon contacting such a
type II topoisomerase charged nucleic acid molecule with a second
nucleotide sequence containing a 3' hydroxyl group, the type II
topoisomerase can ligate the sequences together, and then is
released from the recombinant nucleic acid molecule. As such, type
II topoisomerases also are useful for performing methods of the
invention.
[0252] Structural analysis of topoisomerases indicates that the
members of each particular topoisomerase families, including type
IA, type IB and type II topoisomerases, share common structural
features with other members of the family (Berger, supra, 1998). In
addition, sequence analysis of various type IB topoisomerases
indicates that the structures are highly conserved, particularly in
the catalytic domain (Shuman, supra, 1998; Cheng et al., supra,
1998; Petersen et al., supra, 1997). For example, a domain
comprising amino acids 81 to 314 of the 314 amino acid vaccinia
topoisomerase shares substantial homology with other type IB
topoisomerases, and the isolated domain has essentially the same
activity as the full length topoisomerase, although the isolated
domain has a slower turnover rate and lower binding affinity to the
recognition site (see Shuman, supra, 1998; Cheng et. al., supra,
1998). In addition, a mutant vaccinia topoisomerase, which is
mutated in the amino terminal domain (at amino acid residues 70 and
72) displays identical properties as the full length topoisomerase
(Cheng et al., supra, 1998). In fact, mutation analysis of vaccinia
type IB topoisomerase reveals a large number of amino acid residues
that can be mutated without affecting the activity of the
topoisomerase, and has identified several amino acids that are
required for activity (Shuman, supra, 1998). In view of the high
homology shared among the vaccinia topoisomerase catalytic domain
and the other type IB topoisomerases, and the detailed mutation
analysis of vaccinia topoisomerase, it will be recognized that
isolated catalytic domains of the type IB topoisomerases and type
IB topoisomerases having various amino acid mutations can be used
in the methods of the invention.
[0253] The various topoisomerases exhibit a range of sequence
specificity. For example, type II topoisomerases can bind to a
variety of sequences, but cleave at a highly specific recognition
site (see Andersen et al., J. Biol. Chem. 266:9203-9210, 1991,
which is incorporated herein by reference). In comparison, the type
IB topoisomerases include site specific topoisomerases, which bind
to and cleave a specific nucleotide sequence ("topoisomerase
recognition site"). Upon cleavage of a nucleic acid molecule by a
topoisomerase, for example, a type IB topoisomerase, the energy of
the phosphodiester bond is conserved via the formation of a
phosphotyrosyl linkage between a specific tyrosine residue in the
topoisomerase and the 3' nucleotide of the topoisomerase
recognition site. Where the topoisomerase cleavage site is near the
3' terminus of the nucleic acid molecule, the downstream sequence
(3' to the cleavage site) can dissociate, leaving a nucleic acid
molecule having the topoisomerase covalently bound to the newly
generated 3' end (see FIG. 29).
[0254] A method of the invention for generating a ds recombinant
nucleic acid molecule covalently linked in one strand, can be
performed by contacting 1) a first nucleic acid molecule having a
first end and a second end, wherein the first nucleic acid molecule
has a site-specific topoisomerase recognition site (e.g., a type IA
or a type II topoisomerase recognition site) at or near the 5'
terminus of the first end or the second end or both and,
optionally, comprising one or more recombination sites; 2) at least
a second nucleic acid molecule that has, or can be made to have, a
first end and a second end; and 3) at least one (e.g., 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, etc.) site-specific topoisomerase (e.g., a type
IA or a type IB topoisomerase), under conditions such that all
components are in contact and the at least one topoisomerase can
effect its activity. For example, the topoisomerase can be a type
IA topoisomerase such as E. coli topoisomerase I, E. coli
topoisomerase III, or a eukaryotic topoisomerase III. Upon cleavage
of a nucleic acid molecule, the topoisomerase preferably is stably
bound to the 5' terminus. Upon cleavage by the topoisomerase, the
cleaved nucleic acid molecule often may comprise a 3' overhanging
sequence. Once nucleic acid molecules are joined by the methods
described above, the resulting molecules may then be used in
recombination reactions, such as those described elsewhere
herein.
[0255] A method of the invention for generating a ds recombinant
nucleic acid molecule covalently linked in one strand can be
performed such that any combination of ends are linked, and wherein
one strand at the ends being linked is covalently linked and the
other strand is not covalently linked, but contains a nick. For
example, the first nucleic acid molecule can comprise a coding
sequence, wherein the ATG start codon is at or near the first end
and a poly A signal is encoded at or near the second end; and a
second nucleic acid molecule can comprise a promoter element, which
functions when positioned upstream of a coding sequence, and the
first end is upstream of the second end, the method can be
performed wherein a site-specific topoisomerase recognition site
(e.g., a type IA or a type II topoisomerase recognition site) is at
or near the 5' terminus of the first end of the first nucleic acid
molecule, and wherein the contacting is performed under conditions
such that the topoisomerase (e.g., a type IA or a type II
topoisomerase) can covalently link the 5' terminus of the first end
of the first nucleic acid molecule to the 3' terminus of the first
end of the second nucleic acid molecule, thereby generating a ds
recombinant nucleic acid molecule, in which a polypeptide can be
expressed from the coding sequence. Alternatively, the method can
be performed wherein the topoisomerase recognition site (e.g., a
type IA or a type II topoisomerase recognition site) is at or near
the 5' terminus of the second end of the first nucleic acid
molecule, and wherein the contacting is performed under conditions
such that the topoisomerase (e.g., a type IA or a type II
topoisomerase recognition site) can covalently link the 5' terminus
of the second end of the first nucleic acid molecule to the 3'
terminus of the first end of the second nucleic acid molecule,
thereby generating a ds recombinant nucleic acid molecule from
which an antisense molecule can be expressed. Once nucleic acid
molecules are joined by the methods described above, the resulting
molecules may then be used in recombination reactions, such as
those described elsewhere herein.
[0256] As another example using the first nucleic acid molecule and
second nucleic acid molecule described above, the method can be
performed, wherein the topoisomerase recognition site (e.g., a type
IA or a type II topoisomerase recognition site) is at or near the
5' terminus of each of the first end and the second end of the
first nucleic acid molecule, and wherein the contacting is
performed under conditions such that the type IA topoisomerase can
covalently link the 5' terminus of the first end of the first
nucleic acid molecule to the 3' terminus of the first end of the
second nucleic acid molecule, and the 5' terminus of the second end
of the first nucleic acid molecule to the 3' terminus of the second
end of the second nucleic acid molecule. As such, the ds
recombinant nucleic acid molecule generated by the method is
circularized, and includes a nick in each strand opposite the
location where a strand was covalently linked by a topoisomerase
(e.g., a type IA or a type II topoisomerase). Furthermore, the
promoter of the second nucleic acid molecule can initiate
expression of the first nucleic acid molecule. In one embodiment,
the circularized ds recombinant nucleic acid molecule comprises a
vector. Once nucleic acid molecules are joined by the methods
described above, the resulting molecules may then be used in
combination reactions, such as those described elsewhere
herein.
[0257] As another example using the first nucleic acid molecule and
second nucleic acid molecule described above, the method can be
performed, wherein the topoisomerase recognition site (e.g., a type
IA or a type II topoisomerase recognition site) is at or near the
5' terminus of each of the first end and the second end of the
first nucleic acid molecule, and wherein the contacting is
performed under conditions such that the topoisomerase (e.g., a
type IA or a type II topoisomerase) can covalently link the 5'
terminus of the first end of the first nucleic acid molecule to the
3' terminus of the second end of the second nucleic acid molecule,
and the 5' terminus of the second end of the first nucleic acid
molecule to the 3' terminus of the first end of the second nucleic
acid molecule. As such, the ds recombinant nucleic acid molecule
generated by the method is circularized, and includes a nick in
each strand opposite the location where a strand was covalently
linked by topoisomerase (e.g., a type IA or a type II topoisomerase
recognition site). Furthermore, the promoter of the second nucleic
acid molecule can initiate expression of an antisense sequence. In
one embodiment, the circularized ds recombinant nucleic acid
molecule comprises a vector. Once nucleic acid molecules are joined
by the methods described above, the resulting molecules may then be
used in recombination reactions, such as those described elsewhere
herein.
[0258] As disclosed herein, a method of generating a ds recombinant
nucleic acid molecule covalently linked in one strand, involving a
first nucleic acid molecule and at least a second nucleic acid
molecule, can further include a step for amplifying the ds
recombinant nucleic acid molecule covalently linked in one strand.
The amplification reaction can be carried out by contacting the ds
recombinant nucleic acid molecule with an amplification reaction
primer pair, wherein a first primer of the pair is capable of
binding to the covalently linked strand, at or near one end of the
first or second nucleic acid molecule, and priming an amplification
reaction toward the other nucleic acid molecule to generate a first
extension product that is identical in nucleotide sequence to the
nicked strand of the ds recombinant nucleic acid molecule; and the
second primer of the pair is capable of binding to the first
extension product, typically at or near the 3' terminus, and, in
the presence of the first primer, can generate an amplification
product using the covalently linked strand and the extension
product (or extension products generated therefrom) as templates.
For example, the method can be performed such that the type IA
topoisomerase recognition site is at or near a first end of the
first nucleic acid molecule, and the method further includes
contacting the ds recombinant nucleic acid molecule with an
amplification reaction primer pair, wherein a forward primer is
capable of binding at or near the second end of the first nucleic
acid molecule, and wherein a reverse primer is capable of binding
to a nucleotide sequence complementary to at least a portion of the
second end of the second nucleic acid molecule; and amplifying the
ds recombinant nucleic acid molecule. The first nucleic acid
molecule can include a coding region and the second nucleic acid
molecule can include a regulatory element. Once nucleic acid
molecules are joined by the methods described above, the resulting
molecules may then be used in recombination reactions, such as
those described elsewhere herein.
[0259] A method of generating a ds recombinant nucleic acid
molecule covalently linked in one strand also can be performed by
contacting 1) a first nucleic acid molecule having a first end and
a second end, wherein the first nucleic acid molecule has a
site-specific topoisomerase recognition site (e.g., a type IA or a
type II topoisomerase recognition site) at or near the 5' terminus
of the first end or the second end or both; 2) at least a second
nucleic acid molecule that has, or can be made to have, a first end
and a second end; 3) at least a third nucleic acid molecule which
has, or can be made to have, a first end and a second end, each end
further comprising a 5' terminus and a 3' terminus; and 4) at least
one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) site-specific
topoisomerase (e.g., a type IA or a type II topoisomerase), under
conditions such that all components are in contact and the at least
one topoisomerase can effect its activity. For example, the
topoisomerase can be a type IA topoisomerase such as E. coli
topoisomerase I, E. coli topoisomerase III, or a eukaryotic
topoisomerase III. Upon cleavage of a nucleic acid molecule, the
topoisomerase preferably is stably bound to the 5' terminus.
Preferably, upon cleavage by the topoisomerase, the cleaved nucleic
acid molecule comprises a 3' overhanging sequence. Once nucleic
acid molecules are joined by the methods described above, the
resulting molecules may then be used in recombination reactions,
such as those described elsewhere herein.
[0260] A method of the invention for generating a ds recombinant
nucleic acid molecule covalently linked in one strand, involving a
first nucleic acid molecule that contains a site-specific
topoisomerase recognition site (e.g., a type IA or a type IB
topoisomerase recognition site), or cleavage product thereof, at
least a second nucleic acid molecule, and at least a third nucleic
acid molecule can be performed such that any combination of ends
are linked, and one strand at the ends being linked is covalently
linked and one strand is nicked. According to this embodiment, any
of the ends can contain a type IA, type II, or type IB
topoisomerase recognition site, or can comprise a cleavage product
thereof, provided that the first ds recombinant nucleotide molecule
contains a topoisomerase recognition site (e.g., a type IA or a
type H topoisomerase recognition site) at or near a 5' terminus, or
a cleavage product thereof, and only one topoisomerase or
topoisomerase recognition site is present at the ends that are to
be linked. For example, where the first nucleic acid molecule
comprises a site-specific type IA topoisomerase recognition site at
or near each of the first end and the second end, the method
further can include contacting the first nucleic acid molecule and
the second nucleic acid molecule with at least a third nucleic acid
molecule which has, or can be made to have, a first end and a
second end, each end further comprising a 5' terminus and a 3'
terminus, under conditions such that the topoisomerase (e.g., a
type IA or a type II topoisomerase) can covalently link the 5'
terminus of the first end of the first nucleic acid molecule with
the 3' terminus of the first end of the second nucleotide sequence,
and the 5' terminus of the second end of the first nucleic acid
molecule with the 3' terminus of the first end of the third
nucleotide sequence. It will be recognized that other combinations
of ends and topoisomerase recognition sites, or cleavage products
thereof, can be used to perform such a method of the invention.
Once nucleic acid molecules are joined by the methods described
above, the resulting molecules may then be used in recombination
reactions, such as those described elsewhere herein.
[0261] A method of the invention also can be performed by
contacting a first nucleic acid molecule and a second nucleic acid
molecule with at least a third nucleic acid molecule, which
comprises a first end and a second end, each end further comprising
a 5' terminus and a 3' terminus, wherein the third nucleic acid
molecule comprises a type IB topoisomerase recognition site at or
near the 3' terminus of said first end, or said second end, or both
said first end and said second end; and at least one (e.g., 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, etc.) type IB topoisomerase under
conditions such that the type IB topoisomerase can covalently link
the 3' terminus of the first end or second end of the third nucleic
acid molecule to the 5' terminus of the first end or second end of
the second nucleic acid molecule. In such a method, where the third
nucleic acid molecule comprises a type IB topoisomerase recognition
site at or near the 3' terminus of the first end, the contacting
can be performed under conditions such that the type IB
topoisomerase can covalently link the 3' terminus of the first end
of the third nucleic acid molecule to the 5' terminus of the first
end of the second nucleic acid molecule. It will be recognized that
other combinations of ends and topoisomerase recognition sites, or
cleavage products thereof, can be used to perform such a method of
the invention. Once nucleic acid molecules are joined by the
methods described above, the resulting molecules may then be used
in recombination reactions, such as those described elsewhere
herein.
[0262] In another embodiment, a method for generating a ds
recombinant nucleic acid molecule covalently linked in one strand
can be performed by contacting 1) a first nucleic acid molecule
having a first end and a second end, wherein the first nucleic acid
molecule has a site-specific topoisomerase recognition site (e.g.,
a type IA or a type II topoisomerase recognition site) at or near
the 5' terminus of an end and a type BB topoisomerase recognition
site at or near the 3' terminus of the other end; 2) at least a
second nucleic acid molecule that has, or can be made to have, a
first end and a second end; 3) at least one (e.g., 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, etc.) site-specific topoisomerase (e.g., a type IA
or a type II topoisomerase); and 4) at least one (e.g., 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, etc.) type BB topoisomerase under conditions
such that all components are in contact and the at least one
topoisomerase can effect its activity. For example, the
topoisomerase, for which a recognition site is at or near the 5'
terminus, can be a type IA topoisomerase such as E. coli
topoisomerase I, E. coli topoisomerase III, or a eukaryotic
topoisomerase III. Upon cleavage of a nucleic acid molecule, the
type IA topoisomerase preferably is stably bound to the 5'
terminus, and the type IB topoisomerase preferably is stably bound
at the 3' terminus. Preferably, upon cleavage by the
topoisomerases, the cleaved nucleic acid molecule comprises a 3'
overhanging sequence and a 5' overhanging sequence. The method can
further include contacting the ds recombinant nucleic acid molecule
with a DNA ligase, thereby generating a ds recombinant nucleic acid
molecule covalently linked in both strands. Once nucleic acid
molecules are joined by the methods described above, the resulting
molecules may then be used in recombination reactions, such as
those described elsewhere herein.
[0263] A method of generating a ds recombinant nucleic acid
molecule covalently linked in one strand by contacting a first
nucleic acid molecule, a second nucleic acid molecule, and at least
a third nucleic acid molecule, can further include a step for
amplifying the ds recombinant nucleic acid molecule, particularly
the covalently linked strand. The amplification can be carried out
by contacting the ds recombinant nucleic acid molecule with an
amplification reaction primer pair, wherein a first primer of the
pair can bind selectively to the covalently linked strand at or
near one end of the first or second nucleic acid molecule and prime
an amplification reaction toward the other nucleic acid molecule to
generate a first extension product that is complementary to the
covalently-linked strand; and the second primer of the pair can
bind selectively to the first extension product, typically at or
near the 3' terminus, and, in the presence of the first primer, can
generate an amplification product using the covalently linked
strand and the extension product (or extension products derived
therefrom) as templates. The method can be performed such that the
topoisomerase recognition site (e.g., a type IA or a type IB
topoisomerase recognition site) is at or near the first end of the
first nucleic acid molecule, and can further include contacting the
ds recombinant nucleic acid molecule with an amplification reaction
primer pair, wherein a forward primer is capable of binding to a
nucleotide sequence at or near the second end of the first nucleic
acid molecule and wherein a reverse primer is capable of binding to
a nucleotide sequence complementary to at least a portion of the
third nucleic acid molecule; and amplifying the ds recombinant
nucleic acid molecule. The first nucleic acid molecule can include
a coding region and the third nucleic acid molecule can include a
regulatory element. Furthermore, the ends being linked can contain
complementary overhanging sequences. Once nucleic acid molecules
are joined by the methods described above, the resulting molecules
may then be used in recombination reactions, such as those
described elsewhere herein.
[0264] Representative embodiments of the disclosed methods for
generating a ds recombinant nucleic acid molecule covalently linked
in one strand and, optionally, comprising one or more recombination
sites, are illustrated in FIGS. 11A-11F. In FIG. 11A, one of the
nucleic acid molecules has a topoisomerase attached to the 5'
terminus of one end such that, when this molecule, which has a 3'
overhang, is contacted with a second nucleic acid molecule having a
substantially complementary 3' overhang, under suitable conditions,
the nucleotides comprising the 3' overhangs can hybridize and the
topoisomerases can catalyze ligation. FIG. 11B shows a first
nucleic acid molecule having topoisomerase molecules linked to the
5' terminus and 3' terminus of two different ends of one nucleotide
sequence, and further shows linkage of the first nucleic acid
molecule to two other nucleotide sequences to generate a nucleic
acid molecule which has one strand without any nicks and another
strand with two nicks. FIG. 11C shows a first nucleic acid molecule
having a topoisomerase molecule linked to the 5' terminus of one
end and a second nucleic acid molecule having a topoisomerase
molecule linked to the 5' terminus of one end, and further shows
linkage of the first and second nucleic acid molecule to one other
nucleotide sequence to generate a nucleic acid molecule which has
one strand without any nicks and another strand with two nicks. In
FIG. 11D, one of the nucleic acid molecules to be linked has
site-specific type IA topoisomerases attached to the 5' terminus of
both ends such that, when the nucleotide sequences are contacted
the complementary 3' overhangs can hybridize and the topoisomerases
catalyze ligation. FIG. 11E shows another example of linking three
nucleic acid molecules together, using one nucleic acid molecule
that is topoisomerase-charged with a type IA topoisomerase at a 5'
terminus and another nucleic acid molecule that is
topoisomerase-charged with a type IB topoisomerase at a 3' terminus
of the opposite strand to be linked, such that when the nucleotide
sequences are contacted the complementary 3' overhangs can
hybridize and the topoisomerases catalyze ligation. FIG. 11F
illustrates another example of linking three nucleic acid molecules
together, in this case using one nucleic acid molecule that is
topoisomerase-charged with a topoisomerase (e.g., a type IA or a
type II topoisomerase) at a first 5' terminus and is charged with a
topoisomerase at a second 5'-terminus of the opposite strand, such
that when the nucleotide sequences are contacted under suitable
conditions, the complementary 3' overhangs can hybridize and the
topoisomerases catalyze ligation. Once nucleic acid molecules are
joined by the methods described above, the resulting molecules may
then be used in recombination reactions, such as those described
elsewhere herein.
[0265] The examples set forth in FIGS. 11A-11F show the ends of the
nucleic acid molecules opposite those being linked as having blunt
ends, and shows the being linked as having 3' overhanging
sequences. However, the substrate nucleic acid molecules can have
any ends and overhangs as desired, including both ends being blunt
and/or complementary, or combinations thereof, such that the ends
can be ligated to each other, for example, to form circular
molecules or to other nucleic acid molecules having an appropriate
end. Thus, one or more of the blunt ends as shown in FIGS. 11A-11F
can be substituted with a nucleotide sequence comprising a 5'
overhang or a 3' overhang, either of which can constitute a single
nucleotide such as a thymidine residue or multiple nucleotides
(e.g., two, three, four, five, six, seven, eight, nine, ten,
eleven, twelve, thirteen, fourteen, fifteen, etc. nucleotides),
which can be the same or different. In certain embodiments of the
disclosed methods, a first nucleic acid molecule contains a blunt
end to be linked, and a second nucleic acid molecule contains an
overhang at the end which is to be linked by a site-specific
topoisomerase (e.g., a type IA or a type IB topoisomerase), wherein
the overhang includes a sequence complementary to that comprising
the blunt end, thereby facilitating strand invasion as a means to
properly position the ends for the linking reaction.
[0266] As exemplified in FIGS. 11A-11C, the ds recombinant nucleic
acid molecule generated using the methods of this aspect of the
invention include those in which one strand (not both strands) is
covalently linked at the ends to be linked (i.e. ds recombinant
nucleic acid molecules generated using these methods contain a nick
at each position where two ends were joined). These embodiments are
particularly advantageous in that a polymerase can be used to
replicate the ds recombinant nucleic acid molecule by initially
replicating the covalently linked strand. For example, a
thermostable polymerase such as a polymerase useful for performing
an amplification reaction such as PCR can be used to replicate the
covalently strand, whereas the strand containing the nick does not
provide a suitable template for replication.
[0267] The present invention also provides methods of covalently
ligating the ends of two different nucleic acid molecules or two
ends of the same nucleic acid molecule, such that the product
generated is ligated in both strands and, therefore, does not
contain a nick. Representative embodiments of this aspect of the
invention are illustrated in FIG. 12. For example, in FIG. 12A, one
of the nucleic acid molecules has topoisomerase molecules attached
to the 3' terminus and the 5' terminus of one end such that, when
this molecule, which has a 5' overhang, is contacted with a second
nucleic acid molecule having a substantially complementary 5'
overhang, under suitable conditions, the nucleotides comprising the
5' overhangs can hybridize and the topoisomerases can catalyze
ligation of both strands of the nucleic acid molecules. In FIG.
12B, each end of the nucleic acid molecules to be linked has a
topoisomerase molecule attached to the 3' terminus such that, when
the nucleotide sequences are contacted under suitable conditions,
nucleotides comprising the 5' overhangs can hybridize and the
topoisomerases catalyze ligation (compare FIG. 12C, in which each
of the nucleic acid molecules to be linked has a topoisomerase
attached to the 5' termini of the ends to be linked). FIG. 12D
illustrates linking three nucleic acid molecules together via a
nucleic acid molecule that is topoisomerase-charged at both termini
of both ends. Similarly to FIG. 11, the examples set forth in FIGS.
12A-12D show the ends of the nucleic acid molecules that are not
being linked as having blunt ends. As discussed with respect to
FIG. 11, however, the substrate nucleic acid molecules utilized in
methods as exemplified in FIG. 12 can have any ends as desired,
including topoisomerase-charged ends, such that the ends can be
ligated to each other, for example, to form circular molecules or
to other nucleic acid molecules having an appropriate end, blunt
ends, 5' overhangs, 3' overhangs, and the like, as desired. Once
nucleic acid molecules are joined by the methods described above,
the resulting molecules may then be used in recombination
reactions, such as those described elsewhere herein.
[0268] A covalently bound topoisomerase, in addition to catalyzing
a ligation reaction, also can catalyze the reverse reaction, for
example, religation of the 3' nucleotide of the recognition
sequence, to which the type IB topoisomerase is linked through the
phosphotyrosyl bond, and the nucleotide sequence that, prior to
cleavage, comprised the 3' terminus of the nucleic acid molecule,
and which, following cleavage, contains a free 5' hydroxy group. As
such, methods have been developed for using a type IB topoisomerase
to produce recombinant nucleic acid molecules. For example, cloning
vectors containing a bound type IB topoisomerase have been
developed and are commercially available (Invitrogen Corporation,
Carlsbad, Calif.). Such cloning vectors, when linearized, contain a
covalently bound type IB topoisomerase at each 3' end
("topoisomerase charged"). Nucleotide sequences such as those
comprising a cDNA library, or restriction fragments, or sheared
genomic DNA sequences that are to be cloned into such a vector are
treated, for example, with a phosphatase to produce 5' hydroxyl
termini, then are added to the linearized topoisomerase-charged
vector under conditions that allow the topoisomerase to ligate the
nucleotide sequences at the 5' terminus containing the hydroxyl
group and the 3' terminus of the vector that contains the
covalently bound topoisomerase. A nucleotide sequence such as a PCR
amplification product, which is generated containing 5' hydroxyl
ends, can be cloned into a topoisomerase-charged vector in a rapid
joining reaction (approximately 5 minutes at room temperature). The
rapid joining and broad temperature range inherent to the
topoisomerase joining reaction makes the use of
topoisomerase-charged vectors ideal for high throughput
applications, which generally are performed using automated
systems.
[0269] Type II topoisomerases have not generally been used for
generating recombinant nucleic acid molecules or cloning
procedures, whereas type IB topoisomerases, as indicated above, are
used in a variety of procedures. As disclosed herein, type IA
topoisomerases can be used in a variety of procedures similar to
those described for the type IB topoisomerases. However, previously
described methods of using type IB topoisomerases to ligate two or
more nucleotide sequences have suffered from the disadvantage that
the bound topoisomerase only effects the joining of the 3' end of
the strand to which it is attached and a second strand containing a
5' hydroxyl group. Since the topoisomerase cannot ligate the
complementary strands, the nucleic acid molecules that are
generated contain nicks. While the presence of such nicks does not
prevent the use of the recombinant molecules for transfection of a
host cells, as the nicks generally are resolved intracellularly,
the presence of such nicks in double stranded nucleic acid
molecules significantly limits direct use of the recombinant
molecules. For example, a strand of a nucleic acid molecule
containing a nick cannot be amplified by PCR because the primer
extension reaction terminates at the nick. Thus, nucleic acid
constructs prepared using a topoisomerase according to previously
described methods generally must be further treated, for example,
with a DNA ligase, to obtain a ds recombinant nucleic acid molecule
that is covalently linked in both strands and, therefore, useful
for subsequent manipulations such as PCR.
[0270] Previously described methods for preparing nucleic acid
constructs also generally required numerous steps, particularly
where more than two nucleotide sequences are to be ligated, and
even more so where the sequences must be ligated in a predetermined
orientation. For example, the nucleotide sequences to be linked
generally are ligated sequentially to produce intermediate
constructs, each of which must be cloned, amplified in a host cell,
isolated, and characterized. The constructs containing the correct
sequences then must be isolated in a sufficient quantity and form
such that the next nucleotide sequence can be ligated, and the
process of cloning, amplifying, isolating and characterizing
performed again to identify the proper construct. Clearly, as the
number of different nucleotide sequences to be joined increases, so
do the number of essentially repetitive procedures that must be
performed, thus resulting in an expensive, laborious and lengthy
process.
[0271] As disclosed herein, an advantage of a method of the
invention for generating a ds recombinant nucleic acid molecule
covalently linked in both strands is that there is no need to
perform a separate ligation reaction in order to obtain a
functional ds recombinant nucleic acid molecule covalently linked
in both strands (see FIGS. 8 and 12). In addition, a method of this
aspect of the invention can be performed such that, where a number
of different nucleic acid molecules are to be covalently linked in
a predetermined orientation, there is no requirement that
intermediate constructs be cloned, characterized and isolated
before proceeding to a subsequent step (see Example 1.B). As such,
the methods of this aspect of the invention provide a means to
generate a ds recombinant nucleic acid molecule covalently linked
in both strands much more quickly and at a substantially lower cost
than was possible using previously known methods.
[0272] As an additional advantage, the generated ds recombinant
nucleic acid molecules covalently linked in both strands are in a
form that can be used directly in further procedures, for example,
particular procedures involving extension of a primer such as a PCR
amplification procedure, or other transcription or translation
procedure, because the generated construct does not contain nicks
at the sites where the ds nucleotides sequences have been joined.
As disclosed herein, a method of the invention for generating a ds
recombinant nucleic acid molecule covalently linked in one strand,
in certain embodiments, also is advantageous in that the generated
ds recombinant nucleic acid molecules are in a form that can be
used directly in further procedures, for example, particular
procedures involving extension of a primer such as a PCR
amplification procedure, or other transcription or translation
procedure, because in certain embodiments, the generated ds
recombinant nucleic acid molecule contains one strand that does not
contain a nick at the sites where the ds nucleotides sequences were
joined.
[0273] The term "nucleotide sequence" or "nucleic acid molecule" is
used herein to refer to a discrete nucleic acid molecule. When used
as such, the term "nucleotide sequence" is used merely for
convenience such that the components in a composition or used in a
method of the invention can be clearly distinguished. Thus,
reference is made, for example, to "nucleic acid molecules", which,
in a method of the invention, correspond to the reactants
(substrates) used to produce a recombinant "nucleic acid molecule"
product.
[0274] Certain methods of the invention are exemplified generally
herein with reference to the use of type IB topoisomerase such as
the Vaccinia topoisomerase, or a type IA topoisomerase. However, it
will be recognized that the methods also can be performed using a
topoisomerase other than that exemplified, merely by adjusting the
components accordingly. For example, as described in greater detail
below, methods are disclosed for incorporating a type IB
topoisomerase recognition site at one or both 3' termini of a
linear nucleic acid molecule using a PCR primer comprising, at
least in part, a nucleotide sequence complementary to the
topoisomerase recognition site. In comparison, a topoisomerase
recognition site for a type IA or, if desired, type II
topoisomerase, can be incorporated into a nucleic acid molecule by
using a PCR primer that contains the recognition site.
[0275] Cleavage of a nucleic acid molecule by a site specific type
IB topoisomerase results in the generation of a 5' overhanging
sequence in the strand complementary to and at the same end as that
containing the covalently bound topoisomerase. Furthermore, as
disclosed herein, PCR primers can be designed that can incorporate
a type IB topoisomerase recognition site into a nucleic acid
molecule, and that further can produce, upon cleavage of the
nucleic acid molecule by the topoisomerase, a 5' overhanging
sequence in the complementary strand that has a defined and
predetermined sequence. As such, the methods are readily adaptable
to generating a ds recombinant nucleic acid molecule having the
component nucleic acid molecule operatively linked in a
predetermined orientation. In view of the present disclosure, it
will be recognized that PCR primers also can be designed such that
a type IA topoisomerase recognition site can be introduced into a
nucleic acid molecule, including a library of diverse sequences,
and, if desired, such that upon cleavage by a site-specific
topoisomerase, generates a 3' overhanging sequence.
[0276] A method of generating a ds recombinant nucleic acid
molecule covalently linked in both strands, as disclosed herein,
extends the previously known methods by providing a topoisomerase
at or near the terminus of each nucleic acid molecule to be
covalently linked. For example, with respect to a type IB
topoisomerase, the method provides a topoisomerase recognition
site, or a cleavage product thereof (i.e., a covalently bound type
IB topoisomerase), at or near the 3' terminus of each linear
nucleic acid molecule to be linked. As used herein, the term
"topoisomerase recognition site" means a defined nucleotide
sequence that is recognized and bound by a site specific
topoisomerase. For example, the nucleotide sequence 5'-(C/T)CCTT-3'
is a topoisomerase recognition site that is bound specifically by
most poxvirus topoisomerases, including vaccinia virus DNA
topoisomerase I, which then can cleave the strand after the 3'-most
thymidine of the recognition site to produce a nucleotide sequence
comprising 5'-(C/T)CCTT-PO.sub.4-TOPO, i.e., a complex of the
topoisomerase covalently bound to the 3' phosphate through a
tyrosine residue in the topoisomerase (see Shuman, J. Biol. Chem.
266:11372-11379, 1991; Sekiguchi and Shuman, Nucl. Acids Res.
22:5360-5365, 1994; each of which is incorporated herein by
reference; see, also, U.S. Pat. No. 5,766,891; PCT/US95/16099;
PCT/US98/12372). In comparison, the nucleotide sequence
5'-GCAACTT-3' is the topoisomerase recognition site for type IA E.
coli topoisomerase III.
[0277] Topoisomerase-charged nucleic acid molecules, including
those containing a topoisomerase covalently attached to a 5'
terminus or 3' terminus or both, of one or both ends of the nucleic
acid molecule, can be generated by any of a number of methods. In
some cases and under the appropriate conditions, type I
topoisomerases can cleave a single stranded nucleotide sequence.
For example, a domain comprising the amino-terminal 67 kDa domain
of E. coli topoisomerase I, which is a type IA topoisomerase, can
cleave a single stranded nucleotide sequence containing the
topoisomerase recognition site. Where conditions are such that the
topoisomerases can cleave a single stranded nucleotide sequence,
cleavage of a nucleic acid molecule containing topoisomerase
recognition sites at the 5' and 3' termini of one end of nucleic
acid molecule can be performed in parallel. Alternatively, where
one or both of the topoisomerases requires a nucleic acid molecule
for recognition and cleavage, the reactions are performed serially,
wherein the more terminal (distal) of the topoisomerase recognition
sites is cleaved first, then the more internal (proximal) site,
which remains in a double stranded context, is cleaved. For
example, a nucleic acid molecule containing an E. coli
topoisomerase III recognition site at or near a 5' terminus of an
end and a Vaccinia type IB topoisomerase recognition site at or
near the 3' terminus of the same end, and wherein the type IB
recognition site is closer to the end than the type IA recognition
site, the nucleic acid molecule can be incubated with the Vaccinia
topoisomerase, to produce a type IB topoisomerase charged nucleic
acid molecule, then with the E. coli topoisomerase, to produce a
nucleic acid molecule having the type IA topoisomerase bound to the
5' terminus and the type IB topoisomerase bound to the 3' terminus.
Accordingly, the invention includes methods for producing nucleic
acid molecule comprising a topoisomerase attached to one or both
termini of at least one end, and further provides such
topoisomerase-charged nucleic acid molecules.
[0278] As used herein, the term "cleavage product," when used in
reference to a topoisomerase recognition site, refers to a
nucleotide sequence that has been cleaved by a topoisomerase,
generally at its recognition site, and comprises a complex of the
topoisomerase covalently bound, in the case of type IA or type II
topoisomerase, to the 5' phosphate group of the 5' terminal
nucleotide in the topoisomerase recognition site, or in the case of
a type IB topoisomerase to the 3' phosphate group of the 3'
terminal nucleotide in the topoisomerase recognition site. Such a
complex, which comprises a topoisomerase cleaved nucleic acid
molecule having the topoisomerase covalently bound thereto, is
referred to herein as a "topoisomerase-activated" or a
"topoisomerase-charged" nucleotide sequence.
Topoisomerase-activated nucleic acid molecules can be used in a
method of the invention, as can nucleic acid molecules that contain
an uncleaved topoisomerase recognition site and a topoisomerase,
wherein the topoisomerase can cleave the nucleic acid molecule at
the recognition site and become covalently bound thereto.
[0279] In one embodiment of a method of generating a ds recombinant
nucleic acid molecule covalently linked in both strands, a
topoisomerase recognition site is present at or near the 3'
terminus of the end of each nucleotide sequence to be linked such
that, in the presence of a type IB topoisomerase, each nucleotide
sequence is cleaved to produce a 3' terminus, which contains the
topoisomerase covalently bound thereto (see FIG. 8). The nucleotide
sequences to be covalently linked also can contain a 5' hydroxy
group at the same end as that containing the topoisomerase
recognition site, or a 5' hydroxyl group can be generated using a
phosphatase. Upon contact of such nucleotide sequences, the site
specific topoisomerase can ligate each strand containing a 3'
phosphate to a respective 5' hydroxyl group, thereby generating a
ds recombinant nucleic acid molecule covalently linked in both
strands, which can be produced as a linear, circular, or positively
or negatively supercoiled nucleic acid molecule.
[0280] Preferably, the 5' termini of the ends of the nucleotide
sequences to be linked by a type IB topoisomerase according to a
method of certain aspects of the invention contain complementary 5'
overhanging sequences, which can facilitate the initial association
of the nucleotide sequences, including, if desired, in a
predetermined directional orientation. Alternatively, the 5'
termini of the ends of the nucleotide sequences to be linked by a
type IB topoisomerase according to a method of certain aspects of
the invention contain complementary 5' sequences wherein one of the
sequences contains a 5' overhanging sequence and the other
nucleotide sequence contains a complementary sequence at a blunt
end of a 5' terminus, to facilitate the initial association of the
nucleotide sequences through strand invasion, including, if
desired, in a predetermined directional orientation. The term "5'
overhang" or "5' overhanging sequence" is used herein to refer to a
strand of a nucleic acid molecule that extends in a 5' direction
beyond the terminus of the complementary strand of the nucleic acid
molecule. Conveniently, a 5' overhang can be produced as a result
of site specific cleavage of a nucleic acid molecule by a type IB
topoisomerase (see Example 1).
[0281] Preferably, the 3' termini of the ends of the nucleotide
sequences to be linked by a type IA topoisomerase according to a
method of certain aspects of the invention contain complementary 3'
overhanging sequences, which can facilitate the initial association
of the nucleotide sequences, including, if desired, in a
predetermined directional orientation. Alternatively, the 3'
termini of the ends of the nucleotide sequences to be linked by a
topoisomerase (e.g., a type IA or a type II topoisomerase)
according to a method of certain aspects of the invention contain
complementary 3' sequences wherein one of the sequences contains a
3' overhanging sequence and the other nucleotide sequence contains
a complementary sequence at a blunt end of a 3' terminus, to
facilitate the initial association of the nucleotide sequences
through strand invasion, including, if desired, in a predetermined
directional orientation. The term "3' overhang" or "3' overhanging
sequence" is used herein to refer to a strand of a nucleic acid
molecule that extends in a 3' direction beyond the terminus of the
complementary strand of the nucleic acid molecule. Conveniently, a
3' overhang can be produced upon cleavage by a type IA or type II
topoisomerase.
[0282] The 3' or 5' overhanging sequences can have any sequence,
though generally the sequences are selected such that they allow
ligation of a predetermined end of one nucleic acid molecule to a
predetermined end of a second nucleotide sequence according to a
method of the invention (FIG. 9C, see, also Example 1.B). As such,
while the 3' or 5' overhangs can be palindromic, they generally are
not because nucleic acid molecules having palindromic overhangs can
associate with each other, thus reducing the yield of a ds
recombinant nucleic acid molecule covalently linked in both strands
comprising two or more nucleic acid molecules in a predetermined
orientation. For example, the 5' overhanging sequences of nucleic
acid molecules shown in FIG. 9A are palindrome and, therefore, the
association, for example, of a first CMV element with a second CMV
element through the AGCT overhang is just as likely as the
association of a CMV element with a GFP element through the AGCT
overhang. As such, the efficiency of generating a construct
comprising an operatively covalently linked construct containing,
in order from 5' to 3', a CMV element, a GFP element and a BGH
element would be reduced as compared to the efficiency of
generating such a construct using the elements as shown in FIG. 9C.
The elements shown in FIG. 9B contain palindromic overhangs at one
end of the GFP element and at the end of the BGH element shown and,
therefore, would be less efficient than the elements of FIG. 9C,
but more efficient than those in FIG. 9A, for generating the
desired construct.
[0283] A nucleotide sequence used in the methods and kits of the
current invention can be designed to contain a bridging
phosphorothioate to prevent religation after
topoisomerase-cleavage. For example, where the topoisomerase is E.
coli topoisomerase III, the bridging phosphorothioate can be
incorporated between the two thymidines of the GCAACTT
cleavage/recognition sequence. When cleaved, the clipped sequence
contains a 3'-SH instead of a 3'-OH, thus preventing religation
(see Burgin, et al, Nucl. Acids Res. 23:2973-2979, 1995).
[0284] A nucleic acid molecule useful in a method or kit of an
aspect of the invention can be amplified by an amplification method
such as PCR to contain a topoisomerase recognition site at a 3' or
5' terminus of an end. Furthermore, one or both primers used for
PCR can be designed such that, upon cleavage of an amplified
nucleic acid molecule, the cleaved nucleic acid molecule contains a
5' or 3' overhang at one or both ends. In one embodiment, PCR
primers are designed such that the 5' overhanging sequence on a
first nucleic acid molecule is complementary to a 5' overhanging
sequence on a second (or other) nucleic acid molecule, thereby
facilitating the association of the nucleotide sequences,
preferably in a predetermined orientation, whereupon they can be
covalently linked according to a method of the invention. In
accordance with the invention, by designing unique overhanging
sequences for the different nucleic acid molecule to be linked, any
number of nucleic acid molecules can be linked in a desired order
and/or orientation.
[0285] It should be recognized that PCR is used in two ways with
respect to the methods of the invention. In one aspect, PCR primers
are designed to impart particular characteristics to a desired
nucleic acid molecule, for example, a nucleic acid molecule that
encodes a transcriptional or translational regulatory element or a
coding sequence of interest such as an epitope tag or cell
compartmentalization domain. In this aspect, the PCR primers can be
designed such that, upon amplification, the nucleic acid molecule
contains a topoisomerase recognition site at one or both ends, as
desired. As disclosed herein, the PCR primer also can include an
additional sequence such that, upon cleavage of the amplification
product by a site specific topoisomerase, the cleaved nucleic acid
molecule contains a 5' or 3' overhanging sequence at the
topoisomerase cleaved end. In an embodiment of the invention
involving a topoisomerase that binds and cleaves a 5' terminus
(e.g., an embodiment involving a type IA topoisomerase), the PCR
primers can be designed to contain a bridging phosphorothioate
linkage (see above), which can block religation after topoisomerase
cleavage and can assist in the generation of a topoisomerase
charged amplification product.
[0286] Overhanging sequences generated using PCR can include a
single nucleotide overhang that is generated as an artifact of the
PCR reaction. For example, a polymerase such at Taq, which does not
have a proof-reading function and has an inherent terminal
transferase activity, is commonly used, and produces PCR products
containing a single, non-template derived 3' A overhang at each
end. These amplification products can be linked to topoisomerase
charged nucleic acid molecules containing a single 3' T overhang or
a single 3' dU overhang, which, for a T/A cloning reaction, can be
a vector (see U.S. Pat. Nos. 5,487,993 and 5,856,144, each of which
is incorporated herein by reference), at one or both ends, using
the methods of the invention.
[0287] PCR also is used to amplify a covalently linked ds
recombinant nucleic acid molecule covalently linked in one or both
strands, generated by a method of the invention. For example, as
illustrated in FIG. 13, a method of the invention can generate an
expressible ds recombinant nucleic acid molecule from three
substrate nucleic acid molecules including a nucleotide sequence
comprising a promoter, a nucleotide sequence comprising a coding
sequence, and a nucleotide sequence comprising a polyadenylation
signal. The generation of the ds recombinant nucleic acid molecule
can be facilitated by the incorporation of complementary 3' (or 5')
overhanging sequences at the ends of the ds nucleotides sequences
to be joined. For example, the expressible ds recombinant nucleic
acid molecule can be generated by contacting a first nucleic acid
molecule having a type IA topoisomerase at a 5' terminus of a first
end and a type IB topoisomerase at a 3' terminus of a second end
with a second nucleic acid molecule and a third double stranded
nucleotide sequence. By designing a PCR primer pair containing a
first primer that is specific for a portion of the nucleotide
sequence comprising the promoter that is upstream from the
promoter, and a second primer that is specific for a portion of the
nucleotide sequence comprising the polyadenylation signal that is
down stream of the signal, only a full length functional ds
recombinant nucleic molecule containing the promoter, coding
sequence and polyadenylation signal in the correct (predetermined)
orientation will be amplified. In particular, partial reaction
products, for example, containing only a promoter linked to the
coding sequence, and reaction products containing nicks are not
amplified. Thus, PCR can be used to specifically design a nucleic
acid molecule such that it is useful in a method of the invention,
and to selectively amplify only those reaction products having the
desired components and characteristics.
[0288] As used herein, the term "covalently linked," when used in
reference to a ds recombinant nucleic acid molecule, means that the
nucleic acid molecule is generated from at least two nucleic acid
molecules that are ligated together, in both strands, by a
topoisomerase mediated ligation. It should be recognized, for
example, that a topoisomerase covalently bound to one of the
nucleic acid molecules to be covalently linked can be the same as
or different from the topoisomerase covalently bound to the other
nucleic acid molecule. Thus, a Vaccinia topoisomerase can be
covalently bound to one nucleic acid molecule and another poxvirus
or eukaryotic nuclear type IB topoisomerase can be bound to the
other strand. Generally, however, the topoisomerases, where
different, are members of the same family, for example, type IA or
type IB or type II, although, where the topoisomerases are
covalently bound, for example, to a 5' phosphate and generate
complementary 3' overhangs, the topoisomerase can be from different
families, for example, type IA and type II.
[0289] The term "covalently linked" also is used herein in
reference to a single stranded or double stranded nucleic acid
molecule that is generated from at least two nucleotide sequences
that are ligated together in one strand. For example, a ds
recombinant nucleic acid molecule that is generated when a first
topoisomerase-charged nucleic acid molecule that includes one
topoisomerase bound at or near a 5' terminus contacts a second ds
nucleotide sequence under conditions such that the topoisomerases
can covalently link the 5' terminus of the first nucleic acid
molecule to which it is bound, to the 3' terminus of the second
nucleic acid molecule, can generate a ds recombinant nucleic acid
molecule covalently linked in one strand.
[0290] In one embodiment, a ds recombinant nucleic acid molecule
covalently linked in both strands generated according to a method
of the invention does not contain a nick in either strand at the
site where two nucleotide sequences are ligated, although it can
contain nicks elsewhere in the molecule. In a method for generating
a ds recombinant nucleic acid molecule covalently linked in one
strand, a ds recombinant nucleic acid molecule is generated that
contains a nick at least at the position where ends were linked in
the complementary strands. This nicked ds recombinant nucleic acid
molecule can be converted to a ds recombinant nucleic acid molecule
covalently linked in both strands by introducing the nicked ds
recombinant nucleic acid molecule into a cell, or by subjecting the
ds recombinant nucleic acid molecule to a ligation reaction, such
as using a ligase, as is well known in the art.
[0291] The term "recombinant" is used herein to refer to a nucleic
acid molecule that is produced by linking at least two nucleotide
sequences according to a method of the invention. As such, a ds
recombinant nucleic acid molecule encompassed within the present
invention is distinguishable from a nucleic acid molecule that may
be produced in nature, for example, during meiosis. For example, a
ds recombinant nucleic acid molecule covalently linked in both
strands generated according to a method of certain aspects of the
invention can be identified by the presence of the two
topoisomerase recognition sites, one present in each of the
complementary strands, at or near the site at which the nucleic
acid molecules were joined.
[0292] A method of the invention can be performed by contacting a
first nucleic acid molecule having a first end and a second end,
wherein at the first end or second end or both, the first nucleic
acid molecule has a topoisomerase recognition site, or cleavage
product thereof, at or near the 3' terminus and has (or can be made
to have, for example, by contact with a phosphatase) a hydroxyl
group at the 5' terminus of the same end; at least a second nucleic
acid molecule having a first end and a second end, wherein at the
first end or second end or both, the at least second nucleic acid
molecule has a topoisomerase recognition site, or cleavage product
thereof, at or near the 3' terminus and has (or can be made to
have) a hydroxyl group at the 5' terminus of the same end; and a
topoisomerase, under conditions such that the components are in
contact and the topoisomerase can effect its activity. Upon contact
of the topoisomerase with the first and second (or other) nucleic
acid molecules, and cleavage, where necessary, each nucleotide
sequence comprises at the cleavage site a covalently bound
topoisomerase at the 3' terminus and has, or can have, a hydroxyl
group at the 5' terminus such that, upon contact, the first and at
least second nucleotide sequences are covalently linked in both
strands. Accordingly, the invention provides a ds recombinant
nucleic acid molecule covalently linked in both strands produced by
such a method.
[0293] As used herein, the term "at or near," when used in
reference to the proximity of a topoisomerase recognition site to
the 3' (type IB) or 5' (type IA or type II) terminus of a
nucleotide sequence, means that the site is within about 1 to 100
nucleotides from the 3' terminus or 5' terminus, respectively,
generally within about 1 to 20 nucleotides from the terminus, and
particularly within about 2 to 12 nucleotides from the respective
terminus. An advantage of positioning the topoisomerase recognition
site within about 10 to 15 nucleotides of a terminus is that, upon
cleavage by the topoisomerase, the portion of the sequence
downstream of the cleavage site can spontaneously dissociate from
the remaining nucleotide sequence, which contains the covalently
bound topoisomerase (referred to generally as "suicide cleavage";
see, for example, Shuman, supra, 1991; Andersen et al., supra,
1991). Where a topoisomerase recognition site is greater than about
12 to 15 nucleotides from the terminus, the nucleotide sequence
upstream or downstream of the cleavage site can be induced to
dissociate from the remainder of the sequence by modifying the
reaction conditions, for example, by providing an incubation step
at a temperature above the melting temperature of the portion of
the duplex including the topoisomerase cleavage site.
[0294] An additional advantage of constructing a first or second
(or other) nucleic acid molecule to comprise, for example, a type
IB topoisomerase recognition site about 2 to 15 nucleotides from
one or both ends is that a 5' overhang is generated following
cleavage of the nucleic acid molecule by a site specific
topoisomerase. Such a 5' overhanging sequence, which would contain
2 to 15 nucleotides, respectively, can be designed using a PCR
method as disclosed herein to have any sequence as desired. Thus,
where a cleaved first nucleic acid molecule is to be covalently
linked to a selected second (or other) nucleic acid molecule
according to a method of the invention, and where the selected
sequence has a 5' overhanging sequence, the 5' overhang on the
first nucleic acid molecule can be designed to be complementary to
the 5' overhang on the selected second (or other) ds sequence such
that the two (or more) sequences are covalently linked in a
predetermined orientation due to the complementarity of the 5'
overhangs. As discussed above, similar methods can be utilized with
respect to 3' overhanging sequences generated upon cleavage by, for
example, a type IA or type II topoisomerase.
[0295] As used herein, reference to a nucleotide sequence having "a
first end" and "a second end" means that the nucleotide sequence is
linear. A substrate nucleic acid molecule can be linear or
circular, including supercoiled, although, as a result of cleavage
by one or more topoisomerases, a linear topoisomerase-charged
nucleic acid molecule generally is produced. For example, a
circular nucleic acid molecule containing two type IB topoisomerase
recognition sites within about 100 nucleotides of each other and in
the complementary strands, preferably within about twenty
nucleotides of each other and in the complementary strands, can be
contacted with a site specific type IB topoisomerase such that each
strand is cleaved and the intervening sequence dissociates, thereby
generating a linear nucleic acid molecule having a topoisomrerase
covalently bound to each end.
[0296] It should be recognized that reference to a first end or a
second end of a nucleic acid molecule is not intended to imply any
particular orientation of the nucleotide sequence, and is not
intended to imply a relative importance of the ends with respect to
each other. Where a nucleotide sequence having a first end and
second end is a double stranded nucleotide sequence, each end
contains a 5' terminus and a 3' terminus. Thus, reference is made
herein, for example, to a nucleotide sequence containing a
topoisomerase recognition site at a 3' terminus and a hydroxyl
group at the 5' terminus of the same end, which can be the first
end or the second end.
[0297] A method of the invention can be performed using only a
first nucleic acid molecule and a second nucleic acid molecule, or
can additionally include a third, fourth or more nucleic acid
molecules as desired. Generally, each such nucleotide sequence
contains a topoisomerase recognition site, or a cleavage product
thereof, at or near at least one 3' or 5' terminus, and can contain
a hydroxyl group at the 5' terminus of the same end, or a hydroxyl
group can be generated using a phosphatase. Where a nucleotide
sequence does not contain a topoisomerase recognition site at or
near an end to be linked to a second nucleotide sequence, a
topoisomerase recognition site can be introduced into the
nucleotide sequence using a method as disclosed herein, for
example, by PCR amplification of the sequence using a primer
comprising a complement of the topoisomerase recognition site.
[0298] The terms "first nucleotide sequence," "second nucleotide
sequence," "third nucleotide sequence," and the like, are used
herein only to provide a means to indicate which of several
nucleotide sequences is being referred to. Thus, absent any
specifically defined characteristic with respect to a particular
nucleotide sequence, the terms "first," "second," "third" and the
like, when used in reference to a nucleotide sequence, or a
population or plurality of nucleotide sequences, are not intended
to indicate any particular order, importance or other information
about the nucleotide sequence. Thus, where an exemplified method
refers, for example, to using PCR to amplify a first nucleic acid
molecule such that the amplification product contains a
topoisomerase recognition site at one or both ends, it will be
recognized that, similarly, a second (or other) nucleic acid
molecule also can be so amplified.
[0299] The term "at least a second nucleotide sequence" is used
herein to mean one or more nucleotide sequences in addition to a
first nucleotide sequence. Thus, the term can refer to only a
second nucleotide sequence, or to a second nucleotide sequence and
a third nucleotide sequence (or more). As such, the term "second
(or other) nucleotide sequence" or second (and other) nucleotide
sequences" is used herein in recognition of the fact that the term
"at least a second nucleotide sequence" can refer to a second,
third or more nucleotide sequences. It should be recognized that,
unless indicated otherwise, a nucleotide sequence encompassed
within the meaning of the term "at least a second nucleotide
sequence" can be the same or substantially the same as a first
nucleotide sequence. For example, a first and second nucleic acid
molecule can be the same except for having complementary 5'
overhanging sequences produced upon cleavage by a topoisomerase
such that the first and second nucleic acid molecules can be
covalently linked using a method of the invention. As such, a
method of the invention can be used to produce a concatenate of
first and second nucleic acid molecules, which, optionally, can be
interspersed, for example, by a third nucleic acid molecule such as
a regulatory element, and can contain the covalently linked
sequences in a predetermined directional orientation, for example,
each in a 5' to 3' orientation with respect to each other.
[0300] As disclosed herein, a method of the invention provides a
means to covalently link, two or more ds nucleotides in a
predetermined directional orientation. The term "directional
orientation" or "predetermined directional orientation" or
"predetermined orientation" is used herein to refer to the covalent
linkage, of two or more nucleotide sequences in a particular order.
Thus, a method of the invention provides a means, for example, to
covalently link, a promoter regulatory element upstream of a coding
sequence, and to covalently link a polyadenylation signal
downstream of the coding region to generate a functional
expressible ds recombinant nucleic acid molecule; or to covalently
link two coding sequences such that they can be transcribed and
translated in frame to produce a fusion polypeptide.
[0301] A method of the invention also can be performed by
contacting a first nucleic acid molecule having a first end and a
second end, wherein at the first end or second end or both, the
first nucleic acid molecule has a type IB topoisomerase covalently
bound at the 3' terminus (topoisomerase-charged) and has (or can be
made to have) a hydroxyl group at the 5' terminus of the same end;
and at least a second type IB topoisomerase-charged nucleic acid
molecule, which has (or can be made to have) a hydroxyl group at
the 5' terminus at the same end. Upon contact of the
topoisomerase-activated first and at least second nucleotide
sequences at the ends containing the topoisomerase and a 5'
hydroxyl group, phosphodiester bonds are formed in each strand,
thereby generating a ds recombinant nucleic acid molecule
covalently linked in both strands.
[0302] The invention further provides methods for linking two or
more (e.g., two, three, four, five, six, seven, etc.) nucleotide
sequences, wherein the linked ds recombinant nucleic acid molecule
is covalently linked in one strand, but not both strands, (i.e. the
ds recombinant nucleic acid molecule contains a nick in one strand
at each position where two ends were joined to generate the ds
recombinant nucleic acid molecule). Further, one or more of the
nucleotide sequences may comprise one or more recombination sites.
Using the schematic shown in FIG. 11A for purposes of illustration,
the invention includes methods for linking at least two nucleotide
sequences comprising contacting a first nucleic acid molecule
having a first end and a second end, wherein at the first end at
the second end or at both ends, the first nucleic acid molecule has
a site-specific type IA topoisomerase covalently bound to the 5'
termini; and a second nucleic acid molecule which does not have
topoisomerase covalently bound to either termini of at least one
end. Further, the second nucleotide sequence will typically have
hydroxyl groups at the 3' termini of the end being joined to the
first nucleic acid molecule. In many instances, the two nucleotide
sequences to be joined will have either 3' or 5' overhangs with
sufficient sequence complementarity to allow for hybridization. In
related embodiments, the first and second nucleic acid molecules
described above may be first and second ends of the same nucleic
acid molecule. Thus, connection of the two ends results in the
formation of a circularized molecule. Once nucleic acid molecules
are joined by the methods described above, the resulting molecules
may then be used in recombination reactions, such as those
described elsewhere herein. The invention further includes nucleic
acid molecules prepared by methods of the invention, compositions
comprising such nucleic acid molecules, and methods for using such
nucleic acid molecules.
[0303] Using the schematic shown in FIG. 11B for purposes of
illustration, the invention includes methods for joining three or
more nucleotide sequences. While any number of variations of the
invention are possible, three nucleotide sequences may be joined by
the use of a linker molecule which contains topoisomerases at or
near both the 5' and 3' termini of one strand, and optionally one
or more recombination site. Thus, upon joining of the three
nucleotide sequences, a single nucleotide sequence is formed which
contains a first strand with no nicks at the junction points, and a
second strand with nicks at the junction points. This process-has
the advantage of employing a single topoisomerase modified molecule
to join three nucleotide sequences together. Once nucleic acid
molecules are joined by the methods described above, the resulting
molecules may then be used in recombination reactions, such as
those described elsewhere herein. The invention further includes
nucleic acid molecules prepared by methods of the invention,
compositions comprising such nucleic acid molecules, and methods
for using such nucleic acid molecules.
[0304] The invention further provides methods for covalently
linking both strands of two or more (e.g., two, three, four, five,
six, seven, etc.) nucleic acid molecules. Using the schematic shown
in FIG. 12A for purposes of illustration, the invention includes
methods for linking at least two nucleotide sequences comprising
contacting a first nucleic acid molecule having a first end and a
second end, wherein at the first end, at the second end, or at both
ends, the first nucleic acid molecule has two topoisomerases (e.g.,
a type IA and a type IB topoisomerase) one each covalently bound to
the 3' and 5' termini; and a second nucleic acid molecule which
does not have topoisomerase covalently bound to either termini of
at least one end. Further, the second nucleotide sequence will
often have hydroxyl groups at the 51 and 3' termini of the end
being joined to the first nucleic acid molecule. In many instances,
the two nucleotide sequences to be joined will have either 3' or 5'
overhangs with sufficient sequence complementarity to allow for
hybridization, and, optionally, one or more recombination sites. In
related embodiments, the first and second nucleic acid molecules as
described above can be first and second ends of the same nucleic
acid molecule. Thus, connection of the two ends results in the
formation of a circularized molecule. Once nucleic acid molecules
are joined by the methods described above, the resulting molecules
may then be used in recombination reactions, such as those
described elsewhere herein. The invention further includes nucleic
acid molecules prepared by methods of the invention, compositions
comprising such nucleic acid molecules, and methods for using such
nucleic acid molecules.
[0305] Using the schematic shown in FIG. 12D for purposes of
illustration, the invention includes methods for joining three or
more nucleotide sequences. While any number of variations of the
invention are possible, three nucleotide sequences may be joined by
the use of a linker molecule which contains topoisomerases at or
near both the 5' and 3' termini of each end and, optionally, one or
more recombination sites. Thus, upon joining of the three
nucleotide sequences, a single nucleotide sequence is formed which
contains no nicks at the junction points. This process has the
advantage of employing a single topoisomerase modified molecule to
join three nucleotide sequences together. Once nucleic acid
molecules are joined by the methods described above, the resulting
molecules may then be used in recombination reactions, such as
those described elsewhere herein. The invention further includes
nucleic acid molecules prepared by methods of the invention,
compositions comprising such nucleic acid molecules, and methods
for using such nucleic acid molecules.
[0306] Substrates which particular reagents (e.g., enzymes)
recognize and/or catalyze reactions with can be used in methods of
the invention to produce nucleic acid molecules having particular
characteristics. For example, reagents which catalyze nucleic acid
modifications may recognize termini and/or generate termini having
particular features. One example of such a feature is the presence
or absence of a terminal phosphate group on the 3' or 5' strand.
Such reagents, or combinations of such reagents, may be used to
prepare, for example, nucleic acid molecules (1) from particular
segments and/or (2) having a specific "pattern" of nicks (e.g., a
nick in only one strand where two or more segments are joined,
nicks in alternating strands where three or more segments are
joined, etc.) or having no nicks in either strand.
[0307] Reagents (e.g., enzymes) which can be used in methods of the
invention include, but are not limited to, the following: ligases
(e.g. DNA and RNA Ligases such as T4 DNA Ligase, T4 RNA ligase, E.
coli DNA ligase, etc.), restriction enzymes (e.g., EcoRI, HpaII,
BamHI, etc.), kinases (e.g., T4 polynucleotide kinase, etc.),
phosphatases (e.g., calf intestinal alkaline phosphatase),
topoisomerases, and polymerases (e.g., proof-reading polymerases
such as Pfu, Pfx, THERMALAcE.TM. (Invitrogen Corp., Carlsbad,
Calif.), etc.), and non-proof-reading polymerases such as Taq
polymerase, Tf1 polymerase, Tth polymerase, Thr polymerase,
etc.).
[0308] The cleavage of nucleic acid molecules by many endonucleases
(e.g., restriction endonucleases) results in the formation of two
new ends, wherein a hydroxyl group is present at the 3' terminus of
one end and a phosphate group is present at the 5' terminus of the
other end. Also, when exonucleases (e.g., snake venom
phosphodiesterase, bovine spleen phosphodiesterase, E. coli
exonuclease VII, lambda exonuclease, E. coli exonuclease III, etc.)
digest nucleic acid molecules, they often generate ends with (1) 5'
terminal hydroxyl groups and 3' terminal phosphate groups or (2) 3'
terminal hydroxyl groups and 5' terminal phosphate groups. Further,
exonucleases typically digest only a single stranded of a nucleic
acid molecule but can use either single stranded and/or double
stranded nucleic acids as substrates. In addition, exonucleases
(e.g., exonucleases used in methods of the invention) may digest
nucleic acid molecules from the 3' terminus, 5' terminus, or both
the 3' and 5' termini. Also, kinases (e.g., T4 polynucleotide
kinase, etc.) may be used to replace 5' and/or 3' terminal hydroxyl
groups of nucleic acid molecules with phosphate groups.
[0309] Many polymerases used for the amplification of nucleic acid
molecules, for example, by PCR, generate nucleic acid products
having 3' terminal hydroxyl groups. In addition, the presence or
absence of a phosphate group, or other chemical group, at the 5'
terminus of a PCR product is typically determined by whether the
primer used in the PCR reaction(s) contains a 5' terminal phosphate
or other chemical group. Thus, 5' terminal phosphate groups,
hydroxyl groups, or other groups can be introduced into PCR
products by the use of primers which contain these groups at their
5' termini. As a result, PCR can be used to generate nucleic acid
molecules (i.e., the first nucleic acid molecule referred to below)
which contain a desired arrangement of hydroxyl groups, phosphate
groups and/or other groups on the 5' and/or 3' termini of one or
both ends of a linear nucleic acid molecule (e.g., 5' phosphate
group and a 3' hydroxyl group at one end and a 5' hydroxyl group
and a 3' hydroxyl group at the other end).
[0310] Each of the enzymes types listed above represents a general
class of tools which can be used to generate nucleic acid molecules
having particular characteristics (e.g., having a desired
arrangement of hydroxyl, phosphate and/or other groups on the 3'
and/or 5' termini of one or more ends). For example, double
stranded, linear nucleic acid molecules may be prepared in which
the 5' terminus and the 3' terminus at one end each contain
terminal hydroxyl groups and the 5' terminus and the 3' terminus at
the other end each contain terminal phosphate groups. Such ends may
be prepared using the enzymes discussed above and/or other reagents
and methods known in the art.
[0311] Thus, the present invention contemplates the construction
and use of nucleic acid segments having particular characteristics
(e.g., having a desired arrangement of hydroxyl, phosphate and/or
other groups on the 3' and/or 5' termini of one or more ends). Such
nucleic acids include, but are not limited to, double-stranded,
linear nucleic acid molecules which have first and second ends with
the characteristics set out in Table 4.
TABLE-US-00004 TABLE 4 First End Second End 5' Terminus 3' Terminus
5' Terminus 3' Terminus Phosphate Group Phosphate Group Phosphate
Group Phosphate Group Phosphate Group Phosphate Group Phosphate
Group Hydroxyl Group Phosphate Group Phosphate Group Hydroxyl Group
Phosphate Group Phosphate Group Phosphate Group Hydroxyl Group
Hydroxyl Group Hydroxyl Group Hydroxyl Group Phosphate Group
Phosphate Group Hydroxyl Group Hydroxyl Group Phosphate Group
Hydroxyl Group Hydroxyl Group Hydroxyl Group Hydroxyl Group
Phosphate Group Hydroxyl Group Hydroxyl Group Hydroxyl Group
Hydroxyl Group Hydroxyl Group Phosphate Group Phosphate Group
Phosphate Group Hydroxyl Group Phosphate Group Phosphate Group
Hydroxyl Group Hydroxyl Group Phosphate Group Hydroxyl Group
Phosphate Group Hydroxyl Group Phosphate Group Hydroxyl Group
Hydroxyl Group Phosphate Group Hydroxyl Group Phosphate Group
Phosphate Group Phosphate Group Hydroxyl Group Phosphate Group
Hydroxyl Group Phosphate Group Hydroxyl Group Hydroxyl Group
Phosphate Group Phosphate Group Hydroxyl Group Hydroxyl Group
Hydroxyl Group
[0312] Nucleic acid molecules having a desired arrangement of
hydroxyl, phosphate and/or other groups on the 3' and/or 5' termini
of one or more ends can be directionally linked to other nucleic
acid molecules using linking reactions which require, for example,
the presence of a particular group on one or more termini of the
molecule (e.g., either a 5' hydroxyl group or a 5' phosphate group
and/or a 3' hydroxyl group or a 3' phosphate group).
[0313] A number of reagents which catalyze the linkage of nucleic
acid segments to each other will generally only recognize termini
with particular chemical groups (e.g., a hydroxyl group or a
phosphate group) present. For example, T4 DNA ligase will catalyze
the ligation of the 3' terminus of an end of a nucleic acid
molecule to the 5' terminus of a separate end of the same nucleic
acid molecule or of a different nucleic acid molecule, when the 5'
terminus contains a terminal phosphate group. Further, a number of
topoisomerases (e.g., a type IB topoisomerases) will cleave and
bind to the 3' terminus of the end of a nucleic acid molecule and
catalyze the linkage of this 3' terminus to the 5' terminus of the
end of the same nucleic acid molecule or of a different nucleic
acid molecule, when the 5' end contains a terminal hydroxyl group.
Additionally, a number of topoisomerases (e.g. a type IA
topoisomerases) will cleave and bind to the 5' terminus of the end
of a nucleic acid molecule and catalyze the linkage of this 5'
terminus to the 3' terminus of the end of the same nucleic acid
molecule or of a different nucleic acid molecule, when the 3' end
contains a terminal hydroxyl group.
[0314] One example of such a linking reaction is where a first
nucleic acid molecule having a desired arrangement of groups on one
or more termini (for example, a 5' phosphate on one terminus and a
5' hydroxyl on the other terminus) is linked to a second nucleic
acid molecule that contains a type IB toposiomerase molecule
covalently attached to a phosphate group at the 3' terminus of only
one end of the molecule, i.e., attached to the 3' terminus of one
strand of a double-stranded nucleic acid molecule. In such an
instance, the 3' terminus of the end of the second nucleic acid
molecule that contains the bound toposiomerase can only be joined
to the 5' terminus of the end of the first nucleic acid molecule
that contains the hydroxyl group. Thus, these two nucleic acid
molecules can only be covalently linked in one orientation.
[0315] A linear double stranded nucleic acid molecule which has
phosphate groups at both of the 5' and 3' termini at both ends (see
Table 4) may be generated by any number of methods. One example of
methods which may be used to produce such molecules involves
chemical synthesis of both strands of the double stranded nucleic
acid molecule. These individual strands may then be mixed under
conditions which allow for the formation of the double stranded
molecule.
[0316] Using reagents referred to above, as well as other reagents,
nucleic acid molecules with various chemical groups at their
termini can be covalently linked to each other in one or both
strands. For example, a first nucleic acid segment which contains a
5' terminal phosphate group and a 3' terminal phosphate group with
a type IB toposiomerase bound to it at one end may be linked in
both strands to a second nucleic acid segment which contains 5' and
3' terminal hydroxyl groups at one end. In this instance, the 3'
terminus of first nucleic acid segment which contains the
toposiomerase molecule bound to it may be joined to the 5' terminus
of the end of the second nucleic acid molecule. This linking
reaction may be catalyzed by the bound topoisomerase molecule.
Further, the 5' terminus of the same end of the first nucleic acid
segments may be covalently linked to the 3' terminus of the end of
the second nucleic acid segment to which it is joined by a ligase
(e.g., T4 DNA ligase). As a second example, a first nucleic acid
segments is prepared with a "sticky end" (i.e., an overhang)
generated by digestion with a restriction endonuclease that leaves
a 5' terminal phosphate group present on the "sticky end". The
first nucleic acid segment is contacted with a second nucleic acid
segment which contains a compatible "sticky end" and a
toposiomerase molecule bound to the 5' terminus of this "sticky
end". The result is the covalent connection of these two nucleic
acid segments in a single strand. Further, the nick in the other
strand at the junction point may be sealed by the inclusion of a
ligase, such as T4 DNA ligase, in the reaction mixture.
[0317] Any number of variations of the above are possible depending
on the available ends and the reagents used to prepare nucleic acid
segments with ends for ligation by particular mechanisms or
catalyzed by particular reagents. One example of such a variation
is where the 5' terminus of the "sticky end" of the first nucleic
acid molecule referred contains a hydroxyl group (e.g., the 5'
phosphate is removed by a phosphatase) and the second nucleic acid
molecule contain a type IB topoisomerase bound to the 3' terminus
of the compatible "sticky end".
[0318] Thus, enzymes used to generate termini of nucleic acid
molecules (e.g., by amplification, by cleavage of a larger
molecule, etc.) can be selected such that termini suitable for
"downstream" reactions (e.g., ligation reactions) may be generated.
One example of such a process is shown in the upper portion of FIG.
41 and described as follows. A nucleic acid molecule may be
amplified by PCR using a proofreading polymerase (e.g. Pfx, Pfu,
etc.) which generates amplification products having predominantly
blunt ends (i.e., neither terminus of the amplification product has
an overhanging adenine or other residue) and 3' terminal hydroxyl
groups at both ends. Blunt ended linkers which contain (1) nucleic
acid of a T7 promoter and (2) a molecule of type IB topoisomerase
linked at or near the 3' terminus of the end downstream of the
promoter element (see Figure A). The 5' terminus of the end of the
linker which contains the covalently bound topoisomerase contains a
terminal phosphate. The result of the linking reaction, when
conducted in the presence of T4 DNA ligase, is nucleic acid
molecules which are covalently linked in both strands at the
junction point where the T7 promoter element is joined to the PCR
product. As one skilled in the art would recognize, the process set
out above and in FIG. 41 may be performed with nucleic acid
segments other than promoters and PCR products. In other words,
essentially any nucleic acid segments may be used. Example of
nucleic acid molecules which may be used in methods of the
invention include those which have termini such as those set out in
Table 4. Also, non-proof-reading polymerases (e.g., Taq polymerase)
may be used to generate the PCR product and the linkers containing
the T7 promoter element may have a "T" overhang for use in T/A
cloning.
[0319] Further, the invention is not limited to methods for
connecting two nucleic acid segments. Thus, the invention also
includes methods for connecting two or more nucleic acid segments
to each other, wherein at each connection point the nucleic acid
segments are covalently linked to each other in either one or both
strands. The invention further includes nucleic acid molecules
prepared by methods of the invention, as well as compositions and
reaction mixtures which contain the reaction products and reaction
precursors (e.g., nucleic acid segments which are to be connected
to each other by methods of the invention).
[0320] The process shown in FIG. 41 for the linkage of two nucleic
acid segments is non-directional. In other words, the two segments
will be connected to each other without regard to orientation.
Methods of the invention further include those directed to the
selection, isolation and/or preparation of nucleic acid molecules
which contain two or more (e.g., two, three, four, five, six eight,
ten, etc.) nucleic acid molecules connected in a particular order
and/or orientation. In performing these methods, joining reactions
may be designed, for example, so that nucleic acid segments are
connected to each other (1) in a particular order or orientation or
(2) without regard to orientation and then assembled nucleic acid
molecules which contain two or more segments connected to each
other in a particular order and/or orientation are selected and/or
isolated.
[0321] One example of a method for performing the second process
referred to immediately above is shown in FIG. 41. The process
shown in FIG. 41 involves the connection of two nucleic acid
molecules using methods described elsewhere herein, followed by the
amplification of nucleic acid molecules which contain segments
connected in a particular orientation. The amplification process
employs primers (i.e., primers A and B) which hybridize to
different stands and at opposite ends of the linkage product which
is sought. Thus, when the T7 promoter is connected to the PCR
product in one orientation (e.g., the desired orientation), primers
A and B hybridize to opposite strands and can be used to amplify
the nucleic acid molecule. However, when the T7 promoter is
connected to the PCR product in the other orientation (e.g., the
non-desired orientation), primers A and B hybridize to the same
strand and can not be used to amplify the nucleic acid
molecule.
[0322] Thus, the invention includes, in part, methods for
selectively amplifying nucleic acid molecules based on the order
and/or orientation of nucleic acid segments which are joined by
methods described elsewhere herein. In particular aspects, these
methods involve performing amplification reactions in the presence
of two or more primers which have been selected to amplify one or
more desired nucleic acid molecules assembled using methods
described elsewhere herein. Nucleic acid molecules selectively
amplified by methods of the invention may be assembled by the
joining of two or more nucleic acid segments. As one skilled in the
art would recognize, the selective amplification process described
above can be used to amplify nucleic acid molecules which are
assembled from three, four, five, six, seven, etc. nucleic acid
segments. When three or more nucleic acid segments selectively
amplified by methods described above, only those which contain the
segments corresponding to the primers in the proper orientation
will be amplified. Nucleic acid molecules which contain the correct
segments and segments in the proper order may be selected and/or
isolated by the use of additional processes. For example, if
nucleic acid segments 1, 2, and 3 are connected to each other by
methods of the invention, then assembled nucleic acid segments
containing nucleic acid segments 1 and 3 in the desired orientation
can be selectively amplified using primers corresponding to
sequences present in segments 1 and 3. Further, separation of
nucleic acid molecules to obtain those which are of the size
represented by nucleic acid molecules comprising segments 1, 2, and
3 may be performed to isolate these molecules. In such an instance,
depending on how the nucleic acid segments are assembled, segment 2
could be in either one particular orientation or in both
orientations Any number of such methods may be performed to obtain
assembled nucleic acid molecules which contain nucleic acid
segments connected to each other in a desired orientation and/or
order. The invention further includes reaction mixtures and
compositions for performing the methods described above, as well as
nucleic acid molecules generated by these methods.
[0323] In the embodiment of the invention shown in FIG. 41, it is
not necessary to covalently link both strands at the junction
between the linker containing the T7 Promoter element and the PCR
product. After the first round of amplification, both strands will
be represented in the population because, even if one strand
contains a nick, the first round of amplification will generate a
full-length nucleic acid strand corresponding to the nicked strand.
Thus, both primers will hybridize to nucleic acid strands in the
second and subsequent rounds of amplification. As a result of the
above, the T4 DNA ligase may be omitted from the methods
schematically represented in FIG. 41.
[0324] Again using the process shown in FIG. 41 for reference, when
a nucleic acid molecule is prepared as shown in the upper portion
of FIG. 41, it may be desirable to link both strands of the nucleic
acid segments being joined when the product nucleic acid molecule
is to be directly used (e.g., without one or more additional rounds
of amplification) in a process such as transcription. This is so
because strand separation occurs during the transcription process
and the presence of a nick in one of the strands often interferes
with the transcription process. Thus, when nucleic acid molecules
assembled as shown in FIG. 41 are intended for use for
transcription, it will often be desirable to generate nucleic acid
molecules in which both strands are covalently linked at the
junction between the nucleic acid segments. One exception to the
above is where the template strand does not contain a nick after
linking of the nucleic acid segments being joined. In other words,
in instances where the template strand is not nicked, transcription
will efficiently occur even if a nick is present in the
non-template strand.
[0325] The invention further provides methods for performing
topoisomerase mediated joining reactions and recombination
reactions which can be performed in either a single tube or
multiple tubes. For instance, all of the components necessary to
perform both topoisomerase mediated joining reactions and
recombination reactions can be combined in one tube and both
reactions can occur essentially simultaneously. Examples of
topoisomerase/recombination reactions which can be performed in
either a single tube or in multiple tubes are shown in FIGS. 35-40.
Thus, in particular embodiments, the invention provides single tube
reactions in which (1) one or more nucleic acid molecules or two
ends of one nucleic acid molecule are linked to each other by a
topoisomerase mediated reaction and (2) one or more recombination
sites undergo recombination with one or more other recombination
sites. Any number of toposiomerase mediated joining reaction and/or
recombination reactions may occur in processes of the invention.
Further, these reactions may occur in any order. In particular
embodiments, one or more nucleic acid molecules in reaction
mixtures of the invention will contain (1) one or more
recombination sites and (2) one or more topoisomerases or one or
more topoisomerase recognition sites.
[0326] As explained below in Example 9, in certain instances,
topoisomerases have been found to inhibit particular recombination
reactions. In such instances, nucleic acid molecules which have
undergone toposiomerase mediated joining reaction(s) may be
separated from topoisomerases present in the reaction mixture and
then may used as substrates for recombination reaction(s). Often in
such instances, the topoisomerase mediated joining reaction(s) and
the recombination reaction(s) will occur in separate tubes.
Examples of process by which products of topoisomerase mediated
joining reactions may be separated from topoisomerase include, but
are not limited to, phenol/chloroform extraction, typically
followed by precipitation of the nucleic acid (e.g., ethanol
precipitation), and chromatography (e.g., column
chromatography).
[0327] Alternatively, topoisomerases present in the reaction
mixture may be inactivated, for example, by heating (e.g., heating
to about 65.degree. C. for about 60 min., about 70.degree. C. for
about 60 min., about 75.degree. C. for about 60 min., about
70.degree. C. for about 40 min., about 75.degree. C. for about 40
min., about 80.degree. C. for about 40 min., about 80.degree. C.
for about 30 min., about 85.degree. C. for about 20 min., about
90.degree. C. for about 15 min., about 95.degree. C. for about 5
min. or about 99.degree. C. for about 1 min.) or by the use of
proteases (e.g., proteinase K). In this instance, it will generally
be possible for the topoisomerase mediated joining reaction(s) and
the recombination reaction(s) to occur in the same tube.
[0328] In specific embodiments of single tube reactions, two or
more nucleic acid segments, each comprising one or more
topoisomerases or toposiomerase recognition sites are joined to
each other using a topoisomerase mediated joining reaction (e.g., a
topoisomerase mediated joining reaction). After which, the tube is
heated to about 85.degree. C. for about 20 min. and one or more
recombinases are added. Further, if one or more of the two or more
nucleic acid segments do not comprise recombination sites or if
recombination with additional nucleic acid segments is desired,
then nucleic acid segments which comprise one or more recombination
sites may be added. Typically, the recombination sites present in
the tube will be ones which are capable of recombining with each
other.
[0329] In other specific embodiments of single tube reactions, two
or more nucleic acid segments undergo recombination catalyzed by
one or more recombinases. After recombination has occurred,
toposiomerase is then added to the tube to facilitate topoisomerase
mediated joining of nucleic acid segments. As above, additional
nucleic acid segments may, optionally, be added to the reaction
mixture along with the topoisomerase. Further, when nucleic acid
segments to which one or more toposiomerases are attached are added
to the reaction mixture, it will often not be necessary to add
additional topoisomerase. Thus, in particular embodiments,
topoisomerase modified nucleic segments may be added to the above
reaction mixtures and, depending on the particular reaction
conditions, additional topoisomerase may or may not be added.
[0330] The invention also provides methods for preparing nucleic
acid molecules which contain one or more (e.g., one, two, three,
four, five, six, etc.) multiple cloning sites. For example, one or
more nucleic acid segments used in methods of the invention may
comprise one or more multiple cloning sites. As another example,
multiple cloning sites may be added to nucleic acid segments used
to prepare nucleic acid molecules by methods of the invention or to
nucleic acid molecules prepared by methods of the invention by the
attachment of linkers which contain one or more multiple cloning
sites. In related aspects, the invention includes nucleic acid
molecules prepared by methods of the invention which contain one or
more multiple cloning sites, as well as the use of one or more
these multiple cloning sites to modify nucleic acid molecules
prepared by methods of the invention. The invention also provides
nucleic acid molecules produced by the methods described above, as
well as uses of these molecules and compositions comprising these
molecules.
Viral Vectors
[0331] The invention further provides methods for preparing nucleic
acid molecules having regions of viral nucleic acids, as well as
nucleic acid molecules prepared by such methods and compositions
comprising these nucleic acid molecules.
[0332] Adenoviruses are viral vectors that can be used, for
example, in gene therapy. Adenoviruses are especially attractive
vehicles for delivering genes to respiratory epithelia and the use
of such vectors are included within the scope of the invention.
Adenoviruses naturally infect respiratory epithelia where they
cause a mild disease. Other targets for adenovirus-based delivery
systems are liver, the central nervous system, endothelial cells,
and muscle. Adenoviruses have the advantage of being capable of
infecting non-dividing cells. Kozarsky and Wilson, Current Opinion
in Genetics and Development 3:499-503 (1993), present a review of
adenovirus-based gene therapy. Bout et al., Human Gene Therapy
5:3-10 (1994), demonstrated the use of adenovirus vectors to
transfer genes to the respiratory epithelia of rhesus monkeys.
Other instances of the use of adenoviruses in gene therapy can be
found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et
al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest.
91:225-234 (1993); PCT Publication Nos. WO94/12649 and WO 96/17053;
U.S. Pat. No. 5,998,205; and Wang et al., Gene Therapy 2:775-783
(1995), the disclosures of all of which are incorporated herein by
reference in their entireties.
[0333] Adeno-associated virus (AAV) and Herpes viruses, as well as
vectors prepared from these viruses have also been proposed for use
in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med.
204:289-300; U.S. Pat. No. 5,436,146; Wagstaff et al., Gene Ther.
5:1566-70 (1998)). Herpes viral vectors are particularly useful for
applications where gene expression is desired in nerve cells.
[0334] The invention thus includes methods for preparing nucleic
acid molecules which have one or more functional properties of
viral vectors (e.g., adenoviral vectors, alphaviral vectors, herpes
viral vectors, adeno-associated viral vectors, etc.). In particular
embodiments, methods of the invention include the joining of
nucleic acid segments, wherein one or more of the nucleic acid
segments contains regions which confer upon product nucleic acid
molecules the ability to function as viral vectors (e.g., the
ability to replicate in specific host cells, the ability to be
packaged into viral particles, etc.).
[0335] In particular embodiments, the invention includes methods
for preparing adenoviral vectors by joining at least one (e.g.,
one, two, three, four, etc.) nucleic acid segment which comprises
adenoviral sequences to one or more other nucleic acid segments.
Specific examples of adenoviral vectors, and nucleic acid segments
which can be used to prepare adenoviral vectors are disclosed in
U.S. Pat. Nos. 5,932,210, 6,136,594, and 6,303,362, the entire
disclosures of which are incorporated herein by reference.
Adenoviral vector prepared by methods of the invention may be
replication competent or replication deficient.
[0336] One example of an adenoviral vector may be prepared by
joining a nucleic acid segment comprising adenoviral nucleic acid
to one or more other nucleic acid segments. For example, when a
replication deficient adenoviral vector is desired, the adenoviral
nucleic acid may have deletions of all or part of one or more of
the following regions: the E1a region, the E1b region, and/or the
E3 region. Adenoviral vectors which contain deletions in these
regions are described, for example, in U.S. Pat. No. 6,136,594. The
invention further includes adenoviral vectors prepared by methods
of the invention, as well as uses of these vectors and compositions
comprising these vectors. One example of a use of adenoviral
vectors prepared by methods of the invention include the delivery
of nucleic acid segments to cells of a mammal (e.g., a human).
Thus, the invention provides methods for preparing vector suitable
for use in gene therapy protocols. Typically, such vectors will be
replication deficient.
[0337] In specific embodiments, adenoviral vectors of the invention
will comprise substantially the entire adenoviral genome with the
exception that are deletions of all or part of one or more of the
following regions: the E1a region, the E1b region, and/or the E3
region. In further specific embodiments, non-adenoviral nucleic
acid may be present in one or more of the E1a region, the E1b
region, and/or the E3 region.
[0338] In particular embodiments, adenoviral vectors prepared by
methods of the invention will contain at least one origin of
replication and/or a selection marker which allows for
amplification of the vector in prokaryotic cells, such as E.
coli.
[0339] Adeno-associated viral vectors and Herpes viral vectors may
be prepared by methods of the invention which are similar to those
described above. Thus, the invention further provides methods for
preparing such vectors, as well as vectors produced by these
methods, uses of these vectors, and compositions comprising these
vectors.
[0340] The invention further provides methods for preparing
alphaviral vectors (e.g., Sindbis virus vectors, Semliki Forest
virus vectors, Ross River virus vectors, Venezuelan equine
encephalitis virus vectors, Western equine encephalitis virus
vectors, Eastern equine encephalitis virus vectors, etc.), as well
as alphaviral vectors prepared by such methods, methods employing
these alphaviral vectors and compositions comprising these
alphaviral vectors.
[0341] In particular embodiments, the invention includes methods
for preparing alphaviral vectors by joining at least one nucleic
acid segment which comprises alphaviral sequences to one or more
other nucleic acid segments. Specific examples of alphaviral
vectors and nucleic acids which can be used to prepare alphaviral
vectors are described in U.S. Pat. Nos. 5,739,026 and 6,224,879,
the GibcoBRL's Instruction Manual No. 10179-018, "SFV Gene
Expression System", and Sindbis Expression System manual
(Invitrogen Corporation, Carlsbad, Calif.), catalog no. K750-01
(version E), the entire disclosures of which are incorporated
herein by reference.
[0342] In specific embodiments, alphaviral vector sequences used in
methods of the invention to prepare alphaviral vectors will
comprise one or more of the following components: one or more
packaging signals (which may or may not be of alphaviral origin),
one or more subgenomic promoters, and/or nucleic acid encoding one
or more non-structural protein (e.g., nsp1, nsp2, nsp3, nsp4,
etc.).
[0343] Alphaviral vectors of the invention may be introduced into
cells as DNA or RNA molecules. When DNA forms of such vectors are
introduced into cells, expression control sequences (e.g.,
inducible, repressible or constitutive expression control
sequences) may then be used to generate RNA molecules from which
one or more non-structural proteins may be translated. In specific
embodiments, these non-structural proteins will form an
RNA-dependent RNA polymerase which will amplify RNA molecules
corresponding to all or part of the transcript generated from the
DNA form of the alphaviral vector. Thus, these non-structural
proteins may catalyze the production of additional copies of RNA
molecules from RNA templates, resulting in RNA amplification.
Further, a nucleic acid segment for which high levels of expression
is desired may be operably linked to a subgenomic promoter, thus
resulting in the production of high levels of RNA corresponding to
the nucleic acid segment.
[0344] In one exemplary embodiment, alphaviral vectors prepared by
methods of the invention comprise DNA wherein an inducible promoter
directs transcription of an RNA molecule which encodes nspl, nsp2,
nsp3, and nsp4 of a Sindbis virus and a Sindbis subgenomic promoter
operatively linked to a nucleic acid segment which is not of
Sindbis viral origin. The invention also provides alphaviral
vectors prepared by methods of the invention, methods of using such
alphaviral vectors, and compositions comprising such alphaviral
vectors.
[0345] The invention further provides methods for joining nucleic
acid segments wherein one or more of the nucleic acid segments
contains one or more (e.g., one, two, three, four, etc.) viral
packaging signal (e.g., one or more packaging signal derived from a
virus referred to above). These packaging signals can be used to
direct the packaging of nucleic acid molecules prepared by methods
of the invention. One method for preparing packaged nucleic acid
molecules is by the introduction or expression of nucleic acid
molecules of the invention into packaging cell lines which express
proteins suitable for the production of virus-like particles. The
invention further includes packaged nucleic acid molecules of the
invention, methods for preparing packaged nucleic acid molecules of
the invention, and compositions comprising packaged nucleic acid
molecules of the invention.
[0346] The present invention also provides compositions, and kits
containing such compositions, including kits containing component
useful for performing methods of the invention. In one aspect, a
composition of the invention comprises isolated components
characteristic of a step of a method of the invention. For example,
a composition of the invention can comprise two or more of the same
or different topoisomerase-charged nucleic acid molecules. As used
herein, the term "different," when used in reference to the nucleic
acid molecules of a composition of the invention, means that the
nucleic acid molecules share less than 95% sequence identity with
each when optimally aligned, generally less than 90% sequence
identity, and usually less than 70% sequence identity. Thus,
nucleic acid molecules that, for example, differ only in being
polymorphic variants of each other or that merely contain different
5' or 3' overhanging sequences are not considered to be "different"
for purposes of a composition of the invention. In comparison,
different nucleic acid molecules are exemplified by a first
sequence encoding a polypeptide and second sequence comprising a
regulatory element, or a first sequence encoding a first
polypeptide a second sequence encoding a non-homologous
polypeptide.
[0347] Where a composition of the invention comprises more than two
different isolated nucleic acid molecules or more than two
different topoisomerase-charged nucleic acid molecules, each of the
nucleic acid molecules is different from each other, i.e., they are
all different from each other. However, it will be recognized that
each of the nucleic acid molecules, for example, a sequence
referred to as a first nucleic acid molecule, generally comprises a
population of such nucleotide sequences, which are identical or
substantially identical to each other. Thus, it should be clear
that the term "different" is used in comparing, for example, a
first (or population of first) nucleic acid molecules with a second
(and other) nucleic acid molecule. A composition comprising two or
more different topoisomerase-charged nucleic acid molecules can
further comprise a topoisomerase. Examples of such nucleic acid
molecules comprising the components of a composition of the
invention are disclosed herein and include, for example, coding
sequences, transcriptional regulatory element, translational
regulatory elements, elements encoding a detectable or selectable
markers such as an epitope tag or an antibiotic resistance gene,
elements encoding polypeptide domains such as cell
compartmentalization domains or signal peptides, and the like.
[0348] As used herein, the term "isolated" means that a molecule
being referred to is in a form other than that in which it exists
in nature. In general, an isolated nucleotide sequence, for
example, can be any nucleotide sequence that is not part of a
genome in a cell, or is separated physically from a cell that
normally contains the nucleotide sequence. It should be recognized
that various compositions of the invention comprise a mixture of
isolated nucleic acid molecules. As such, it will be understood
that the term "isolated" only is used in respect to the isolation
of the molecule from its natural state, but does not indicate that
the molecule is an only constituent.
[0349] A composition of the invention can comprise two different
nucleic acid molecules, each of which contains a topoisomerase
recognition site at or near one or both ends, and a site specific
topoisomerase, which can bind to and cleave the nucleic acid
molecules at the topoisomerase recognition site. Optionally, at
least one of the different nucleic acid molecules can be a
topoisomerase-charged nucleic acid molecule. Preferably, the
topoisomerase covalently bound to the topoisomerase-charge nucleic
acid molecule is of the same family as the topoisomerase in the
composition.
[0350] Various combinations of components can be used in a method
of the invention. For example, the method can be performed by
contacting a topoisomerase-activated first nucleic acid molecule,
which optionally comprises one or more recombination sites; a
second nucleic acid molecule having a first end and a second end,
wherein at the first end or second end or both, the second
nucleotide sequence has a topoisomerase recognition site at or near
the 3' terminus, and a hydroxyl group at the 5' terminus of the
same end; and a topoisomerase. Where the 5' terminus of one or both
ends to be linked has a 5' phosphate group, a phosphatase also can
be contacted with the components of the reaction mixture. Upon such
contacting, the topoisomerase can cleave the second nucleotide
sequence to produce a topoisomerase-activated second nucleic acid
molecule, the phosphatase, if necessary, can generate a 5' hydroxyl
group at the same end, and the second nucleic acid molecule then
can be covalently linked to the topoisomerase-activated first
nucleic acid molecule. As such, it will be recognized that a
composition of the invention can comprise any of various
combinations of components useful for performing a method of the
invention. Once nucleic acid molecules are joined by the methods
described above, the resulting molecules may then be used in
recombination reactions, such as those described elsewhere herein.
The invention further includes nucleic acid molecules prepared by
methods of the invention, compositions comprising such nucleic acid
molecules, and methods for using such nucleic acid molecules.
[0351] In general, a method of the invention for generating a ds
recombinant nucleic acid molecule covalently linked in both strands
is based on the determination that a ds recombinant nucleic acid
molecule covalently linked in both strands can be produced by
contacting a first nucleic acid molecule with a second nucleic acid
molecule, wherein the first and second sequences each have, at the
ends to be linked, a topoisomerase recognition site, for example,
5'-(C/T)CCTT-3' (Shuman, supra, 1991; U.S. Pat. No. 5,766,891).
Upon cleavage, the site specific topoisomerase is covalently bound
at the 3' terminus. Where the cleaved nucleotide sequences also
contain a 5' hydroxy group at the same end as the bound
topoisomerase, and the ends of the two nucleotide sequences
associate, the topoisomerase on each 3' terminus can covalently
link that terminus to a 5' hydroxyl group on the associated
nucleotide sequence (see FIG. 12B).
[0352] As used herein, reference to contacting a first nucleotide
sequence and at least a second nucleotide sequence "under
conditions such that all components are in contact" means that the
reaction conditions are appropriate for the topoisomerase-cleaved
ends of the nucleotide sequences to come into sufficient proximity
such that a topoisomerase can effect its enzymatic activity and
covalently link the 3' or 5' terminus of a first nucleotide
sequence to a 5' or 3' terminus, respectively, of a second
nucleotide sequence. Examples of such conditions, which include the
reaction temperature, ionic strength, pH, and the like, are
disclosed herein, and other appropriate conditions as required, for
example, for particular 5' overhanging sequences of the termini
generated upon topoisomerase cleavage, can be determined
empirically or using formulas that predict conditions for specific
hybridization of nucleotide sequences, as is well known in the art
(see, for example, (Sambrook et al., Molecular Cloning: A
laboratory manual (Cold Spring Harbor Laboratory Press 1989);
Ausubel et al., Current Protocols in Molecular Biology, John Wiley
and Sons, Baltimore, Md. (1987, and supplements through 1995), each
of which is incorporated herein by reference).
[0353] In one embodiment, a method of the invention provides a
means to render an open reading from a cDNA or an isolated genomic
DNA sequence expressible by operatively linking one or more
regulatory elements to the putative coding sequence. Accordingly, a
first nucleic acid molecule comprising an open reading frame can be
amplified by PCR using a primer pair that generates an amplified
first nucleic acid molecule having a topoisomerase recognition site
at one or both ends and, optionally, one or more recombination
sites, as desired, such that, upon cleavage by the site specific
topoisomerase, one or both ends contains a defined 5' or 3'
overhang or is blunt. Where both ends of the amplified first
nucleic acid molecule are so constructed, the 5' or 3' overhanging
sequences generally, but not necessarily, are different from each
other. The amplified first nucleic acid molecule then can be
contacted with a second nucleic acid molecule comprising a desired
regulatory element such as a promoter and, in certain embodiments,
(a) one or more topoisomerase recognition sites, and with a
topoisomerase and/or (b) one or more recombination sites, under
conditions which facilitate recombination, such that the second
nucleotide sequence is operatively covalently linked to the 5' end
of the coding sequence according to a method of the invention.
[0354] In such a method, a second (or other) nucleic acid molecule
also can comprise two or more regulatory elements, for example, a
promoter, an internal ribosome entry site and an ATG initiator
methionine codon, or the like, or other sequence of interest, for
example, an sequence encoding an epitope tag, in operative linkage
with each other, and which can be operatively covalently linked to
the 5' end of a first nucleic acid molecule comprising a coding
sequence. Such a method can further include contacting a third
nucleic acid molecule comprising, for example, a polyadenylation
signal, which can be operatively covalently linked according to a
method of the invention to the 3' end of the coding sequence,
thereby generating an expressible ds recombinant nucleic acid
molecule. As such, a method of the invention provides a means for
generating a functional ds recombinant nucleic acid molecule that
can be transcribed, translated, or both as a functional unit. As
disclosed herein, the inclusion of complementary 5' or 3'
overhanging sequences generated by topoisomerase cleavage at the
termini of the nucleic acid molecules to be linked together by the
site specific topoisomerase facilitates the generation of a ds
recombinant nucleic acid molecule having a desired directional
orientation of the nucleotide sequences in the construct.
[0355] In another embodiment, a method of the invention is
performed such that the first nucleic acid molecule or a second (or
other) nucleic acid molecule, or combination thereof, is one of a
plurality of nucleotide sequences. As used herein, the term
"plurality," when used in reference to a first or at least a second
nucleotide sequence, means that the nucleotide sequences are
related but different. For purposes of the present invention, the
nucleotide sequences of a plurality are "related" in that each
nucleotide sequence in the plurality contains at least a
topoisomerase recognition site, or a cleaved form thereof, at one
or more termini and/or at least one recombination site.
Furthermore, the nucleotide sequences of a plurality are
"different" in that they can comprise, for example, a cDNA library,
a combinatorial library of nucleotide sequences, a variegated
population of nucleotide sequences, or the like. Methods of making
cDNA libraries, combinatorial libraries, libraries comprising
variegated populations of nucleotide sequences, and the like are
well known in the art (see, for example, U.S. Pat. No. 5,837,500;
U.S. Pat. No. 5,622,699; U.S. Pat. No. 5,206,347; Scott and Smith,
Science 249:386-390, 1992; Markland et al., Gene 109:13-19, 1991;
O'Connell et al., Proc. Natl. Acad. Sci., USA 93:5883-5887, 1996;
Tuerk and Gold, Science 249:505-510, 1990; Gold et al., Ann. Rev.
Biochem. 64:763-797, 1995; each of which is incorporated herein by
reference).
[0356] The present invention further provides a method of
generating a ds recombinant nucleic acid molecule covalently linked
in both strands by amplifying a portion of a first nucleotide
sequence using a PCR primer pair, wherein at least one primer of
the primer pair encodes a topoisomerase recognition site or a
complement thereof and, optionally, one or more recombination
sites, thereby producing a first nucleic acid molecule having a
first end and a second end, wherein the first end or second end or
both has a topoisomerase recognition site at the 3' terminus and/or
the 5' terminus; and contacting the first nucleic acid molecule
with at least a second nucleic acid molecule having a first end and
a second end, wherein the first end or second end or both has a
topoisomerase recognition site at the 3' terminus and/or the 5'
terminus, or a cleavage product thereof; and a topoisomerase (see
FIG. 12). When contacted under conditions such that an end of the
first nucleic acid molecule having a topoisomerase recognition site
and an end of the at least second nucleic acid molecule having a
topoisomerase recognition site can associate, a ds recombinant
nucleic acid molecule covalently linked in both strands is
generated. Once nucleic acid molecules are joined by the methods
described above, the resulting molecules may then be used in
recombination reactions, such as those described elsewhere herein.
The invention further includes nucleic acid molecules prepared by
methods of the invention, compositions comprising such nucleic acid
molecules, and methods for using such nucleic acid molecules.
[0357] As disclosed herein, a PCR method using primers designed to
incorporate one or more topoisomerase recognition sites and,
optionally, one or more recombination sites at one or both ends of
an amplified nucleic acid molecule provides a convenient means for
producing nucleic acid molecules useful in a method of the
invention. In certain embodiments, at least one of the primers of a
primer pair is designed such that it comprises, in a 5' to 3'
orientation, a nucleotide sequence complementary to a topoisomerase
recognition site, such that PCR introduces a functional recognition
site in the opposite strand (see primer sequences in FIG. 9D), and
a nucleotide sequence complementary to the 3' end of a target
nucleic acid molecule to be amplified (i.e., a target specific
region). In addition, the primer can contain, in a position 5' to
the complement of the topoisomerase recognition site, a desired
nucleotide sequence of any length (generally about 1 to 100
nucleotide, usually about 2 to 20 nucleotides, and particularly
about 4 to 12 nucleotides), which, upon cleavage of the
amplification product by a site specific topoisomerase, forms a
desired 5' overhang. The second primer of the PCR primer pair can
be complementary to a desired sequence of the nucleotide sequence
to be amplified, and can comprise a complement to a topoisomerase
recognition site, a sequence that would generate a 5' overhang upon
cleavage by a site specific topoisomerase, or any other sequence,
as desired.
[0358] Such a primer can comprise or encode any other sequence of
interest, including, for example, a site specific integration
recognition site such as an att site, a lox site, or the like, or,
as discussed above, can simply be used to introduce a topoisomerase
recognition site into a nucleic acid molecule comprising such a
sequence of interest. A ds recombinant nucleic acid molecule
generated according to a method of the invention and containing a
site specific integration recognition site such as an att site or
lox site can be integrated specifically into a desired locus such
as into a vector, a gene locus, or the like, that contains the
required integration site, for example, an att site or lox site,
respectively, and upon contact with the appropriate enzymes
required for the site specific event, for example, lambda Int and
IHF proteins or Cre recombinase, respectively. The incorporation,
for example, of attB or attP sequences into a ds recombinant
nucleic acid molecule covalently linked in both strands according
to a method of the invention allows for the convenient manipulation
of the nucleic acid molecule using the GATEWAY.TM. Cloning System
(Invitrogen Corporation, Carlsbad, Calif.).
[0359] In one embodiment, a construct generated according to a
method of the invention is further amplified by a PCR reaction or
other amplification reaction. Direct PCR of a ds recombinant
nucleic acid molecule generated according to a method of the
invention is possible because the construct is covalently linked in
at least one strand. As such, PCR can be used to generate a large
amount of the construct. More importantly, as indicated above, PCR
provides an in vitro selection method for obtaining only a desired
product generated according to a method of the invention, without
obtaining partial reaction products. For example, a method of the
invention can be used to generate a ds recombinant nucleic acid
molecule covalently linked in both strands comprising, operatively
linked in a 5' to 3' orientation, a first nucleic acid molecule
comprising a promoter, a second nucleic acid molecule comprising a
coding region, and a third nucleic acid molecule comprising a
polyadenylation signal.
[0360] As disclosed herein, a construct having a predetermined
orientation can be generated by including complementary 5'
overhanging sequences on the ends of the nucleic acid molecules to
be joined. By selecting a PCR primer pair including a first primer
complementary to the first nucleic acid molecule and upstream of
the promoter sequence, and a second primer complementary to the
third nucleic acid molecule and downstream of the polyadenylation
signal, a functional amplification product comprising the promoter,
coding region and polyadenylation signal can be generated. In
contrast, partial reaction products that lack either the first
nucleic acid molecule or third ds nucleotide is not amplified
because either the first or second primer, respectively, would not
hybridize to the partial product. In addition, a construct lacking
the second nucleic acid molecule would not be generated due to the
lack of complementarity of the 5' overhanging sequences of the
first and third nucleic acid molecules. As such, a method of the
invention provides a means to obtain a desired functional ds
recombinant nucleic acid molecule covalently linked in both
strands.
[0361] The use of PCR in such a manner further provides a means to
screen a large number of nucleic acid molecules generated according
to a method of the invention in order to identify constructs of
interest. Since methods for utilizing PCR in automated high
throughput analyses are routine and well known, it will be
recognized that the methods of the invention can be readily adapted
to use in a high throughput system. Using such a system, a large
number of constructs can be screened in parallel, and partial or
incomplete reaction products can be identified and disposed of,
thereby preventing a waste of time and expense that would otherwise
be required to characterize the constructs or examine the
functionality of the constructs in further studies.
[0362] The methods of the invention have broad application to the
field of molecular biology. As discussed in greater detail below,
the methods of the invention can be used, for example, to label DNA
or RNA probes, to perform directional cloning (see Example 1.B), to
generate sense or antisense RNA molecules (see Example 2.A), to
prepare bait or prey constructs for performing a two hybrid assay
(see Example 2.C), to prepare linear expression elements (see
Examples 2.A and 2.B), and to prepare constructs useful for coupled
in vitro transcription/translation assays (see Example 2.B). For
example, a method of generating ds recombinant nucleic acid
molecules covalently linked in both strands provides a means to
generate linear expression elements (LEEs), which consist of a
linear nucleic acid molecule comprising two or more nucleotide
sequences such as a promoter or other regulatory element linked to
an open reading frame (see Example 1). LEEs have been reported to
efficiently transfect cells, thus bypassing a requirement for
cloning the expression element in a vector (Sykes and Johnston,
Nat. Biotechnol. 17:355-359, 1999). The components of a LEE can be
noncovalently linked, or can be covalently linked via a ligation
reaction. The preparation of noncovalently linked LEEs requires
using PCR primers containing deoxyuridine residues to amplify each
nucleotide sequence component, then treating the PCR products with
uracil-DNA glycosylase to generate overhanging ends that can
hybridize. However, the efficiency of transfection using such
noncovalently linked LEEs is variable, and, in some cases, much
lower than the efficiency of covalently linked LEEs (Sykes and
Johnston, supra, 1999). Furthermore, such LEEs are not suitable for
use as templates for PCR amplification because the primer extension
reaction cannot proceed past nicks in the template and, therefore,
is terminated producing incomplete reaction products.
[0363] A method of the invention provides a straightforward and
simple means to generate covalently linked LEEs, thereby avoiding
the inconvenient and additional steps previously described for
preparing a LEE, as well as reducing variability in transfection
efficiency as observed using noncovalently linked LEEs. For
example, a first nucleic acid molecule, which encodes an open
reading frame of interest, can be amplified by PCR as disclosed
herein to contain a topoisomerase recognition site, or cleavage
product thereof, on one or both ends. Furthermore, the PCR primers
can be designed such that, upon cleavage of the amplified first
nucleic acid molecule by a site specific topoisomerase, the
cleavage product contains a predetermined and desired 5'
overhanging sequence. A second nucleotide sequence (and a third or
more, as desired), in addition to containing a topoisomerase
recognition site, or cleavage product thereof, can include or
encode a regulatory element, for example, a promoter, an enhancer,
a silencer, a splice acceptor site, a translation start site, a
ribosome recognition site or internal ribosome entry site, a
polyadenylation signal, an initiator methionine codon, or a STOP
codon, or can encode any other desired sequence such as an epitope
tag or cell compartmentalization domain. Preferably, the second (or
other) nucleic acid molecule to be covalently linked to the first
nucleic acid molecule has a 5' overhanging sequence that is
complementary to the 5' overhang at the end of the first nucleic
acid molecule to which it is to be linked. Upon contact of such
nucleotide sequences in presence of a topoisomerase a promoter, for
example, can be operatively covalently linked to the 5' terminus of
the open reading frame, and a polyadenylation signal can be
operatively covalently linked to the 3' terminus of the open
reading frame, thereby generating a covalently linked functional
LEE (see Example 1).
[0364] Examples of regulatory elements useful in the present
invention are disclosed herein and include transcriptional
regulatory elements, translational regulatory elements, elements
that facilitate the transport or localization of a nucleotide
sequence or polypeptide in (or out of) a cell, elements that confer
a detectable phenotype, and the like. Transcriptional regulatory
elements include, for example, promoters such as those from
cytomegalovirus, Moloney leukemia virus, and herpes virus, as well
as those from the genes encoding metallothionein, skeletal actin,
phosphoenolpyruvate carboxylase, phosphoglycerate, dihydrofolate
reductase, and thymidine kinase, as well as promoters from viral
long terminal repeats (LTRs) such as Rous sarcoma virus LTR and
operators; enhancers, which can be constitutively active such as an
immunoglobulin enhancer, or inducible such as SV40 enhancer; and
the like. For example, a metallothionein promoter is a
constitutively active promoter that also can be induced to a higher
level of expression upon exposure to a metal ion such as copper,
nickel or cadmium ion. In comparison, a tetracycline (tet)
inducible promoter is an example of a promoter that is induced upon
exposure to tetracycline, or a tetracycline analog, but otherwise
is inactive. A transcriptional regulatory element also can be a
tissue specific regulatory element, for example, a muscle cell
specific regulatory element, such that expression of an encoded
product is restricted to the muscle cells in an individual, or to
muscle cells in a mixed population of cells in culture, for
example, an organ culture. Muscle cell specific regulatory elements
including, for example, the muscle creatine kinase promoter
(Sternberg et al., Mol. Cell. Biol. 8:2896-2909, 1988, which is
incorporated herein by reference) and the myosin light chain
enhancer/promoter (Donoghue et al., Proc. Natl. Acad. Sci., USA
88:5847-5851, 1991, which is incorporated herein by reference) are
well known in the art. Other tissue specific promoters, as well as
regulatory elements only expressed during particular developmental
stages of a cell or organism are well known in the art.
[0365] In additional embodiments, the regulatory elements contained
in the nucleotide sequences used in or produced by the practice of
the invention can be one or more operators. A number of operators
are known in the art. An example of an operator suitable for use
with the invention is the tryptophan operator of the tryptophan
operon of E. coli. The tryptophan repressor, when bound to two
molecules of tryptophan, binds to the E. coli tryptophan operator
and, when suitably positioned with respect to the promoter, blocks
transcription. Another example of an operator suitable for use with
the invention is operator of the E. coli tetracycline operon.
Components of the tetracycline resistance system of E. coli have
also been found to function in eukaryotic cells and have been used
to regulate gene expression. For example, the tetracycline
repressor, which binds to tetracycline operator in the absence of
tetracycline and represses gene transcription, has been expressed
in plant cells at sufficiently high concentrations to repress
transcription from a promoter containing tetracycline operator
sequences (Gatz et al., Plants 2:397-404 (1992)). The tetracycline
regulated expression systems are described, for example in U.S.
Pat. No. 5,789,156, the entire disclosure of which is incorporated
herein by reference. Additional examples of operators which can be
used with the invention include the Lac operator and the operator
of the molybdate transport operator/promoter system of E. coli
(see, e.g., Cronin et al., Genes Dev. 15:1461-1467 (2001) and
Grunden et al., J. Biol. Chem., 274:24308-24315 (1999)).
[0366] Thus, in particular embodiments, the invention provides
methods for preparing nucleic acid molecules that contain one or
more operators which can be used to regulate expression in
prokaryotic or eukaryotic cells. As one skilled in the art would
recognize, when a nucleic acid molecule which contains an operator
is placed under conditions in which transcriptional machinery is
present, either in vivo or in vitro, regulation of expression will
often be modulated by contacting the nucleic acid molecule with a
repressor and one or more metabolites which facilitate binding of
an appropriate repressor to the operator. Thus, the invention
further provides methods for preparing nucleic acid molecules which
encode repressors which modulate the function of operators, as well
as nucleic acid molecules produced by these methods, compositions
comprising these molecules, and uses of these molecules and
compositions.
[0367] Regulatory or other elements useful in generating a
construct according to a method of the invention can be obtained in
various ways. In particular, many of the elements are included in
commercially available vectors and can be isolated therefrom and
can be modified to contain a topoisomerase recognition site at one
or both ends, for example, using a PCR method as disclosed herein.
In addition, the sequences of or encoding the elements useful
herein generally are well known and disclosed in publications. In
many cases, the elements, for example, many transcriptional and
translational regulatory elements, as well as cell
compartmentalization domains, are relatively short sequences and,
therefore, are amenable to chemical synthesis of the element or a
nucleotide sequence encoding the element. Thus, in one embodiment,
an element comprising a composition of the invention, useful in
generating a ds recombinant nucleic acid molecule according to a
method of the invention, or included within a kit of the invention,
can be chemically synthesized and, if desired, can be synthesized
to contain a topoisomerase recognition site at one or both ends of
the element and, further, to contain an overhanging sequence
following cleavage by a site specific topoisomerase.
[0368] A topoisomerase-charged vector can be generated in the
following manner (Genome Res. 9: 383-392, 1999): A vector is
linearized with a restriction enzyme that leaves "sticky ends".
Using a ligase such as T4 DNA ligase, adapter oligonucleotides are
ligated to both ends, and both strands, of the linearized DNA. The
adapter oligonucleotides contain and position a 5'-CCCTT-3'
Vacccinia topoisomerase type I recognition sequence such that it
can be cleaved by topoisomerase and trap the covalent
topoisomerase-DNA complex at each 3' end of the vector. The adapted
vector is then incubated with purified Vaccinia topoisomerase and
an annealing oligonucleotide that complete the "topoisomerase
sites" at each end of the vector. The annealing oligonucleotide
acts to leave a break, or nick, in the "bottom" strand opposite the
last T in the 5'-CCCTT-3' containing oligonucleotide. The
oligonucleotide adapter fragments that are "downstream" of the
topoisomerase cleavage site (the "leaving groups") are released
upon topoisomerase cleavage and are removed in the
topoisomerase-vector purification process. In the absence of the 5'
hydroxyl from the "leaving group", topoisomerase is trapped in a
covalent complex with the DNA ends to produce a
topoisomerase-charged vector.
[0369] Where nucleic acid molecules are to be covalently linked
according to a method of the invention, the nucleotide sequences
generally are operatively linked such that the recombinant nucleic
acid molecule that is generated has a desired structure and
performs a desired function or encodes a desired expression
product. As used herein, the term "operatively linked" means that
two or more nucleotide sequences are positioned with respect to
each other such that they act as a unit to effect a function
attributable to one or both sequences or a combination thereof. The
term "operatively covalently linked" is used herein to refer to
operatively linked nucleotide sequences generated according to a
method of the invention for generating a ds recombinant nucleic
acid molecule covalently linked in one or both strands. For
example, a nucleotide sequence containing an open reading frame can
be operatively linked to a promoter such that the promoter confers
its regulatory effect on the open reading frame similarly to the
way in which it would effect expression of an open reading frame
that it normally is associated with in a genome in a cell.
Similarly, two or more nucleotide sequences comprising open reading
frames can be operatively linked in frame such that, upon
transcription and translation, a chimeric fusion polypeptide is
produced.
[0370] Although a ds recombinant nucleic acid molecule covalently
linked in one or both strands, generated according to a method of
the invention generally is linear, the construct generated also can
be a circularized ds recombinant nucleic acid molecule.
Furthermore, a circular ds recombinant nucleic acid molecule can be
generated such that it has the characteristics of a vector, and
contains, for example, regulatory elements required for replication
in a prokaryotic host cell, a eukaryotic host cell, or both, and
can contain a nucleotide sequence encoding a polypeptide that
confers antibiotic resistance or the like. An advantage of such a
method is that the generated ds recombinant nucleic acid molecule,
which is circularized according to a method of the invention, can
be transformed or transfected into an appropriate host cell,
wherein the construct is amplified. Thus, in addition to an in
vitro method such as PCR, which can be used to generate large
amounts of a linear ds recombinant nucleic acid molecule generated
according to a method of the invention, an in vivo method using a
host cell can be used for obtaining a large amount of a
circularized product generated according to a method of the
invention. Such elements including bacterial origins of
replication, antibiotic resistance genes, and the like, which
comprise a topoisomerase recognition site according to the present
invention, can be useful components to include in a kit of the
invention as disclosed herein.
[0371] It should be recognized that a linear ds recombinant nucleic
acid molecule covalently linked in one or both strands, also can be
cloned into a vector, which can be a plasmid vector or a viral
vector such as a bacteriophage, baculovirus, retrovirus,
lentivirus, adenovirus, vaccinia virus, semliki forest virus and
adeno-associated virus vector, all of which are well known and can
be purchased from commercial sources (Promega, Madison Wis.;
Stratagene, La Jolla Calif.; GIBCO/BRL, Gaithersburg Md.). If
desired, the vector can be linearized and modified according to a
method of the invention, for example, using a PCR method, to
contain a topoisomerase recognition site, or cleavage product
thereof, at one or both 3' termini, or can be constructed by one
skilled in the art (see, generally, Meth. Enzymol., Vol. 185,
Goeddel, ed. (Academic Press, Inc., 1990); Jolly, Canc. Gene Ther.
1:51-64, 1994; Flotte, J. Bioenerg. Biomemb. 25:37-42, 1993;
Kirshenbaum et al., J. Clin. Invest. 92:381-387, 1993; each of
which is incorporated herein by reference).
[0372] Viral expression vectors can be particularly useful where a
method of the invention is practiced for the purpose of generating
a ds recombinant nucleic acid molecule covalently linked in one or
both strands, that is to be introduced into a cell, particularly a
cell in a subject. Viral vectors provide the advantage that they
can infect host cells with relatively high efficiency and can
infect specific cell types or can be modified to infect particular
cells in a host.
[0373] Viral vectors have been developed for use in particular host
systems and include, for example, baculovirus vectors, which infect
insect cells; retroviral vectors, other lentivirus vectors such as
those based on the human immunodeficiency virus (HIV), adenovirus
vectors, adeno-associated virus (AAV) vectors, herpesvirus vectors,
vaccinia virus vectors, and the like, which infect mammalian cells
(see Miller and Rosman, BioTechniques 7:980-990, 1992; Anderson et
al., Nature 392:25-30 Suppl., 1998; Verma and Somia, Nature
389:239-242, 1997; Wilson, New Engl. J. Med. 334:1185-1187 (1996),
each of which is incorporated herein by reference). For example, a
viral vector based on an HIV can be used to infect T cells, a viral
vector based on an adenovirus can be used, for example, to infect
respiratory epithelial cells, and a viral vector based on a
herpesvirus can be used to infect neuronal cells. Other vectors,
such as AAV vectors can have greater host cell range and,
therefore, can be used to infect various cell types, although viral
or non-viral vectors also can be modified with specific receptors
or ligands to alter target specificity through receptor mediated
events.
[0374] A method of the invention can be used to operatively
covalently link a first nucleic acid molecule containing an open
reading frame to a second (and other) nucleic acid molecule
containing an open reading frame such that a nucleic acid molecule
encoding a chimeric polypeptide is generated. The chimeric
polypeptide comprises a fusion polypeptide, in which the two (or
more) encoded peptides (or polypeptides) are translated into a
single product, i.e., the peptides are covalently linked through a
peptide bond. For example, a first nucleic acid molecule can encode
a cell compartmentalization domain, such as a plasma membrane
localization domain, a nuclear localization signal, a mitochondrial
membrane localization signal, an endoplasmic reticulum localization
signal, or the like, or a protein transduction domain such as the
human immunodeficiency virus TAT protein transduction domain, which
can facilitate translocation of a peptide linked thereto into a
cell (see Schwarze et al., Science 285:1569-1572, 1999; Derossi et
al., J. Biol. Chem. 271:18188, 1996; Hancock et al., EMBO J.
10:4033-4039, 1991; Buss et al., Mol. Cell. Biol. 8:3960-3963,
1988; U.S. Pat. No. 5,776,689 each of which is incorporated herein
by reference). Such a domain can be useful to target a fusion
polypeptide comprising the domain and a polypeptide encoded by a
second nucleic acid molecule, to which it is covalently linked
according to a method of the invention, to a particular compartment
in the cell, or for secretion from or entry into a cell. As such,
the invention provides a means to generate ds recombinant nucleic
acid molecules covalently linked in both strands that encode a
chimeric polypeptide.
[0375] A fusion polypeptide expressed from a nucleic acid molecule
generated according to a method of the invention also can comprise
a peptide having the characteristic of a detectable label or a tag
such that the express fusion polypeptide can be detected, isolated,
or the like. For example, a nucleic acid molecule containing a
topoisomerase recognition site, or cleavage product thereof, as
disclosed herein, can encode an enzyme such as alkaline
phosphatase, .beta.-galactosidase, chloramphenicol
acetyltransferase, luciferase, or other enzyme; or can encode a
peptide tag such as a polyhistidine sequence (e.g., hexahistidine),
a V5 epitope, a c-myc epitope; a hemagglutinin A epitope, a FLAG
epitope, or the like. Expression of a fusion polypeptide comprising
a detectable label can be detected using the appropriate reagent,
for example, by detecting light emission upon addition of luciferin
to a fusion polypeptide comprising luciferase, or by detecting
binding of nickel ion to a fusion polypeptide comprising a
polyhistidine tag. Similarly, isolation of a fusion polypeptide
comprising a tag can be performed, for example, by passing a fusion
polypeptide comprising a myc epitope over a column having an
anti-c-myc epitope antibody bound thereto, then eluting the bound
fusion polypeptide, or by passing a fusion polypeptide comprising a
polyhistidine tag over a nickel ion or cobalt ion affinity column
and eluting the bound fusion polypeptide. Methods for detecting or
isolating such fusion polypeptides will be well known to those in
the art, based on the selected detectable label or tag (see, for
example, Hopp et al., BioTechnology 6:1204, 1988; U.S. Pat. No.
5,011,912; each of which is incorporated herein by reference).
[0376] A method of the invention also can be used to detectably
label a nucleotide sequence with a chemical or small organic or
inorganic moiety such that the nucleotide sequence is useful as a
probe. For example, a nucleic acid molecule, which has a
topoisomerase recognition site, or cleavage product thereof, at a
3' terminus, can have bound thereto a detectable moiety such as a
biotin, which can be detected using avidin or streptavidin, a
fluorescent compound (e.g., Cy3, Cy5, Fam, fluorescein, or
rhodamine), a radionuclide (e.g., sulfur-35, technicium-99,
phosphorus-32, or tritium), a paramagnetic spin label (e.g.,
carbon-13), a chemiluminescent compound, or the like, such that,
upon generating a covalently linked double stranded recombinant
nucleic acid molecule according to a method of the invention, the
generated nucleic acid molecule will be labeled. Methods of
detectably labeling a nucleotide sequence with such moieties are
well known in the art (see, for example, Hermanson, "Bioconjugate
Techniques" (Academic Press 1996), which is incorporated herein by
reference). Furthermore, a detectable label can be used to allow
capture of a ds nucleic acid molecule that is generated by the
present invention. Finally, a detectable label, for example biotin,
can be used to block ligation of a topoisomerase-charged end of a
first nucleic acid molecule to a labeled end of a second nucleic
acid molecule, thus providing a method to direct ligation to the
unlabelled end of the second nucleic acid molecule. It should be
recognized that such elements as disclosed herein or otherwise
known in the art, including nucleotide sequences encoding cell
compartmentalization domains, or detectable labels or tags, or
comprising transcriptional or translation regulatory elements can
be useful components of a kit as disclosed herein.
[0377] A method of the invention provides a means to conveniently
generate ds recombinant nucleic acid molecules that encode chimeric
polypeptides useful, for example, for performing a two hybrid
assay. In such a method, the first nucleic acid molecule encodes a
polypeptide, or a relevant domain thereof, that is suspected of
having or being examined for the ability to interact specifically
with one or more other polypeptides. The first nucleic acid
molecule is modified as disclosed herein to contain a topoisomerase
recognition site at one or both ends and, if desired, a 5'
overhanging sequence. The second nucleic acid molecule, to which
the first nucleic acid molecule is to be covalently-linked
according to a method of the invention, can encode a transcription
activation domain or a DNA binding domain (Example 2.C), and
contains a topoisomerase recognition site, or cleavage product
thereof, and a 5' overhanging sequence complementary to that at the
end of the first nucleic acid molecule to which it is to be linked.
Upon contact with a topoisomerase, if the nucleotide sequences are
not already topoisomerase-charged, a first hybrid useful for
performing a two hybrid assay (see, for example, Fields and Song,
Nature 340:245-246, 1989; U.S. Pat. No. 5,283,173; Fearon et al.,
Proc. Natl. Acad. Sci., USA 89:7958-7962, 1992; Chien et al., Proc.
Natl. Acad. Sci., USA 88:9578-9582, 1991; Young, Biol. Reprod.
58:302-311 (1998), each of which is incorporated herein by
reference), or modified form of a two hybrid assay such as the
reverse two hybrid assay (Leanna and Hannink, Nucl. Acids Res.
24:3341-3347, 1996, which is incorporated herein by reference), the
repressed transactivator system (U.S. Pat. No. 5,885,779, which is
incorporated herein by reference), the protein recruitment system
(U.S. Pat. No. 5,776,689, which is incorporated herein by
reference), and the like, is generated. Similar methods are used to
generate the second hybrid protein, which can comprise a plurality
of polypeptides to be tested for the ability to interact with the
polypeptide, or domain thereof, of the first hybrid protein.
[0378] Similarly, such a method of generating a chimeric protein
can be performed according to a method of the current invention for
generating a ds recombinant nucleic acid molecule covalently linked
in one strand, using first and second nucleic acid molecules
comprising a site-specific topoisomerase recognition site (e.g., a
type IA or a type II topoisomerase recognition site), or cleavage
product thereof, at least at one 5' terminus of an end to be
joined, wherein the nucleic acid molecules can further comprise
complementary 3' overhangs upon cleavage by the topoisomerase.
[0379] Similarly, such a method of generating a chimeric protein
can be performed according to a method of the current invention for
generating a ds recombinant nucleic acid molecule covalently linked
in both strands using first and second nucleic acid molecules
comprising a topoisomerase recognition site, or cleavage product
thereof, at least at the 5' terminus of the ends to be joined,
wherein the nucleic acid molecules can further comprise
complementary 3' overhangs upon cleavage by the topoisomerase; or
one of the first or second nucleic acid molecules can comprise
topoisomerase recognition sites, or cleavage products thereof, at
the 5' terminus and the 3' terminus of at least one end, and the
other nucleic acid molecule can contain a 3' hydroxyl group and a
5' hydroxyl group at the end to be joined, and wherein, upon
cleavage by the topoisomerases, the topoisomerase-charged nucleic
acid molecule can contain a 5' or 3' overhang that is complementary
to, and facilitates hybridization to, a 5' or 3' overhang,
respectively, or a blunt end, at the end of the other nucleic acid
molecule to be joined.
[0380] In an alternative embodiment, the present invention also
provides a method for the directional insertion of DNA fragments
into cloning or expression vectors with the ease and efficiency of
topoisomerase-mediated cloning. This invention also has advantages
over current cloning systems because it decreases the laborious
screening process necessary to identify cloned inserts in the
desired orientation. This aspect of the invention consists, in its
simplest form, of a linearized expression vector having a single
topoisomerase molecule covalently attached at both 3' ends. At
least one end of the linearized vector contains a 5'
single-stranded overhang, while the opposite end can be either
blunt, possess a single 3' T extension for T/A cloning, or may
itself contain a second 5' single-stranded overhang sequence. These
single-stranded sequence overhangs are alternatively referred to
herein as "SSS" and may consist of any convenient sequence.
[0381] Construction of a topoisomerase-charged cloning vector
according to this aspect of the invention may be accomplished, for
example, by endonuclease digestion of the vector (which may be a
pDONR vector (see FIG. 32) or a pDEST vector (see FIG. 33)),
followed by complementary annealing of synthetic oligonucleotides
and site-specific cleavage of the heteroduplex by Vaccinia
topoisomerase I. Digestion of a vector with any compatible
endonuclease creates specific sticky ends. Custom oligonucleotides
may be annealed to these sticky ends, and possess sequences that,
following topoisomerase I modification, form custom ends of the
vector (see FIGS. 32 and 33). The sequence and length of the SSS
will vary based on the desires of the user.
[0382] In one use of the TOPO SSS vectors provided by this aspect
of the present invention, the DNA fragment to be inserted into the
vector is a PCR product. Following PCR amplification with custom
primers, the product can be directionally inserted into a
topoisomerase I charged cloning vector having a SSS on one or both
ends of the insertion site. The custom primers may be designed such
that at least one primer of a given primer pair contains an
additional sequence at its 5' end. The added sequence may be
designed to be complementary to the sequence of the single-stranded
overhang in the vector. The complementarity between the 5'
single-stranded overhang in the vector and the 5' end of the PCR
product mediates the directional insertion of the PCR product into
the topoisomerase-mediated vector. Specifically, since only one end
of the vector and one end of the PCR product possess complimentary
SSS regions, the insertion of the product is directional.
Topoisomerase I catalyzes the ligation of the PCR product to the
vector.
[0383] This aspect of the invention also provides a modified
cloning vector, having an overhanging single stranded piece of DNA,
(the SSS) charged with topoisomerase, or "TOPO SSvector". The
modified vector allows the directional insertion of PCR amplified,
or otherwise suitable, open reading frames (ORF) for subsequent
expression, and takes advantage of the efficiency of
topoisomerase-mediated cloning.
[0384] As noted above, topoisomerases are a class of enzymes that
modify the topological state of DNA via the breakage and rejoining
of DNA strands, (Shuman et al., U.S. Pat. No. 5,766,891,
incorporated herein by reference). Vaccinia virus encodes a 314 aa
type I topoisomerase enzyme capable of site-specific single-strand
nicking of double stranded DNA, as well as 5' hydroxyl driven
religation. Site-specific type I topoisomerases include, but are
not limited to, viral topoisomerases such as pox virus
topoisomerase. Examples of pox virus topoisomerases include shope
fibroma virus and ORF virus. Other site-specific topoisomerases are
well known to those skilled in the art and can be used to practice
this invention.
[0385] Shuman teaches that Vaccinia topoisomerase binds to duplex
DNA and cleaves the phosphodiester backbone of one strand while
exhibiting a high level of sequence specificity. Cleavage occurs at
a consensus pentapyrimidine element 5'-(C/T)CCTT-3' or related
sequences in the scissile strand. In one embodiment the scissile
bond is situated in the range of 2-12 bp from the 3' end of the
duplex DNA. In another embodiment cleavable complex formation by
Vaccinia topoisomerase requires six duplex nucleotides upstream and
two nucleotides downstream of the cleavage site. Examples of
Vaccinia topoisomerase cleavable sequences include, but are not
limited to, +6/-6 duplex GCCCTTATTCCC (SEQ ID NO: 29), +8/-4 duplex
TCGCCCTTATTC (SEQ ID NO: 30), +10/-2 duplex TGTCGCCCTTAT (SEQ ID
NO: 31), +11/-1 duplex GTGTCGCCCTTA (SEQ ID NO: 32).
[0386] Examples of other site-specific type I topoisomerases are
well known in the art. These enzymes are encoded by many organisms
including, but not limited to Saccharomyces cerevisiae,
Saccharomyces pombe and Tetrahymena, however these species'
topoisomerase I enzymes have less specificity for a consensus
sequence than does Vaccinia's. (Lynn, R. M., Bjornsti, M., Caron,
P. R. and Wang, J. C., (1989) Peptide sequencing and site-directed
mutagenesis identify tyrosine-727 as the active site tyrosine of
Saccharomyces cerevisiae DNA topoisomerase I, Proc. Natl. Acad.
Sci. USA, 86: 3559-3563), (Eng, W., Pandit, S. D., and Sternglanz,
R., (1989) Mapping of the active site tyrosine of eukaryotic DNA
topoisomerase I, J. Biol. Chem., 264: 13373-13376) and (Busk, H.,
Thomsen, B., Bonven, B. J., Nielsen, O. F., and Westergaard, O.
(1987) Preferential relaxation of supercoiled DNA containing a
hexadecameric recognition for topoisomerase I, Nature, 327:
638-640), respectively.
[0387] As used herein with regard to this aspect of the invention,
the term donor signifies a duplex DNA which contains a 5'-CCCTT
cleavage site near the 3' end, and the term acceptor signifies a
duplex DNA which contains a 5'-OH terminus. Once covalently
activated by topoisomerase the donor will be transferred to those
acceptors to which it has SSS complementation.
[0388] According to this aspect of the present invention,
topoisomerase-modified vectors are further adapted to contain at
least one 5' single-stranded overhang sequence to facilitate the
directional insertion of DNA segments. In a preferred embodiment,
the segment to be cloned is a PCR product constituting an open
reading frame (ORF) which will be expressed from the resultant
recombinant vector. The primers used for amplifying the ORF are
designed such that at least one primer of the primer pair contains
an additional sequence at its 5' end. This sequence is designed to
be complementary to the sequence of the 5' single-stranded overhang
present in the topoisomerase-modified vector of the present
invention.
[0389] Certain preferred, but non-exclusive, embodiments according
to this aspect of the present invention are described in detail
below in Examples 5-8.
[0390] Nucleic acid molecules assembled using methods of the
invention either may be used directly or may be amplified and then
used for any number of purposes. With reference to FIG. 34, nucleic
acid segments to be assembled using methods of the invention may be
generated by any number of methods. For example, these segments may
be obtained by any method known in the art. In instances where the
nucleic acid segments do not have one or more (e.g., one, two,
three, four, etc.) termini and/or regions suitable for assembly
using methods of the invention, such termini and/or regions may be
added. Suitable termini and/or regions may be added, for example,
by amplifying nucleic acids using PCR or by the addition of one or
more (e.g., one, two, three, four, etc.) adapter linkers (e.g.,
adapter linkers which contain one or more topoisomerase recognition
sites). Nucleic acid segments having suitable termini and/or
regions may then be assembled using methods of the invention
described elsewhere herein.
[0391] As shown in FIG. 34, once assembled, the linked nucleic acid
segments may be amplified (e.g., in vivo or in vitro) and then used
in any number of methods or processes, many of which are described
elsewhere herein. Alternatively, the assembled nucleic acid
segments may be used directly for applications such as in vitro
transcription/translation, recombinational cloning, or for
transforming or transfecting cells. The invention thus provides
versatile compositions and methods for manipulating nucleic
acids.
[0392] As also indicated in FIG. 34, the invention further provides
methods for linking nucleic acid segments which then may be used in
any number of methods or processes. As one example of such a
method, the initial ligation products generated by the first step
set out in Figure A, which is referred to here solely for
illustrative purposes, are directly transcribed (e.g., used for in
vitro transcription). This process is facilitated by the fact that
the nucleic acid which is transcribed is covalently linked in both
strands at the junction point between the linker containing the T7
promoter element and the PCR product. Further, transcription of the
linkage products from the T7 promoter results in the production of
both sense and antisense RNA which can be used to form, for
example, double stranded RNA. This double stranded RNA can be used
for inhibiting gene expression. In particular, methods of the
invention may be used to produce double stranded RNA for RNAi
applications. Such RNAi molecules may be prepared from RNA
molecules prepared in two separate tubes and then mixed or in the
same tube. In the first case, transcription of sense strand and
antisense strand RNA may occur after DNA molecules which encode
these strand have been separated and placed in separate tubes. In
the second case, transcription of both sense strand and antisense
strand RNA may occur in the same tube. Thus, the invention also
provides one and two tube methods for the preparation RNA for, for
example, the preparation of RNAi.
[0393] As one skilled in the art would recognize, any number of
variations of the above are possible and within the scope of the
invention. For example, a promoter other than a T7 promoter may be
used. Further, any of the nucleic acid molecules described above,
as well as elsewhere herein, may be designed to contain one or more
recombination sites which can then be used to connect these
molecules with other nucleic acid molecules (e.g., other nucleic
acid molecules with cognate recombination sites).
[0394] The invention provides compositions and methods for linking
nucleic acid molecules using topoisomerase and recombination. In
particular embodiments of the invention, nucleic acid molecules
undergo one or more (e.g., one, two, three, four, five, six, seven,
eight, nine, ten, etc.) recombination reactions and are then linked
to one or more (e.g., one, two, three, four, five, six, seven,
eight, nine, ten, etc.) other nucleic acid molecules by methods
involving covalent linking of strands catalyzed by one or more
(e.g. one, two, three, four, etc.) topoisomerases. In other
embodiments, nucleic acid molecules are linked to other nucleic
acid molecules by methods involving covalent linking of strands
catalyzed by one or more (e.g., one, two, three, four, etc.)
topoisomerases and then undergo one or more (e.g., one, two, three,
four, five, six, seven, eight, nine, ten, etc.) recombination
reactions. As one skilled in the art would recognize, the invention
is not tied to any particular order of topoisomerase-mediated
linkage of nucleic acid molecules or recombination reactions. Thus,
in general, the invention is directed to compositions and methods
for performing both recombination reactions and linking nucleic
acid segments using topoisomerases.
[0395] The invention thus also provides adapter-linker molecules
for use in accordance with the methods and compositions of the
invention. The adapter linkers that are provided by, and that may
be used in connection with, the present invention can contain both
a topoisomerase site and a recombination site. One example of a
process of the invention is set out schematically in FIG. 35. FIG.
35 shows a process which involves the connection of a
topoisomerase-adapted nucleic acid segment ("adapter linker") which
contains a single recombination site to another nucleic acid
segment, referred to as an insert. These two nucleic acid segments
may be connected by any topoisomerase-mediated process described
herein.
[0396] Adapter linkers of the invention may comprise (1) one or
more recombination sites and/or (2) one or more topoisomerase
recognition sites or one or more topoisomerases. In particular
embodiments, at least one of the one or more recombination sites of
the adapter linkers will be located within zero, one, two, three,
four, five, six, seven, eight, nine, ten, fifteen, or twenty
nucleotides of at least one of the one or more topoisomerase
recognition site or one or more topoisomerase. In specific
embodiments, recombination sites present in adapter linkers of the
invention are attL, attB, attP, or attL recombination sites. In
additional specific embodiments, the topoisomerase recognition
sites recognition are recognition sites for type IB topoisomerases,
type IA topoisomerases or type II topoisomerases, or the
topoisomerases are type IB topoisomerases, type IA topoisomerases
or type II topoisomerases. In addition, topoisomerase recognition
sites or topoisomerases may be located, with respect to
recombination sites, in adapter linkers of the invention such that
upon recombination, particular recombination sites become
associated with the product molecules. For example, a topoisomerase
recognition site may be located on either end of an attL site in an
adapter linker such that when the linker is attached to a nucleic
acid molecule and recombination occurs, either an attB or an attP
site is generated on the nucleic acid molecule to which the adapter
linker was attached. Thus, adapter linkers may contain
toposiomerase recognition sites and/or topoisomerases positioned,
with respect to recombination sites, such that upon ligation to a
nucleic acid molecule and recombination any number of variations of
recombination sites are present on the product nucleic acid
molecules. Examples of such recombination sites include attL, attB,
attP, and attR recombination sites.
[0397] The invention further provides methods for linking any
number of nucleic acid segments using adapter linkers which contain
recombination sites having the same or different specificities, as
well as adapter linkers which contain recombination sites having
the same or different specificities and kits which contain such
adapter linkers. For example, three separate PCR products, referred
to as segments A, B, and C, may be linked to adapter linkers such
that attL1 and attL3 sites are present at the ends of segment A,
attR3 and attR4 sites are present at the ends of segment B, and
attL4 and attL2 sites are present at the ends of segment C. Thus,
upon recombination with a linearized vector which contains attRl
and attR2 recombination sites at or near the termini, all three PCR
products are joined to each other and inserted into the vector to
generate a circularized nucleic acid molecule. Any number of
variations of the above are possible and are within the scope of
the invention.
[0398] The invention further includes sets of two or more (e.g.,
two, three, four, five, six, seven eight, nine, etc.) adapter
linkers which contain (1) one or more recombination sites having
the same or different specificities and/or (2) one or more
topoisomerases or toposiomerase recognition sites, as well as
methods for using these sets of adapter linkers to generate nucleic
acid molecules which contain one or more recombination sites,
compositions comprising such adapter linker sets or individual
member of these sets, nucleic acid molecules which have been
adapted with one or more adapter linkers of these sets, and methods
for using these nucleic acid molecules.
[0399] After topoisomerase-mediated assembly, the assembled nucleic
acid molecule may be recombined with another nucleic acid segment
which contains one or more (e.g., one, two, three, four, etc.)
suitable recombination sites. The recombination sites shown in FIG.
35 are attL1 and attR1 sites but any suitable recombination sites
may be used (e.g. lox sites, attR sites, attL sites, attB sites,
attP sites, etc.). Additional suitable recombination sites are
described elsewhere herein.
[0400] The invention thus includes methods for generating nucleic
acid molecules using topoisomerase recognition sites and
recombination sites with recombine with each other. The invention
also includes nucleic acid molecules prepared by and used in
methods of the invention, as well as methods for using nucleic acid
molecules generated by methods described herein.
[0401] The invention further includes methods for generating
nucleic acid molecules using multiple (e.g., two, three, four,
five, six, seven, eight, nine, ten, etc.) recombination sites and
topoisomerase recognition sites, as well as nucleic acid molecules
prepared by and used in such methods. Further, these recombination
sites may have multiple (e.g., two, three, four, five, six, seven,
eight, nine, ten, etc.) specificities. In addition, the
topoisomerase recognition sites may be designed to generate termini
which will result in the connection of these termini to different
nucleic acid segments. For example, these termini may be designed
to generate different "sticky ends" upon cleavage with a
topoisomerase.
[0402] Another example of methods described above is shown in FIG.
36. FIG. 36 shows a process in which two nucleic acid segments are
connected using a process which involves topoisomerase-mediated
covalent linkage of strands of the termini of the nucleic acid
segments. The resulting nucleic acid molecule then undergoes
recombination, which results in (1) the topoisomerase assembled
nucleic acid molecule becoming linked to a nucleic acid segment
which contains an origin of replication and (2) replacement of a
negative selection marker (e.g., a ccdB gene) with a promoter. The
recombined nucleic acid product is then connected to a nucleic acid
segment which is topoisomerase adapted at both termini and contains
a positive selection marker. This last step results in the nucleic
acid molecule being circularized.
[0403] The circularized nucleic acid end product shown in FIG. 36
may be introduced into host cells, which may be prokaryotic (e.g.,
bacterial) or eukaryotic (e.g., yeast, plant, animal (including
mammalian, such as human)) cells such as those described elsewhere
herein. Further, cells which contain this end product can be
selected for using positive and negative selection. Thus, for
example, cells which have acquired a nucleic acid molecule wherein
the negative selection marker has not been replaced by the promoter
will be selected against. The invention further includes methods
and compositions similar to those set out in FIGS. 35 and 36 in
which any number of the steps and components are varied. Examples
of steps and components which may be varied are described elsewhere
herein. The invention further includes methods for using nucleic
acid molecules generated by methods described above.
[0404] As one skilled in the art would recognize, nucleic acid
segments used in processes such as those shown in FIGS. 35 and 36
could contain any number of different elements. For example, a
positive selection marker could be substituted for the promoters
shown in FIG. 36. Further, the insert shown in FIG. 35 may contain
nucleic acid which has any number of functionalities. In
particular, when the insert contains a regions which is
transcribed, the transcript can be a mRNA or an RNA which serves a
function in the absence of translation. Examples of RNA which
serves a function in the absence of translation include transfer
RNAs (e.g., suppressor tRNAs), antisense RNAs, ribosomal RNAs, and
ribozymes. Additionally, more than one of the nucleic acid segments
connected and/or recombined by methods of the invention may contain
all or part of one or more (e.g., one, two, three, four, five, six,
seven, etc.) open reading frames. In such instances, nucleic acid
segments may be connected to each other such that transcription and
translation result in the production of one or more fusion
proteins. Additional nucleic acid elements which can be used in
methods of the invention are described elsewhere herein.
[0405] Once a nucleic acid molecule, such as the end product of the
process shown in FIG. 35, has been generated by methods of the
invention, the nucleic acid molecule may optionally be connected to
one or more (e.g., one, two, three, four, etc.) other nucleic acid
molecules or may be circularized by joining of the termini to each
other. Further, when three or more nucleic acid molecules are
connected to each other by methods of the invention, the termini of
various intermediate molecules or the end product may be joined to
each other to circularize these molecules.
[0406] The invention further provides compositions and methods for
performing homologous recombination and for producing transgenic
animals. Gene targeting by homologous recombination between an
exogenous DNA construct and cognate chromosomal sequences allows
precise modifications to be made at predetermined sites in the
genome. Gene targeting is well-established in, e.g., mouse
embryonic stem (ES) cells, and has been used to effect
modifications in a large number of murine genes. (See e.g., Brandon
et al., Curr. Biol. 5:625-634, 758-765, 873-881 (1995)). Gene
targeting can also be accomplished in somatic cells. (See e.g.,
Itzhaki et al., Nat. Genet. 15:258-265 (1997)). Cells that have
been modified by gene targeting via homologous recombination can
then be manipulated by methods known in the art to establish
transgenic animals.
[0407] One example of a composition of the invention that can be
used in homologous recombination applications is the end product
nucleic acid molecule set out in FIG. 37. FIG. 37 further shows an
example of a method for preparing such compositions. In particular,
FIG. 37 shows the linkage of topoisomerase adapted nucleic acid
segments to a non-topoisomerase adapted nucleic acid segment. In
this instance, the nucleic acid segment which the designer of the
nucleic acid end product seeks to integrate into a chromosome,
referred to here as an insert, is flanked by regions which contain
(1) a positive selection marker and (2) a negative selection marker
positioned between two recombination sites. Recombination may then
be used to replace the two negative selection markers with nucleic
acid having homology to a chromosomal region into which the end
product is to integrate (labeled "HR1" and "HR2" in FIG. 37).
[0408] Regions of homology used in the practice of the invention
will vary with the chromosomes of cells into which nucleic acid
molecules are to integrate. Further, in many instances, regions of
homology will be selected to facilitate integration into cells of a
particular organism. Such an organism may be unicellular organism
(e.g., a yeast, a protozoan, etc.) or multicellular organism (e.g.,
a plant, an animal, etc.).
[0409] The invention thus provides nucleic acid molecules and
compositions for performing homologous recombination and cells
produced via homologous recombination involving these molecules and
compositions. Methods of the present invention can be used in the
linking of multiple nucleic acid segments. FIG. 38, for example,
shows a schematic representation of the linking of four nucleic
acid segments using toposiomerase to generate a linear nucleic acid
molecule with recombination sites (labeled "L1" and "L2") located
near the termini. In the first step, topoisomerase adapted nucleic
acid segment which contains an attL1 recombination site and an
attL2 recombination site are linked to two other nucleic acid
segments using topoisomerase. In this particular instance, each
strand of the termini which are joined to each other is covalently
linked to a topoisomerase molecule. Thus, upon toposiomerase
mediated linkage of the nucleic acid strands, no nicks are present
at the junction points. In the second step, the topoisomerase
assembled nucleic acid segments are contacted with another nucleic
acid segment which contains an origin of replication (labeled
"ori"), a positive selection marker (labeled "PM"), an attR1
recombination site, and an attR2 recombination site in the presence
of LR CLONASE.TM. under conditions which allow for recombination
between the attL and attR recombination sites. In certain such
methods, for example, TOPO-adapted vectors are incubated with one
or more nucleic acid segments (e.g., one or more PCR products) at
room temperature (e.g., about 20-20.degree. C.) for about 5-30 (and
preferably about 10) minutes; the reaction is then heat-treated by
incubation at about 80.degree. C. for about 20 minutes, and the
reaction mixture then used in a standard LR reaction according to
manufacturer's instructions (Invitrogen Corporation, Carlsbad,
Calif.), except the incubation time for the LR reaction is
increased to about 3 hours. Recombination results in the formation
of a circular nucleic acid molecule which contains the various
starting nucleic acid segments separated from the origin and
selection marker by attB1 and attB2 recombination sites. As one
skilled in the art would recognize, any suitable recombination
sites could be used in place of the aut recombination sites shown
in this figure. The invention thus also provides compositions
comprising such nucleic acids, compositions used for producing such
nucleic acids, and uses of such nucleic acids and compositions in
the recombination and topoisomerase-mediated joining methods of the
invention described elsewhere herein.
[0410] The invention further provides nucleic acid molecules
suitable for performing cloning reactions in which a first nucleic
acid molecule, which shares one or more region of homology with a
second nucleic acid molecule, is used to insert nucleic acid from
the second nucleic acid molecule into the first nucleic acid
molecule. The invention further provides compositions and methods
for performing such cloning reactions.
[0411] One example of a process referred to above is RecE/T
cloning, which is described in PCT Publication WO 01/04288, the
entire disclosure of which is incorporated herein by reference.
Typically, in RecE/T cloning, a linear first nucleic acid molecule
(e.g., a vector) is introduced into a cell which contains (1)
regions at the termini that share homology with two separate,
nearby regions (e.g., nucleic acid regions which are about 20 to
about 30, about 20 to about 40, about 20 to about 50, about 30 to
about 40, about 40 to about 50, about 40 to about 60, about 40 to
about 80, about 50 to about 90, etc. nucleotides in length) of a
nucleic acid molecule present in the cell (e.g., a plasmid, a
bacterial artificial chromosome, a natural chromosome, etc.),
referred to here as "a second nucleic acid molecule", (2) a
selection marker, and (3) an origin of replication. The linear
first nucleic acid molecule will generally only replicate if it
becomes circularized. Further, the first nucleic acid molecule will
typically become circularized when it has undergone recombination
with the second nucleic acid molecule and acquired nucleic acid
from the second nucleic acid molecule which is intervening between
the regions of homology. In such embodiments, the regions of
homology in the first nucleic acid molecule will typically be in a
reverse orientation as compared to the second nucleic acid
molecule. Generally, the cell in which recombination occurs will be
one which expresses a recombinase such as RecE/T or RecAlpha/Beta.
Thus, the invention provides, in part, methods for performing
RecE/T cloning, nucleic acid molecules prepared by such methods,
compositions comprising such nucleic acid molecules, and methods
for using such nucleic acid molecules and compositions.
[0412] Modifications of the RecE/T process may be employed to
generate a number of different end products. For example, when the
regions of homology are arranged in various ways, the first nucleic
acid molecule can be designed to (1) insert into the second nucleic
acid molecule, or (2) delete nucleic acid from the second nucleic
acid molecule. Typically, when insertion of the second nucleic acid
molecule into the second nucleic acid molecule is desired, the
regions of homology of the first nucleic acid molecule will be in
the same orientation with respect to the regions of homology in the
second nucleic acid molecule. Further, when deletion of nucleic
acid from the second nucleic acid molecule is desired, the regions
of homology of the first nucleic acid molecule will generally be in
an inverse orientation with respect to the regions of homology in
the second nucleic acid molecule. Also, when insertion of the first
nucleic acid molecule into the second nucleic acid molecule is
desired, typically the first nucleic acid molecule will not contain
an origin of replication. The invention provides methods for
performing the above processes. The invention also provides nucleic
acid molecules and compositions for use in the above processes.
[0413] The present invention can also be used to link two nucleic
acid segments in a single step process using topoisomerase and
recombination sites to generate a circular nucleic acid molecule.
An example of this embodiment is depicted in FIG. 39 where one of
the nucleic acid segments contains an attL1 recombination site
(labeled "L1"), a promoter (labeled "P"), and toposiomerase
molecule covalently linked to one terminus. The other nucleic acid
segment contains an attRl recombination site (labeled "R1"), an
open reading frame (labeled "ORF"), an origin of replication
(labeled "ORI"), a positive selection marker (labeled "PM"), and
topoisomerase molecule covalently linked to one terminus. Thus,
when these two nucleic acid segments are contacted with each other
in the presence of LR CLONASE.TM. under conditions which allow for
recombination between the attL and attR recombination sites and
topoisomerase mediated linkage of nucleic acid strands, a circular
molecule is formed having the structure indicated. In certain such
methods, for example, TOPO-adapted vectors are incubated with one
or more nucleic acid segments (e.g., one or more PCR products) at
room temperature (e.g., about 20-20.degree. C.) for about 5-30 (and
preferably about 10) minutes; the reaction is then heat-treated by
incubation at about 80.degree. C. for about 20 minutes, and the
reaction mixture then used in a standard LR reaction according to
manufacturer's instructions (Invitrogen Corporation, Carlsbad,
Calif.), except the incubation time for the LR reaction is
increased to about 3 hours. As one skilled in the art would
recognize, any suitable recombination sites could be used in place
of the att recombination sites shown in this figure.
[0414] The present invention can also be used to link two nucleic
acid segments using toposiomerase mediated methods to generate a
circular nucleic acid molecule. A schematic representation of one
embodiment of this aspect of the invention is illustrated in FIG.
40. As shown in FIG. 40, the circular molecule contains an open
reading frame (labeled "ORF") positioned between attL1 and attL2
recombination site (labeled "L1" and "L2"). The topoisomerase
assembled product then undergoes recombination with another
circular molecule which contains attR1 and attR2 recombination
sites to generate a third circular nucleic acid molecule which
contains the open reading frame positioned between attB1 and attB2
recombination sites. Further, the open reading frame is operably
linked to a promoter. Thus, the final nucleic acid molecule
produced by this process is an expression construct. As one skilled
in the art would recognize, any suitable recombination sites could
be used in place of the att recombination sites shown in this
figure.
[0415] As disclosed herein, a first nucleic acid molecule can be
one of a plurality of nucleotide sequences, for example, a cDNA
library, a combinatorial library of nucleotide sequences, or a
population of variegated nucleotide sequences. As such, a
particularly useful embodiment of a method of the invention is in
generating recombinant polynucleotides encoding chimeric
polypeptides for performing a high throughput two hybrid assay for
identifying protein-protein interactions that occur among
populations of polypeptides (see U.S. Pat. No. 6,057,101 and U.S.
Pat. No. 6,083,693, each of which is incorporated herein by
reference). In such a method, two populations (pluralities) of
nucleotide sequences encoding polypeptides are examined, each
plurality having a complexity of from a few related but different
nucleotide sequences to as high as tens of thousands of such
sequences. By performing a method of the invention, for example,
using a PCR primer pair to amplify each nucleotide sequence in the
plurality, wherein at least one primer of the PCR primer pair
comprises (a) at least one topoisomerase recognition site or
complement thereof or (b) at least one recombination site,
covalently linked recombinant polynucleotides encoding a population
of chimeric bait polypeptides and a population of chimeric prey
polypeptides readily can be generated by contacting the amplified
pluralities of nucleotide sequences, each of which comprises (a) at
least one topoisomerase recognition site, with at least one
topoisomerase and a nucleotide sequence, which contains at least
one topoisomerase recognition site and encodes a transcription
activation domain or a DNA binding domain or (b) at least one
recombination site site, with at least one topoisomerase and a
nucleotide sequence, which contains at least one recombination site
and encodes a transcription activation domain or a DNA binding
domain.
[0416] In practicing a method of the invention, a first nucleic
acid molecule also can encode a ribonucleic acid (RNA) molecule,
which can function, for example, as a riboprobe, an antisense
nucleotide sequence, a ribozyme, or a triplexing nucleotide
sequence, or can be used in an in vitro translation reaction, and
the second nucleic acid molecule can encode a regulatory element
useful for expressing an RNA from the first nucleotide sequence
(see Example 2.A). For example, where it is desired to produce a
large amount of RNA, a second nucleic acid molecule component for
performing a method of the invention can comprise an RNA polymerase
promoter such as a T7, T3 or SP6 RNA polymerase promoter. Where the
RNA molecule is to be expressed in a cell, for example, an
antisense molecule to be expressed in a mammalian cell, the second
(or other) nucleic acid molecule can include a promoter that is
active in a mammalian cell, particularly a tissue specific
promoter, which is active only in a target cell. Furthermore, where
the RNA molecule is to be translated, for example, in a coupled in
vitro transcription/translation reaction, the first nucleotide
sequence or second (or other) nucleotide sequence can contain
appropriate translational regulatory elements (see Example
2.B).
[0417] Methods of the invention may also be used to produce
constructs which allow for silencing of genes in vivo. One method
of silencing genes involves the production of double-stranded RNA,
termed RNA interference (RNAi). (See, e.g., Mette et al., EMBO J,
19:5194-5201 (2000)). The mechanism by which RNAi is believed to
function, which is reviewed in Fjose et al., Biotechnol. Annu. Rev.
7:31-57 (2001), appears to be based on the ability of double
stranded RNA to induce the degradation of specific RNA molecules.
This mechanism is reported to involve the conversion of
double-stranded RNA into short RNAs that direct ribonucleases to
homologous RNA targets (e.g., mRNA targets). Methods of the
invention can be used in a number of ways to produce molecules such
as RNAi. Thus, expression products of nucleic acid molecules of the
invention can be used to silence gene expression.
[0418] One example of a nucleic acid molecule designed to produce
RNAi is a molecule in which a nucleic acid segment is linked to one
or more promoters such that RNA corresponding to both strands are
produced as two separate transcripts or as part of the same
transcript. For example, a nucleic acid molecule could be prepared
using methods of the invention wherein two copies of an open
reading frame are connected by an intervening nucleic acid segment
with two promoters that drive transcription in different
directions. Thus, one of the promoters drives transcription of
sense strand mRNA and the other promoter drives transcription of
antisense mRNA. Another example of a nucleic acid molecule which
could be used to produce RNAi is one in which an open reading frame
is flanked on each end by promoters which, drive transcription of
the open reading frame in opposing directions. As a third example,
doubles stranded RNA can be produced from a nucleic acid molecule
which encode RNA having a "snapback" region (e.g., a region that is
six, seven, eight, nine ten, etc. nucleotides in length) at one
terminus. Thus, an RNA transcript of this type will form a hairpin
turn at or near one terminus. When such an RNA molecule is
incubated, under appropriate conditions, in the presence of an RNA
dependent RNA polymerase, the double stranded region formed by the
hairpin can be used to prime second strand synthesis to form double
stranded RNA molecule.
[0419] Nucleic acid segments designed to produce RNAi, such as the
nucleic acid molecules described above, need not correspond to the
full-length gene or open reading frame. For example, when the
nucleic acid segment corresponds to all or part of an ORF or encode
an RNA molecule which does not correspond to all or part of an ORF,
the segment may only correspond to part of the ORF (e.g., about 15,
about 16, about 17, about 18, about 19, about 20, about 21, about
22, about 23, about 24, about 25, about 26, about 27, about 28,
about 29, about 30, about 40, about 50, about 60, etc. nucleotides
at the 5' or 3' end of the ORF).
[0420] Thus, in particular embodiments, the invention provides
methods for preparing nucleic acid molecules comprising at least
three segments. In some embodiments, at least two of these segments
share at least one region of sequence identity (e.g., a region at
least about 15, at least about 16, at least about 17, at least
about 18, at least about 19, at least about 20, at least about 21,
at least about 22, at least about 23, at least about 24, at least
about 25, at least about 26, at least about 27, at least about 28,
at least about 29, at least about 30, at least about 40, at least
about 50, at least about 60, at least about 70, at least about 80,
at least about 90, at least about 100 nucleotides, etc. nucleotides
in length). In other embodiments, one nucleic acid segment is
flanked by a region which can confer transcription of the interior
portion of the molecule in opposing directions (e.g., to produce
sense and antisense transcripts). The invention further provides
nucleic acid molecules prepared by methods of the invention and the
use of such molecules to either inhibit gene expression or
facilitate the degradation of specific RNA molecules.
[0421] The invention further includes methods for preparing nucleic
acid molecules which express one or more RNA molecules which can be
used to prepare double stranded RNA having overhangs on one or both
ends. For example, methods of the invention can be used to express
two single stranded RNA molecules which are 21 nucleotides in
length and share sequence complementarity over 19 of their
nucleotides. Thus, when these two single stranded RNA molecules
hybridize to each other, there will be a two nucleotide overhang on
each end. Double stranded RNA molecules similar to those described
above have been shown to be capable of inhibiting gene expression
when introduced into mammalian cells (Elbashir et al., Nature
411:494-498 (2001)).
[0422] The invention thus includes methods for generating nucleic
acid molecules which can be used to produce short RNA molecules, as
well as RNA molecules produced by nucleic acid molecules prepared
by these methods and methods for preparing these RNA molecules.
These short RNA molecules will typically be about 15, about 16,
about 17, about 18, about 19, about 20, about 21, about 22, about
23, about 24, about 25, about 26, about 27, about 28, about 29,
about 30 nucleotides in length. Further, these short RNA molecules
will typically be between from about 15 to about 30, from about 15
to about 25, from about 15 to about 24, from about 23 to about 22,
from about 15 to about 21, from about 15 to about 20, from about 15
to about 19, from about 15 to about 18, from about 20 to about 30,
from about 20 to about 28, from about 20 to about 25, from about 20
to about 24, from about 20 to about 23, from about 20 to about 22,
or from about 20 to about 21 nucleotides in length.
[0423] The invention further includes methods for generating
nucleic acid molecules which can be used to produce short double
stranded RNA molecules, as well as RNA molecules produced by
nucleic acid molecules prepared by these methods. These short
double stranded RNA molecules may comprise a double stranded region
which is about 10, about 12, about 14, about 15, about 16, about
17, about 18, about 19, about 20, about 21, about 22, about 23,
about 24, about 25, about 26, about 27, about 28, about 29, about
30 nucleotides in length. Further, the double stranded region of
these RNA molecules may be between from about 10 to about 30, from
about 10 to about 25, from about 10 to about 20, from about 10 to
about 18, from about 10 to about 17, from about 15 to about 30,
from about 15 to about 25, from about 15 to about 24, from about 23
to about 22, from about 15 to about 21, from about 15 to about 20,
from about 15 to about 19, from about 15 to about 18, from about 20
to about 30, from about 20 to about 28, from about 20 to about 25,
from about 20 to about 24, from about 20 to about 23, from about 20
to about 22, or from about 20 to about 21 nucleotides in length.
Further, these double stranded RNA molecules may comprise overhangs
at one or both termini which are about 1, about 2, about 3, about
4, about 5, about 6, about 7, or about 8 nucleotides in length
and/or are between from about 1 to about 10, from about 1 to about
8, from about 1 to about 6, from about 1 to about 4, from about 1
to about 2, from about 2 to about 10, from about 2 to about 8, from
about 2 to about 6, or from about 2 to about 4 nucleotides in
length.
[0424] The invention also provides methods for preparing nucleic
acid molecules which can be used to express antisense RNA (e.g.,
antisense mRNA). Methods similar to those described above for the
production of nucleic acid molecules which can be used for RNAi may
be employed; however, only the antisense strand will typically be
transcribed in molecules prepared by methods of the invention which
may be used to generate antisense RNA.
[0425] In related embodiments, promoters which drive transcription
of the sense RNA or antisense RNA can be either constitutive (e.g.,
CMV promoter, SV40 promoter, etc.), inducible (e.g., a
metallothionein promoter, etc.), or repressible. Thus, for example,
two different inducible promoters can be used to drive
transcription of sense RNA and antisense RNA. In such an instance,
promoter activation can be used to induce production of sense RNA,
antisense RNA, or both sense RNA and antisense RNA. Further, the
amount of sense RNA and/or antisense RNA produced can be related by
using, for example, graduated induction and/or derepression of the
promoters.
[0426] Gene silencing methods involving the use of compounds such
as RNAi and antisense RNA, for examples, are particularly useful
for identifying gene functions. More specifically, gene silencing
methods can be used to reduce or prevent the expression of one or
more genes in a cell or organism. Phenotypic manifestations
associated with the selective inhibition of gene functions can then
be used to assign role to the "silenced" gene or genes. As an
example, Chuang et al., Proc. Natl. Acad. Sci. (USA) 97:4985-4990
(2000), have demonstrated that in vivo production of RNAi can alter
gene activity in Arabidopsis thaliana. Thus, the invention provides
methods for regulating expression of nucleic acid molecules in
cells and tissues comprising the expression of RNAi and antisense
RNA. The invention further provides methods for preparing nucleic
acid molecules which can be used to produce RNA corresponding to
one or both strands of a DNA molecule.
[0427] The invention thus provides methods for regulating
expression of nucleic acid molecules in vivo (e.g., in cells and
tissues) and/or in vitro comprising the expression of sense RNA
and/or antisense RNA. The invention further provides methods for
preparing nucleic acid molecules which can be used to produce RNA
corresponding to one or both strands of a nucleic acid molecule
(e.g., a DNA molecule). The invention also provides compositions
for performing the methods described above and nucleic acid
molecules produced by the above methods (e.g., RNA and DNA
molecules).
[0428] The invention also relates to compounds and methods for gene
silencing involving ribozymes. In particular, the invention
provides antisense RNA/ribozymes fusions, which comprise 1)
antisense RNA corresponding to a target gene and 2) one or more
ribozymes that cleave RNA (e.g., hammerhead ribozyme, hairpin
ribozyme, delta ribozyme, Tetrahymena L-21-ribozyme, etc.). Further
provided by the invention are vectors that express such fusions,
methods for producing such vectors, and methods for using such
vector to suppress gene expression.
[0429] Expression of antisense molecules fused to ribozymes can be
used, for example, to cleave specific RNA molecules in a cell
because the antisense RNA portion of the transcript can be designed
to hybridize to particular "mRNA molecules. Further, the ribozyme
portion of the transcript can be designed to cleave the RNA
molecule to which it has hybridized. For example, the ribozyme can
be one which cleaves double stranded RNA (e.g., a Tetrahymena L-21
ribozyme).
[0430] A method of the invention can be particularly useful for
generating an expressible ds recombinant nucleic acid molecule that
can be inserted in a site specific manner into a target DNA
sequence. The target DNA sequence can be any DNA sequence,
particularly a genomic DNA sequence, and preferably a gene for
which some or all of the nucleotide sequence is known. The method
can be performed utilizing a first nucleic acid molecule, which has
a first end and a second end and encodes a polypeptide, for
example, a selectable marker, wherein the first nucleic acid
molecule comprises at least one topoisomerase recognition site
and/or at least one recombination site or cleavage product thereof
at the 3' terminus of each end and, optionally, a hydroxyl group at
the 5' terminus of each end, and wherein, preferably, the 5'
termini comprise 5' overhanging sequences, which are different from
each other; and covalently linking the first nucleic acid molecule
to first and second PCR amplification products according to a
method of the invention. The first and second amplification
products are generated from sequences upstream and downstream of
the site at which the construct is to be inserted, and each
amplification product contains at least one topoisomerase
recognition site and optionally at least one recombination site,
preferably, a 5' overhanging sequence, which is generated following
contact with the site specific topoisomerase. Preferably, the first
and second amplification products have different 5' overhanging
sequences such that each can be linked to a predetermined end of
the first nucleic acid molecule. Such a method similarly can be
performed using a ds amplification product comprising at least one
topoisomerase recognition site and, optionally, at least one
recombination site, or cleavage product thereof, at the 5' terminus
of one or both ends, wherein, upon cleavage by the topoisomerase,
the topoisomerase-charged molecule can comprise a 3' overhang at
one or both ends containing the topoisomerase. In addition, the
method can be performed using a ds amplification product comprising
topoisomerase recognition sites and, optionally, recombination
sites, or cleavage products thereof, at or near the 5' terminus and
the 3' terminus of one or both ends, wherein, upon cleavage by the
topoisomerases, the topoisomerase-charged nucleic acid molecule
preferably contains a 5' or 3' overhang at one or both ends
containing the topoisomerases. Once nucleic acid molecules are
joined by the methods described above, the resulting molecules may
then be used in recombination reactions, such as those described
elsewhere herein.
[0431] The first and second amplification products may be generated
using two sets of PCR primer pairs. The two sets of PCR primer
pairs may be selected such that, in the presence of an appropriate
polymerase such as Taq polymerase and a template comprising the
sequences to be amplified, the primers amplify portions of a target
DNA sequence that are upstream of and adjacent to, and downstream
of and adjacent to, the site for insertion of the selectable
marker. In addition, the sets of PCR primer pairs may be designed
such that the amplification products contain a topoisomerase
recognition site and, following cleavage by the site specific
topoisomerase, a 5' overhanging sequence at the end to be
covalently linked to the selectable marker. As such, the first PCR
primer pair includes 1) a first primer, which comprises, in an
orientation from 5' to 3', a nucleotide sequence complementary to a
5' overhanging sequence of the end of the selectable marker to
which the amplification product is to be covalently linked, a
nucleotide sequence complementary to a topoisomerase recognition
site, such that PCR introduces a functional recognition site in the
opposite strand (see primer sequences in FIG. 9D), and a nucleotide
sequence complementary to a 3' sequence of a target DNA sequence
upstream of the insertion site; and 2) a second primer, which
comprises a nucleotide sequence of the target genomic DNA upstream
of the 3' sequence to which the first primer is complementary,
i.e., downstream of the insertion site. The second PCR primer pair
includes 1) a first primer, which comprises, from 5' to 3', a
nucleotide sequence complementary to the 5' overhanging sequence of
the end of the selectable marker to which it is to be covalently
linked, a nucleotide sequence complementary to a topoisomerase
recognition site, such that PCR introduces a functional recognition
site in the opposite strand (see primer sequences in FIG. 9D), and
a nucleotide sequence of a 5' sequence of a target DNA sequence,
wherein the 5' sequence of the target genomic DNA is downstream of
the 3' sequence of the target DNA sequence to which the first
primer of the first PCR primer pair is complementary; and the
second primer of the second primer pair comprises a nucleotide
sequence complementary to a 3' sequence of the target DNA sequence
that is downstream of the 5' sequence of the target genomic DNA
contained in the first primer. The skilled artisan will recognize
that the sequences of the primer that are complementary to the
target genomic DNA are selected based on the sequence of the target
DNA. These primers may further comprise one or more recombination
sites.
[0432] Upon contact of the nucleic acid molecule comprising the
selectable marker, the first and second amplification products, and
a topoisomerase (if the molecules are not topoisomerase-charged), a
ds recombinant nucleic acid molecule covalently linked in both
strands is generated according to a method of the invention. The
generated ds recombinant nucleic acid molecule can be further
amplified, if desired, using PCR primers that are specific for an
upstream and downstream sequence of the target genomic DNA, thus
ensuring that only functional constructs are amplified. The
generated ds recombinant nucleic acid molecule is useful for
performing homologous recombination in a genome, for example, to
knock-out the function of a gene in a cell, or to confer a novel
phenotype on the cell containing the generated recombinant nucleic
acid molecule. The method can further be used to produce a
transgenic non-human organism having the generated ds recombinant
nucleic acid molecule stably maintained in its genome.
[0433] A method of the invention also is useful for covalently
linking, an adapter or linker sequence to one or both ends of a
nucleic acid molecule of interest, including to each of a plurality
of nucleic acid molecules. For example, where it is desired to put
linkers on both ends of a first nucleic acid molecule, the method
can be performed by contacting a topoisomerase with a first nucleic
acid molecule, which has a topoisomerase recognition site, or
cleavage product thereof, at one or both 3' or 5' termini and which
can include hydroxyl groups at both 5' termini and one or more
recombination sites; and a second nucleic acid molecule and at
least a third double stranded nucleotide sequence, each of which
can include a topoisomerase recognition site, or cleavage product
thereof at the appropriate 3' or 5' terminus and which can also
include, where desirable, a 5' hydroxyl group at the same terminus
and one or more recombination sites. An appropriate terminus is the
terminus to which the linker is to be covalently linked in at least
one strand to the first nucleotide sequence. In one embodiment, one
or both linker sequences contain an overhanging sequence that is
complementary to a sequence at the 5' terminus of the end of the
first nucleic acid molecule to which the linker is to be covalently
linked, thereby facilitating the initial association of the
nucleotide sequences in the proper (predetermined) orientation
(see, for example, FIG. 9 and Example 1.B). In performing such a
method, the linker sequences comprising the second and at least
third nucleotide sequence can be the same or different.
[0434] FIG. 14 shows one example of a process for preparing a
nucleic acid molecule containing a topoisomerase (e.g., a type IA
topoisomerase) bound to the 5' terminus of one end of the sequence,
and wherein the same end further comprise a 3' overhang (see (4) in
FIG. 14). In step A, a nucleotide sequence to be modified with
topoisomerase is digested with a restriction enzyme that generates
a "sticky" end. The restricted nucleotide sequence is then
contacted in step B with a linear, single stranded nucleotide
sequence which contains a topoisomerase attached the 5' terminus
and a ligase (e.g., a DNA ligase such as T4 DNA ligase). The
linear, single stranded nucleotide sequence also contains a region
at the 3' terminus which shares sufficient sequence complementarity
to the "sticky" end generated by the restriction enzyme, such that
the two molecules will hybridize. Thus, in step B, the two
nucleotide sequences are ligated to each other. In step C, the
product of the second step is contacted with a third nucleotide
sequence which shares sequence complementarity to portions of the
linear, single stranded nucleic acid molecule generated in step B,
and a ligase. The product of step C, shown in (4), is a nucleic
acid molecule containing a topoisomerase attached to the 5'
terminus of one end and a 3' overhang on the same end. It will be
recognized that numerous variations of the exemplified method are
within the scope of the invention. For example, similar processes
can be performed to prepare nucleic acid molecules which comprise
topoisomerase attached to the 3' terminus of one end or which have
a 5' overhang or are blunt ended at the end to which a
topoisomerase is attached. In another example, the nucleotide
sequence labeled number 3 in FIG. 14 can be produced in the
following manner: a nucleic acid molecule can be digested with a
restriction enzyme to generate a nucleic acid molecule with a
single-stranded 5' overhang that includes a type IA topoisomerase
recognition site. The nucleic acid molecule with the single
stranded overhang can then be contacted with type IA topoisomerase
to generate a type IA topoisomerase-charged nucleic acid
molecule.
[0435] FIG. 15 shows two embodiments of the invention in which
single stranded or double stranded DNA is covalently linked to
single stranded RNA. Where single stranded DNA is joined to single
stranded RNA, the 3' end of the ribonucleotide sequence is
covalently linked to the 5' end of the deoxyribonucleotide
sequence. Where double stranded DNA is joined to single stranded
RNA, the 3' terminus of the ribonucleotide sequence shares
sufficient sequence complementarity to the 3' overhang of the
deoxyribonucleotide sequence such that the two molecules hybridize.
As above, the 3' end of the ribonucleotide sequence is also
covalently linked to the 5' end of the deoxyribonucleotide
sequence. As will be recognized, numerous variations of the above
are within the scope of the invention. For example, the RNA
molecule can be double stranded. In another example, all of the
nucleotide sequences can be deoxyribonucleotide sequences and/or
can comprise one or more recombination sites.
[0436] The present invention provides a ds recombinant nucleic acid
molecule having, or which can be made to have, a first end and a
second end, each end including a 5' terminus and a 3' terminus,
wherein the molecule comprises a site-specific type IA
topoisomerase recognition site at or near a 5' terminus of the
first end, the second end, or both the first end and the second
end. The ds recombinant nucleic acid molecule can further include a
type IB topoisomerase recognition site at or near a 3' termini of
an end that does not include a type IA topoisomerase recognition
site. The ds recombinant nucleic acid molecule can be a vector.
[0437] The present invention further provides a
topoisomerase-charged ds recombinant nucleic acid molecule having a
first end and a second end, each end having a 5' terminus and a 3'
terminus, wherein a site-specific type IA topoisomerase is bound at
the 5' terminus of the first end, the second end, or both the first
end and the second end. For example, the topoisomerase-charged ds
recombinant nucleic acid molecule can include a type IA
topoisomerase bound at the 5' termini of each of the first and
second ends. The topoisomerase-charged nucleic acid ds recombinant
nucleic acid molecule can include a type IB topoisomerase bound at
a 3' termini of an end not bound by a type IA topoisomerase. The
topoisomerase-charged ds recombinant nucleic acid molecule can be a
vector.
Kits
[0438] The present invention also provides kits, which contain
components useful for conveniently practicing the methods of the
invention. In one embodiment, a kit of the invention contains a
first nucleic acid molecule, which encodes a polypeptide,
particularly a selectable marker, and contains a topoisomerase
recognition site at each end. Preferably, the first nucleotide
sequence comprises a topoisomerase-activated nucleotide sequence.
More preferably, the topoisomerase-charged first nucleotide
sequence comprises a 5' overhanging sequence at each end, and most
preferably the 5' overhanging sequences are different from each
other. Optionally, each of the 5' termini comprises a 5' hydroxyl
group.
[0439] In addition, the kit can contain at least a nucleotide
sequence (or complement thereof) comprising a regulatory element,
which can be an upstream or downstream regulatory element, or other
element, and which contains a topoisomerase recognition site at one
or both ends. Preferably, the kit contains a plurality of nucleic
acid molecules, each comprising a different regulatory element or
other element, for example, a sequence encoding a tag or other
detectable molecule or a cell compartmentalization domain. The
different elements can be different types of a particular
regulatory element, for example, constitutive promoters, inducible
promoters and tissue specific promoters, or can be different types
of elements including, for example, transcriptional and
translational regulatory elements, epitope tags, and the like. Such
nucleic acid molecules can be topoisomerase-activated, and can
contain 5' overhangs or 3' overhangs that facilitate operatively
covalently linking the elements in a predetermined orientation,
particularly such that a polypeptide such as a selectable marker is
expressible in vitro or in one or more cell types.
[0440] The kit also can contain primers, including first and second
primers, such that a primer pair comprising a first and second
primer can be selected and used to amplify a desired ds recombinant
nucleic acid molecule covalently linked in one or both strands,
generated using components of the kit. For example, the primers can
include first primers that are complementary to elements that
generally are positioned at the 5' end of a generated ds
recombinant nucleic acid molecule, for example, a portion of a
nucleic acid molecule comprising a promoter element, and second
primers that are complementary to elements that generally are
positioned at the 3' end of a generated ds recombinant nucleic acid
molecule, for example, a portion of a nucleic acid molecule
comprising a transcription termination site or encoding an epitope
tag. Depending on the elements selected from the kit for generating
a ds recombinant nucleic acid molecule covalently linked in both
strands, the appropriate first and second primers can be selected
and used to amplify a full length functional construct.
[0441] In another embodiment, a kit of the invention contains a
plurality of different elements, each of which can comprise one or
more recombination sites and/or can be topoisomerase-activated at
one or both ends, and each of which can contain a 5' overhanging
sequence or a 3' overhanging sequence or a combination thereof. The
5' or 3' overhanging sequences can be unique to a particular
element, or can be common to plurality of related elements, for
example, to a plurality of different promoter element. Preferably,
the 5' overhanging sequences of elements are designed such that one
or more elements can be operatively covalently linked to provide a
useful function, for example, an element comprising a Kozak
sequence and an element comprising a translation start site can
have complementary 5' overhangs such that the elements can be
operatively covalently linked according to a method of the
invention.
[0442] The plurality of elements in the kit can comprise any
elements, including transcription or translation regulatory
elements; elements required for replication of a nucleotide
sequence in a bacterial, insect, yeast, or mammalian host cell;
elements comprising recognition sequences for site specific nucleic
acid binding proteins such as restriction endonucleases or
recombinases; elements encoding expressible products such as
epitope tags or drug resistance genes; and the like. As such, a kit
of the invention provides a convenient source of different elements
that can be selected depending, for example, on the particular
cells that a construct generated according to a method of the
invention is to be introduced into or expressed in. The kit also
can contain PCR primers, including first and second primers, which
can be combined as described above to amplify a ds recombinant
nucleic acid molecule covalently linked in one or both strands,
generated using the elements of the kit. Optionally, the kit
further contains a site specific topoisomerase in an amount useful
for covalently linking in at least one strand, a first nucleic acid
molecule comprising a topoisomerase recognition site to a second
(or other) nucleic acid molecule, which can optionally be
topoisomerase-activated nucleic acid molecules or nucleotide
sequences that comprise a topoisomerase recognition site.
[0443] In still another embodiment, a kit of the invention contains
a first nucleic acid molecule, which encodes a selectable marker,
and contains a topoisomerase recognition site and/or a
recombination site at each end; a first and second PCR primer pair,
which can produce a first and second amplification products that
can be covalently linked in one or both strands, to the first
nucleic acid molecule in a predetermined orientation according to a
method of the invention. Such a generated construct can be
introduced into a cell and can incorporate into the genome of the
cell by homologous recombination in a site specific manner, where
it can be stably maintained and can express a heterologous
polypeptide in the cell or can knock-out a target gene function. A
target gene to be knocked-out, for example, can be any gene for
which at least part of the sequence is known or can be readily
determined and the function of which it is desired to disrupt, for
example, an oncogene, a gene involved in apoptosis, a gene encoding
a serine/threonine or a tyrosine kinase, or any other gene.
[0444] The first PCR primer pair in a kit of the invention useful
for generating a ds recombinant nucleic acid molecule covalently
linked in both strands, includes a first primer that comprises, in
an orientation from 5' to 3', a nucleotide sequence complementary
to a 5' overhanging sequence of a nucleic acid molecule to which it
is to be covalently linked (for example, an end of the nucleic acid
molecule encoding the selectable marker), a nucleotide sequence
complementary to a topoisomerase recognition site, such that PCR
introduces a functional recognition site in the opposite strand
(see primer sequences in FIG. 9D), and/or a recombination site, and
a nucleotide sequence complementary to a 3' sequence of the target
DNA sequence. The first PCR primer pair also includes a second
primer that comprises a nucleotide sequence of the target DNA
sequence upstream of the 3' sequence to which the first primer is
complementary.
[0445] The second PCR primer pair of a kit useful for generating a
ds recombinant nucleic acid molecule covalently linked in both
strands, includes a first primer that comprises, from 5' to 3', a
nucleotide sequence complementary to a 5' overhanging sequence of a
nucleic acid molecule to which it is to be covalently linked, a
nucleotide sequence complementary to a topoisomerase recognition,
such that PCR introduces a functional recognition site in the
opposite strand (see primer sequences in FIG. 9D), site and/or a
recombination site, and a nucleotide sequence of a 5' sequence of
the target DNA sequence, wherein the 5' sequence of the target gene
is downstream of the 3' sequence of the target DNA sequence to
which the first primer of the first primer pair is complementary.
The second PCR primer pair also includes a second primer that
comprises a nucleotide sequence complementary to a 3' sequence of
the target gene that is downstream of the 5' sequence of the target
DNA sequence contained in the first primer.
[0446] In another embodiment, a kit of the invention useful for
generating a ds recombinant nucleic acid molecule covalently linked
in both strands contains a first nucleic acid molecule, which
encodes a transcription activation domain and comprises a
topoisomerase recognition site, or cleavage product thereof, at a
3' terminus; and a second nucleic acid molecule, which encodes a
DNA binding domain and comprises a topoisomerase recognition site
and/or a recombination site, or cleavage product thereof, at a 3'
terminus. Upon cleavage by the site specific topoisomerase, the
first or second nucleic acid molecule can have a 5' overhang, or
both sequences can have 5' overhangs, which are the same or are
different from each other. Where the nucleic acid molecules have a
5' overhang, the overhang generally is complementary to a nucleic
acid molecule to which first or second nucleic acid molecule is to
be covalently linked according to a method of the invention. The
kit also can contain one or a pair of adapters, linkers or the
like, which can comprise a topoisomerase recognition site, or
cleavage product thereof, at one or both 3' termini, and,
optionally, a hydroxyl group at the same terminus/termini. Such
adapters, linkers, or the like are selected such that they contain
a 5' overhang that is complementary to one or the other of the two
nucleic acid molecules described above and part of the kit.
[0447] Similarly, a kit of the invention can contain one or a pair
of adapters, linkers or the like, which comprise a topoisomerase
recognition site and/or a recombination site, or cleavage product
thereof, at one or both 5' termini, and, optionally, a hydroxyl
group at the same terminus (or termini). Such adapters, linkers, or
the like are selected such that they contain a 3' overhang that is
complementary to one or the other of the two nucleic acid molecules
described above and part of the kit. In addition, the kit can
contain one or a pair of adapters, linkers or the like, which
comprise a topoisomerase recognition site, or cleavage product
thereof, at one or both 5' and/or 3' termini, and, optionally, a
hydroxyl group at the same terminus/termini.
[0448] Adapters, linkers, or the like generally are selected such
that they contain a 5' and/or a 3' overhang that is complementary
to one or the other of the two nucleic acid molecules as disclosed
herein and part of the kit. Such adapters, linkers, or the like can
be joined to the ends of nucleic acid molecules that are to
covalently linked to one or the other of the first or second
nucleic acid molecules provided with the kit, thus facilitating the
construction of chimeric polynucleotides encoding the bait and prey
polypeptides useful in a two hybrid assay. Such a kit also can
contain a PCR primer or primer pair, which can be used to prepare
an amplified plurality of nucleotide sequences comprising a
topoisomerase recognition site, or cleavage product thereof (see
Example 1).
[0449] A PCR primer pair in a kit of the invention, which can be
used for generating a ds recombinant nucleic acid molecule
covalently linked in one strand, can include a first primer that
comprises, in an orientation from 5' to 3', a nucleotide sequence
of a 5' overhanging sequence of a nucleic acid molecule to which it
is to be linked (for example, an end of the nucleic acid molecule
encoding the selectable marker), a topoisomerase recognition site
(e.g., a type IA or type II topoisomerase recognition site) and,
optionally, a recombination site, and a nucleotide sequence
complementary to a 5' sequence of the target DNA sequence. The PCR
primer pair also includes a second primer that comprises a
nucleotide sequence of the target DNA sequence downstream of the 5'
sequence to which the first primer is complementary.
[0450] In another embodiment, a kit of the invention contains a
first nucleic acid molecule, which encodes a transcription
activation domain and comprises a site-specific topoisomerase
recognition site (e.g., a type IA or a type II topoisomerase
recognition site) and, optionally, a recombination site, or
cleavage product thereof, at a 5' terminus; and a second nucleic
acid molecule, which encodes a DNA binding domain and comprises a
site-specific topoisomerase recognition site (e.g., a type IA or a
type II topoisomerase recognition site), or cleavage product
thereof, at a 5' terminus. Upon cleavage by the site specific
topoisomerase, the first or second nucleic acid molecule can have a
3' overhang, or both sequences can have 3' overhangs, which are the
same or are different from each other. Where the nucleic acid
molecules have a 3' overhang, the overhang generally is
complementary to a nucleic acid molecule to which first or second
nucleic acid molecule is to be linked according to a method of the
invention. The kit also can contain one or a pair of adapters,
linkers or the like, which may comprise a site-specific
topoisomerase recognition site (e.g., a type IA or a type II
topoisomerase recognition site), a cleavage product thereof, and/or
a recombination site, at one or both 5' and/or 3' termini and/or
located internally, and which can contain a 5' overhang that is
complementary to one or the other of the two nucleic acid molecules
of the kit.
[0451] A ds recombinant nucleic acid molecule covalently linked in
one or both strands, and generated according to a method of the
invention, can be used for various purposes, including, for
example, for expressing a polypeptide in a cell, for diagnosing or
treating a pathologic condition, or the like. As such, the present
invention provides a medicament, which can be useful for treating a
pathologic condition by expressing a polypeptide in one or more
cells or by expressing an antisense molecule, or the like. Such a
ds recombinant nucleic acid molecule can be provided to a cell by
contacting the cell ex vivo, then administering the cell to the
subject, such a method also allowing for selection and/or expansion
of the cells containing the ds recombinant nucleic acid molecule
prior to such administration, or can be provided directly to the
subject. For administration to a living subject, the ds recombinant
nucleic acid molecule, which is covalently linked in one or both
strands, generally is formulated in a composition suitable for
administration to the subject. Thus, the invention provides
compositions containing a ds recombinant nucleic acid molecule
covalently linked in one or both strands, generated according to a
method of the invention. As disclosed herein, such nucleic acid
molecules are useful as medicaments for treating a subject
suffering from a pathological condition.
[0452] A composition for administration generally is formulated
using one or more pharmaceutically acceptable carriers as well
known in the art and include, for example, aqueous solutions such
as water or physiologically buffered saline or other solvents or
vehicles such as glycols, glycerol, oils such as olive oil or
injectable organic esters. A pharmaceutically acceptable carrier
can contain physiologically acceptable compounds that act, for
example, to stabilize or to increase the absorption of the
conjugate. Such physiologically acceptable compounds include, for
example, carbohydrates, such as glucose, sucrose or dextrans,
antioxidants, such as ascorbic acid or glutathione, chelating
agents, low molecular weight proteins or other stabilizers or
excipients. One skilled in the art would know that the choice of a
pharmaceutically acceptable carrier, including a physiologically
acceptable compound, depends, for example, on the route of
administration of the composition, which can be, for example,
orally or parenterally such as intravenously, and by injection,
intubation, or other such method known in the art. A composition of
the invention also can contain a second reagent such as a
diagnostic reagent, nutritional substance, toxin, or therapeutic
agent, for example, a cancer chemotherapeutic agent.
[0453] The ds recombinant nucleic acid molecule covalently linked
in one or both strands, can be incorporated within an encapsulating
material such as into an oil-in-water emulsion, a microemulsion,
micelle, mixed micelle, liposome, microsphere or other polymer
matrix (see, for example, Gregoriadis, Liposome Technology, Vol. I
(CRC Press, Boca Raton, Fla. 1984); Fraley, et al., Trends Biochem.
Sci., 6:77 (1981), each of which is incorporated herein by
reference). Liposomes, for example, which consist of phospholipids
or other lipids, are nontoxic, physiologically acceptable and
metabolizable carriers that are relatively simple to make and
administer. "Stealth" liposomes (see, for example, U.S. Pat. Nos.
5,882,679; 5,395,619; and 5,225,212, each of which is incorporated
herein by reference) are an example of such encapsulating materials
particularly useful for preparing a pharmaceutical composition, and
other "masked" liposomes similarly can be used, such liposomes
extending the time that a nucleic acid molecule remains in the
circulation. Cationic liposomes, for example, also can be modified
with specific receptors or ligands (Morishita et al., J. Clin.
Invest., 91:2580-2585 (1993), which is incorporated herein by
reference). The nucleic acid molecule also can be introduced into a
cell by complexing it with an adenovirus-polylysine complex (see,
for example, Michael et al., J. Biol. Chem. 268:6866-6869 (1993),
which is incorporated herein by reference). Such compositions can
be particularly useful for introducing a nucleic acid molecule into
a cell in vivo or in vitro, including ex vivo, wherein the cell
containing the nucleic acid molecule is administered back to the
subject (see U.S. Pat. No. 5,399,346, which is incorporated herein
by reference). A nucleic acid molecule generated according to a
method of the invention also can be introduced into a cell using a
biolistic method (see, for example, Sykes and Johnston, supra,
1999).
Host Cells
[0454] The invention also relates to host cells, or derivatives
thereof, comprising one or more of the nucleic acid molecules or
vectors of the invention, particularly those nucleic acid molecules
and vectors described in detail herein. Representative host cells
that may be used according to this aspect of the invention include,
but are not limited to, bacterial cells, yeast cells, plant cells
and animal cells, and derivatives thereof. Preferred bacterial host
cells include Escherichia spp. cells (particularly E. coli cells
and most particularly E. coli strains DH10B, Stb12, DH5a, DB3,
DB3.1 (preferably E. coli LIBRARY EFFICIENCY.RTM. DB3.PM Competent
Cells; Invitrogen Corporation, Carlsbad, Calif.), DB4, DB5, JDP682
and ccdA-over (see U.S. application Ser. No. 09/518,188, filed Mar.
2, 2000, and U.S. provisional Application No. 60/475,004, filed
Jun. 3, 2003, by Louis Leong et al., entitled "Cells Resistant to
Toxic Genes and Uses Thereof," the disclosures of which are
incorporated by reference herein in their entireties); Bacillus
spp. cells (particularly B. subtilis and B. megaterium cells);
Streptomyces spp. cells; Erwinia spp. cells; Klebsiella spp. cells;
Serratia spp. cells (particularly S. marcessans cells); Pseudomonas
spp. cells (particularly P. aeruginosa cells); and Salmonella spp.
cells (particularly S. typhimurium and S. typhi cells). Preferred
animal host cells include insect cells (most particularly
Drosophila melanogaster cells, Spodoptera frugiperda Sf9 and Sf2!
cells and Trichoplusa High-Five cells), nematode cells
(particularly C. elegans cells), avian cells, amphibian cells
(particularly Xenopus laevis cells), reptilian cells, and mammalian
cells (most particularly NIB3T3, CHO, COS, VERO, BHK and human
cells). Preferred yeast host cells include Saccharomyces cerevisiae
cells and Pichia pastoris cells. In addition, derivatives of such
host cells are suitable for use in accordance with the present
invention. These and other suitable host cells are available
commercially, for example from Invitrogen Corporation (Carlsbad,
Calif.), American Type Culture Collection (Manassas, Va.), and
Agricultural Research Culture Collection (NRRL; Peoria, Ill.).
[0455] Methods for introducing the nucleic acid molecules and/or
vectors of the invention into the host cells described herein, to
produce host cells comprising one or more of the nucleic acid
molecules and/or vectors of the invention, will be familiar to
those of ordinary skill in the art. For instance, the nucleic acid
molecules and/or vectors of the invention may be introduced into
host cells using well known techniques of infection, transduction,
electroporation, transfection, and transformation. The nucleic acid
molecules and/or vectors of the invention may be introduced alone
or in conjunction with other the nucleic acid molecules and/or
vectors and/or proteins, peptides or RNAs. Alternatively, the
nucleic acid molecules and/or vectors of the invention may be
introduced into host cells as a precipitate, such as a calcium
phosphate precipitate, or in a complex with a lipid.
Electroporation also may be used to introduce the nucleic acid
molecules and/or vectors of the invention into a host. Likewise,
such molecules may be introduced into chemically competent cells
such as E. coli. If the vector is a virus, it may be packaged in
vitro or introduced into a packaging cell and the packaged virus
may be transduced into cells. Hence, a wide variety of techniques
suitable for introducing the nucleic acid molecules and/or vectors
of the invention into cells in accordance with this aspect of the
invention are well known and routine to those of skill in the art.
Such techniques are reviewed at length, for example, in Sambrook,
J., et al., Molecular Cloning, a Laboratory Manual, 2nd Ed., Cold
Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, pp.
16.30-16.55 (1989), Watson, J. D., et al., Recombinant DNA, 2nd
Ed., New York: W.H. Freeman and Co., pp. 213-234 (1992), and
Winnacker, E.-L., From Genes to Clones, New York: VCH Publishers
(1987), which are illustrative of the many laboratory manuals that
detail these techniques and which are incorporated by reference
herein in their entireties for their relevant disclosures.
Polymerases
[0456] Polymerases for use in the invention include but are not
limited to polymerases (DNA and RNA polymerases), and reverse
transcriptases. DNA polymerases include, but are not limited to,
Thermus thermophilus (Tth) DNA polymerase, Thermus aquaticus (Taq)
DNA polymerase, Thermotoga neopolitana (Tne) DNA polymerase,
Thermotoga maritima (Tma) DNA polymerase, Thermococcus litoralis
(Tli or VENT.TM.) DNA polymerase, Pyrococcus furiosus (Pfi) DNA
polymerase, DEEPVENT.TM. DNA polymerase, Pyrococcus woosii (Pwo)
DNA polymerase, Pyrococcus sp KOD2 (KOD) DNA polymerase, Bacillus
sterothermophilus (Bst) DNA polymerase, Bacillus caldophilus (Bca)
DNA polymerase, Sulfolobus acidocaldarius (Sac) DNA polymerase,
Thermoplasma acidophilum (Tac) DNA polymerase, Thermus flavus
(Tfl/Tub) DNA polymerase, Thermus ruber (Tru) DNA polymerase,
Thermus brockianus (DYNAZYME.TM.) DNA polymerase, Methanobacterium
thermoautotrophicum (Mth) DNA polymerase, mycobacterium DNA
polymerase (Mtb, Mlep), E. coli pol I DNA polymerase, T5 DNA
polymerase, T7 DNA polymerase, and generally pol I type DNA
polymerases and mutants, variants and derivatives thereof. RNA
polymerases such as T3, T5, T7 and SP6 and mutants, variants and
derivatives thereof may also be used in accordance with the
invention.
[0457] The nucleic acid polymerases used in the present invention
may be mesophilic or thermophilic, and are preferably thermophilic.
Preferred mesophilic DNA polymerases include Pol I family of DNA
polymerases (and their respective Klenow fragments) any of which
may be isolated from organism such as E. coli, H. influenzae, D.
radiodurans, H. pylori, C. aurantiacus, R. prowazekii, T. pallidum,
Synechocystis sp., B. subtilis, L. lactis, S. pneumoniae, M
tuberculosis, M. leprae, M. smegmatis, Bacteriophage L5, phi-C31,
T7, T3, T5, SP01, SP.sub.02, mitochondrial from S. cerevisiae
MIP-1, and eukaryotic C. elegans, and D. melanogaster (Astatke, M.
et al., 1998, J. Mol. Biol. 278, 147-165), pol III type DNA
polymerase isolated from any sources, and mutants, derivatives or
variants thereof, and the like. Preferred thermostable DNA
polymerases that may be used in the methods and compositions of the
invention include Taq, Tne, Tma, Pfu, KOD, Tfl, Tth, Stoffel
fragment, VENT.TM. and DEEPVENT.TM. DNA polymerases, and mutants,
variants and derivatives thereof (U.S. Pat. No. 5,436,149; U.S.
Pat. No. 4,889,818; U.S. Pat. No. 4,965,188; U.S. Pat. No.
5,079,352; U.S. Pat. No. 5,614,365; U.S. Pat. No. 5,374,553; U.S.
Pat. No. 5,270,179; U.S. Pat. No. 5,047,342; U.S. Pat. No.
5,512,462; WO 92/06188; WO 92/06200; WO 96/10640; WO 97/09451;
Barnes, W. M., Gene 112:29-35 (1992); Lawyer, F. C., et al., PCR
Meth. Appl. 2:275-287 (1993); Flaman, J.-M, et al., Nucl. Acids
Res. 22(15):3259-3260 (1994)).
[0458] Reverse transcriptases for use in this invention include any
enzyme having reverse transcriptase activity. Such enzymes include,
but are not limited to, retroviral reverse transcriptase,
retrotransposon reverse transcriptase, hepatitis B reverse
transcriptase, cauliflower mosaic virus reverse transcriptase,
bacterial reverse transcriptase, Tth DNA polymerase, Taq DNA
polymerase (Saiki, R. K., et al., Science 239:487-491 (1988); U.S.
Pat. Nos. 4,889,818 and 4,965,188), Tne DNA polymerase (WO 96/10640
and WO 97/09451), Tma DNA polymerase (U.S. Pat. No. 5,374,553) and
mutants, variants or derivatives thereof (see, e.g., WO 97/09451
and WO 98/47912). Preferred enzymes for use in the invention
include those that have reduced, substantially reduced or
eliminated RNase H activity. By an enzyme "substantially reduced in
RNase H activity" is meant that the enzyme has less than about 20%,
more preferably less than about 15%, 10% or 5%, and most preferably
less than about 2%, of the RNase H activity of the corresponding
wildtype or RNase H.sup.+ enzyme such as wildtype Moloney Murine
Leukemia Virus (M-MLV), Avian Myeloblastosis Virus (AMV) or Rous
Sarcoma Virus (RSV) reverse transcriptases. The RNase H activity of
any enzyme may be determined by a variety of assays, such as those
described, for example, in U.S. Pat. No. 5,244,797, in Kotewicz, M.
L., et al., Nucl. Acids Res. 16:265 (1988) and in Gerard, G. F., et
al., FOCUS 14(5):91 (1992), the disclosures of all of which are
fully incorporated herein by reference. Particularly preferred
polypeptides for use in the invention include, but are not limited
to, M-MLV H.sup.- reverse transcriptase, RSV H.sup.- reverse
transcriptase, AMV H.sup.- reverse transcriptase, RAV
(rous-associated virus) H.sup.- reverse transcriptase, MAV
(myeloblastosis-associated virus) H.sup.- reverse transcriptase and
HIV H.sup.- reverse transcriptase. (See U.S. Pat. No. 5,244,797 and
WO 98/47912). It will be understood by one of ordinary skill,
however, that any enzyme capable of producing a DNA molecule from a
ribonucleic acid molecule (i.e., having reverse transcriptase
activity) may be equivalently used in the compositions, methods and
kits of the invention.
[0459] The enzymes having polymerase activity for use in the
invention may be obtained commercially, for example from Invitrogen
Corporation (Carlsbad, Calif.), Perkin-Elmer (Branchburg, N.J.),
New England BioLabs (Beverly, Mass.) or Boehringer Mannheim
Biochemicals (Indianapolis, Ind.). Enzymes having reverse
transcriptase activity for use in the invention may be obtained
commercially, for example from Invitrogen Corporation (Carlsbad,
Calif.), Pharmacia (Piscataway, N.J.), Sigma (Saint Louis, Mo.) or
Boehringer Mannheim Biochemicals (Indianapolis, Ind.).
Alternatively, polymerases or reverse transcriptases having
polymerase activity may be isolated from their natural viral or
bacterial sources according to standard procedures for isolating
and purifying natural proteins that are well-known to one of
ordinary skill in the art (see, e.g., Houts, G. E., et al., J.
Virol. 29:517 (1979)). In addition, such polymerases/reverse
transcriptases may be prepared by recombinant DNA techniques that
are familiar to one of ordinary skill in the art (see, e.g.,
Kotewicz, M. L., et al., Nucl. Acids Res. 16:265 (1988); U.S. Pat.
No. 5,244,797; WO 98/47912; Soltis, D. A., and Skalka, A. M., Proc.
Natl. Acad. Sci. USA 85:3372-3376 (1988)). Examples of enzymes
having polymerase activity and reverse transcriptase activity may
include any of those described in the present application.
Methods of Nucleic Acid Synthesis, Amplification and Sequencing
[0460] The present invention may be used in combination with any
method involving the synthesis of nucleic acid molecules, such as
DNA (including cDNA) and RNA molecules. Such methods include, but
are not limited to, nucleic acid synthesis methods, nucleic acid
amplification methods and nucleic acid sequencing methods. Such
methods may be used to prepare molecules (e.g., starting molecules)
used in the invention or to further manipulate molecules or vectors
produced by the invention.
[0461] Nucleic acid synthesis methods according to this aspect of
the invention may comprise one or more steps. For example, the
invention provides a method for synthesizing a nucleic acid
molecule comprising (a) mixing a nucleic acid template (e.g., a
nucleic acid molecules or vectors of the invention) with one or
more primers and one or more enzymes having polymerase or reverse
transcriptase activity to form a mixture; and (b) incubating the
mixture under conditions sufficient to make a first nucleic acid
molecule complementary to all or a portion of the template.
According to this aspect of the invention, the nucleic acid
template may be a DNA molecule such as a cDNA molecule or library,
or an RNA molecule such as a mRNA molecule. Conditions sufficient
to allow synthesis such as pH, temperature, ionic strength, and
incubation times may be optimized by those skilled in the art. If
desired, recombination sites and/or topoisomerase recognition sites
may be added to such synthesized molecules during or after the
synthesis process (see for sample, U.S. patent application Ser. No.
09/177,387 filed Oct. 23, 1998 based on U.S. provisional patent
application No. 60/065,930 filed Oct. 24, 1997).
[0462] In accordance with the invention, the target or template
nucleic acid molecules or libraries may be prepared from nucleic
acid molecules obtained from natural sources, such as a variety of
cells, tissues, organs or organisms. Cells that may be used as
sources of nucleic acid molecules may be prokaryotic (bacterial
cells, including those of species of the genera Escherichia,
Bacillus, Serratia, Salmonella, Staphylococcus, Streptococcus,
Clostridium, Chlamydia, Neisseria, Treponema, Mycoplasma, Borrelia,
Legionella, Pseudomonas, Mycobacterium, Helicobacter, Erwinia,
Agrobacterium, Rhizobium, and Streptomyces) or eukaryotic
(including fungi (especially yeast's), plants, protozoans and other
parasites, and animals including insects (particularly Drosophila
spp. cells), nematodes (particularly Caenorhabditis elegans cells),
and mammals (particularly hurrian cells)).
[0463] Of course, other techniques of nucleic acid synthesis which
may be advantageously used will be readily apparent to one of
ordinary skill in the art.
[0464] In other aspects of the invention, the invention may be used
in combination with methods for amplifying or sequencing nucleic
acid molecules. Nucleic acid amplification methods according to
this aspect of the invention may include the use of one or more
polypeptides having reverse transcriptase activity, in methods
generally known in the art as one-step (e.g., one-step RT-PCR) or
two-step (e.g., two-step RT-PCR) reverse
transcriptase-amplification reactions. For amplification of long
nucleic acid molecules (i.e., greater than about 3-5 Kb in length),
a combination of DNA polymerases may be used, as described in WO
98/06736 and WO 95/16028.
[0465] Amplification methods according to the invention may
comprise one or more steps. For example, the invention provides a
method for amplifying a nucleic acid molecule comprising (a) mixing
one or more enzymes with polymerase activity with one or more
nucleic acid templates; and (b) incubating the mixture under
conditions sufficient to allow the enzyme with polymerase activity
to amplify one or more nucleic acid molecules complementary to all
or a portion of the templates. The invention also provides nucleic
acid molecules amplified by such methods. If desired, recombination
sites may be added to such amplified molecules during or after the
amplification process (see for example, U.S. patent application
Ser. No. 09/177,387 filed Oct. 23, 1998, based on U.S. provisional
patent application No. 60/065,930 filed Oct. 24, 1997, the
disclosures of which are incorporated herein by reference in their
entireties).
[0466] General methods for amplification and analysis of nucleic
acid molecules or fragments are well known to one of ordinary skill
in the art (see, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; and
4,800,159; Innis, M. A., et al., eds., PCR Protocols: A Guide to
Methods and Applications, San Diego, Calif.: Academic Press, Inc.
(1990); Griffin, H. G., and Griffin, A. M., eds., PCR Technology:
Current Innovations, Boca Raton, Fla.: CRC Press (1994)). For
example, amplification methods which may be used in accordance with
the present invention include PCR (U.S. Pat. Nos. 4,683,195 and
4,683,202), Strand Displacement Amplification (SDA; U.S. Pat. No.
5,455,166; EP 0 684 315), and Nucleic Acid Sequence-Based
Amplification (NASBA; U.S. Pat. No. 5,409,818; EP 0 329 822).
[0467] Typically, these amplification methods comprise: (a) mixing
one or more enzymes with polymerase activity with the nucleic acid
sample in the presence of one or more primer sequences, and (b)
amplifying the nucleic acid sample to generate a collection of
amplified nucleic acid fragments, preferably by PCR or equivalent
automated amplification technique.
[0468] Following amplification or synthesis by the methods of the
present invention, the amplified or synthesized nucleic acid
fragments may be isolated for further use or characterization. This
step is usually accomplished by separation of the amplified or
synthesized nucleic acid fragments by size or by any physical or
biochemical means including gel electrophoresis, capillary
electrophoresis, chromatography (including sizing, affinity and
immunochromatography), density gradient centrifugation and
immunoadsorption. Separation of nucleic acid fragments by gel
electrophoresis is particularly preferred, as it provides a rapid
and highly reproducible means of sensitive separation of a
multitude of nucleic acid fragments, and permits direct,
simultaneous comparison of the fragments in several samples of
nucleic acids. One can extend this approach, in another preferred
embodiment, to isolate and characterize these fragments or any
nucleic acid fragment amplified or synthesized by the methods of
the invention. Thus, the invention is also directed to isolated
nucleic acid molecules produced by the amplification or synthesis
methods of the invention.
[0469] In this embodiment, one or more of the amplified or
synthesized nucleic acid fragments are removed from the gel which
was used for identification (see above), according to standard
techniques such as electroelution or physical excision. The
isolated unique nucleic acid fragments may then be inserted into
standard vectors, including expression vectors, suitable for
transfection or transformation of a variety of prokaryotic
(bacterial) or eukaryotic (yeast, plant or animal including human
and other mammalian) cells. Alternatively, nucleic acid molecules
produced by the methods of the invention may be further
characterized, for example by sequencing (i.e., determining the
nucleotide sequence of the nucleic acid fragments), by methods
described below and others that are standard in the art (see, e.g.,
U.S. Pat. Nos. 4,962,022 and 5,498,523, which are directed to
methods of DNA sequencing).
[0470] Nucleic acid sequencing methods according to the invention
may comprise one or more steps. For example, the invention may be
combined with a method for sequencing a nucleic acid molecule
comprising (a) mixing an enzyme with polymerase activity with a
nucleic acid molecule to be sequenced, one or more primers, one or
more nucleotides, and one or more terminating agents (such as a
dideoxynucleotides) to form a mixture; (b) incubating the mixture
under conditions sufficient to synthesize a population of molecules
complementary to all or a portion of the molecule to be sequenced;
and (c) separating the population to determine the nucleotide
sequence of all or a portion of the molecule to be sequenced.
[0471] Nucleic acid sequencing techniques which may be employed
include dideoxy sequencing methods such as those disclosed in U.S.
Pat. Nos. 4,962,022 and 5,498,523.
Kits
[0472] In another aspect, the invention provides kits which may be
used in conjunction with the invention. Kits of the invention may
contain any number of components but typically will contain at
least two components. Kits according to this aspect of the
invention may comprise one or more containers, which may contain
one or more components selected from the group consisting of one or
more nucleic acid molecules or vectors of the invention, one or
more primers, the molecules and/or compounds of the invention,
supports of the invention, one or more polymerases, one or more
reverse transcriptases, one or more recombination proteins (or
other enzymes for carrying out the methods of the invention), one
or more topoisomerases, one or more buffers, one or more
detergents, one or more restriction endonucleases, one or more
nucleotides, one or more terminating agents (e.g., ddNTPs), one or
more transfection reagents, pyrophosphatase, and the like. The kits
of the invention may also comprise instructions for carrying out
methods of the invention.
[0473] For example, a kit of the invention may comprise (1) a first
nucleic acid molecule which comprises one or more (e.g., 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, etc.) recombination sites and/or one or more
(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) toposiomerase
recognition sites and (2) instructions for covalently linking the
first nucleic molecule to another nucleic acid molecule using
methods described herein. In particular embodiments, the
instructions describe methods for linking two or more nucleic
molecules in either one or both strands. In a related embodiment,
the first nucleic acid molecule is topoisomerase adapted prior to
inclusion in the kit.
[0474] Additional kits of the invention can contain, for example,
one or more topoisomerase-charged nucleic acid molecule substrates,
which can include one or more control nucleic acid sequences which
can be useful, for example, to test the accuracy or fidelity of the
components of the kit; one or more topoisomerases; one or more
compositions comprising one or more topoisomerases; one or more
recombinases (or recombination proteins); one or more compositions
comprising one or more recombinases (or recombination proteins);
one or more primers, which can comprise at least one topoisomerase
recognition site and/or at least one recombination site, a
nucleotide sequence complementary to at least one topoisomerase
recognition site and/or at least one recombination site, or both at
least one topoisomerase recognition site and at least one
nucleotide sequence complementary to at least one topoisomerase
recognition site; one or more cells, which can contain or be useful
for containing a nucleic acid molecule of the kit or generated
using the kit; one or more reagents, polymers, buffers, or the
like, for performing a method using the kit; instructions for
performing a method using the kit; and the like.
[0475] In another aspect, a kit of the invention may contain a
nucleic acid molecule having a first end and a second end, and
encoding a polypeptide to be expressed, for example, a selectable
marker, wherein the nucleic acid molecule comprises a topoisomerase
recognition site or cleavage product thereof at the 3' terminus of
one or both ends. Optionally, the nucleic acid molecule contains a
hydroxyl group at the 5' terminus of one or both of the other ends,
i.e., at the ends that do not contain a topoisomerase recognition
site or that are not topoisomerase-charged. Further, one or both 5'
termini may comprise overhanging sequences, which are different
from each other. A kit of the invention also can contain a nucleic
acid molecule having a first end and a second end, and encoding a
polypeptide to be expressed, for example, a selectable marker,
wherein the nucleic acid molecule comprises a topoisomerase
recognition site or cleavage product thereof at the 5' terminus of
one or both ends. Optionally, the nucleic acid molecule contains a
hydroxyl group at the 3' terminus of one or both ends, and
preferably, one or both 3' termini comprise overhanging sequences,
which are different from each other. In addition, a kit of the
invention can contain a nucleic acid molecule having a first end
and a second end, and encoding a polypeptide to be expressed, for
example, a selectable marker, wherein the nucleic acid molecule
comprises a topoisomerase recognition site or cleavage product
thereof at the 5' terminus and the 3' terminus of one or both ends.
As such, it should be recognized that a kit of the invention can
include any of various combinations of such nucleic acid molecules
comprising one or more topoisomerase recognition sites or
topoisomerase-charged nucleic acid molecules.
[0476] A kit of the invention also can contain a nucleic acid
molecule comprising a regulatory element or other nucleotide
sequence, for example, a coding sequence, and a topoisomerase
recognition site and/or a recombination site, or cleavage product
thereof, at a 3' terminus of at least a first end and, optionally,
a hydroxyl group at the 5' terminus of an end containing the
recognition site; or comprising a topoisomerase recognition site or
cleavage product thereof at a 5' terminus of at least a first end,
and, optionally, a hydroxyl group at the 3' terminus of the end
containing the recognition site; or comprising a topoisomerase
recognition site at the 5' terminus and 3' terminus of at least a
first end. In certain embodiments, the kit may contain a variety of
upstream regulatory elements, a variety of downstream regulatory
elements, a variety of elements useful detecting or identifying a
molecule containing the element, and combinations thereof. For
example, the kit can contain a variety of gene promoter elements,
which are active constitutively or inducibly and in a few or many
different types of cells, elements that permit ribosome binding
such as an internal ribosome entry site, an element encoding a
Kozak sequence or an initiator methionine, or the like. In
addition, or alternatively, the kit can contain a variety of
downstream regulatory elements such a polyadenylation signal
sequences, sequences that terminate transcription or translation,
or the like. Similarly, the kit can contain elements encoding
detectable markers such as epitope tags, or the like. In certain
such aspects of the invention, the kit contains a variety of such
elements, each of which contains at least one topoisomerase
recognition site and/or at least one recombination site. In certain
other such aspects, these elements may contain an overhanging
sequence such that they can be operably covalently linked to each
other or to a nucleic acid molecule encoding a polypeptide such as
a selectable marker according to a method of the invention.
[0477] Optionally, the kit contains element specific primers, which
can amplify a construct containing one of the variety of elements
included in the kit. Where the kit contains such primers, the
nucleic acid molecules comprising the regulatory or other element
has a nucleotide sequence that can be specifically recognized by
the primer and that results in extension of the primer through and
including the regulatory element. In particular, the kit can
contain element specific forward and reverse primers, which can be
combined to produce a primer pair that amplifies, for example, a
construct containing a particular 5' regulatory element and a
particular 3' regulatory element of the kit. Such a primer pair can
selectively amplify a desired functional covalently linked ds
nucleic acid molecule generated according to a method of the
invention, but does not amplify partial reaction products.
[0478] In another embodiment, a kit of the invention contains a
first nucleic acid molecule, which has a first end and a second
end, contains a topoisomerase recognition site, or cleavage product
thereof, and/or a recombination site, at or near one or both 3'
termini, and encodes a transcription activation domain; and a
second nucleic acid molecule, which has a first end and a second
end, contains a topoisomerase recognition site, or cleavage product
thereof, at or near one or both 3' termini, and encodes a DNA
binding domain; or contains a first nucleic acid molecule, which
has a first end and a second end, contains a topoisomerase
recognition site, or cleavage product thereof, and/or a
recombination site, at or near one or both 5' termini, and encodes
a transcription activation domain; and a second nucleic acid
molecule, which has a first end and a second end, contains a
topoisomerase recognition site, or cleavage product thereof, and/or
a recombination site, at or near one or both 5' termini, and
encodes a DNA binding domain. A kit of the invention also can
contain a first nucleic acid molecule, which has a first end and a
second end, and encodes a transcription activation domain, and a
second nucleic acid molecule, which has a first end and a second
end, and encodes a DNA binding domain, wherein at least the first
nucleic acid molecule- or the second nucleic acid molecule contains
a topoisomerase recognition site, or cleavage product thereof, at
or near a 5' terminus and at or near 3' terminus of at least one
end, and wherein the other ds nucleotide contains a 3' hydroxyl and
5' hydroxyl at the end to be covalently linked to the end of the
nucleic acid molecule comprising the recognition sites. Such a kit
is useful, for example, for generating covalently linked ds
recombinant nucleic acid molecules encoding chimeric polypeptides
for performing a two hybrid assay. The kit can further contain a
primer pair, which can amplify a nucleotide sequence to be operably
linked to the first or second nucleic acid molecule, wherein at
least one primer of the primer pair comprises a topoisomerase
recognition site, a complement of a topoisomerase recognition site,
or both. Preferably, an amplification product generated using such
a primer pair contains, following cleavage by a site-specific
topoisomerase, a 3' or 5' overhanging sequence that is
complementary to the first or second nucleic acid molecule to which
it is to be covalently linked. Such a kit can facilitate the
generation of recombinant polynucleotides that comprise a first or
second nucleotide sequence of the kit and encode a chimeric
polypeptide useful for performing a two hybrid assay.
[0479] The present invention also relates to additional kits for
carrying out the methods of the invention, and particularly for use
in creating the product nucleic acid molecules of the invention.
The invention also relates to kits for carrying out homologous
recombination (particularly gene targeting) according to the
methods of the invention. Such kits of the invention may also
comprise further components for further manipulating the
recombination site-containing molecules and/or compounds produced
by the methods of the invention. The kits of the invention may
comprise one or more nucleic acid molecules of the invention
(particularly starting molecules comprising one or more
recombination sites and optionally comprising one or more reactive
functional moieties), one or more molecules and/or compounds of the
invention, one or more supports of the invention and/or one or more
vectors of the invention. Such kits may optionally comprise one or
more additional components selected from the group consisting of
one or more host cells or derivatives thereof, one or more
nucleotides, one or more polymerases and/or reverse transcriptases,
one or more suitable buffers, one or more primers, one or more
terminating agents, one or more populations of molecules for
creating combinatorial libraries and one or more combinatorial
libraries.
[0480] In another embodiment, a kit of the invention contains a
first nucleic acid molecule, which encodes a polypeptide,
particularly a selectable marker, and contains a topoisomerase
recognition site at each end. In certain preferred such
embodiments, the first nucleic acid molecule is a circular molecule
(for example, a plasmid, vector, etc.) and comprises at least one
recombination site, and more preferably at least two recombination
sites, flanking the one or more, preferably two or more,
topoisomerase recognition sites on the molecule. Preferably, the
first nucleotide sequence comprises a topoisomerase-activated
nucleotide sequence. More preferably, the topoisomerase-charged
first nucleotide sequence comprises a 5' overhanging sequence at
each end, and most preferably the 5' overhanging sequences are
different from each other. Optionally, each of the 5' termini
comprises a 5' hydroxyl group.
[0481] Kits according to this aspect of the invention may also
contain at least a nucleotide sequence comprising a regulatory
element, which can be an upstream or downstream regulatory element,
or other element, which contains one or more topoisomerase
recognition sites and, optionally, contains one or more
recombination sites at one or both ends. Preferably, the kit
contains a plurality of nucleic acid molecules, each comprising a
different regulatory element or other element, for example, a
sequence encoding a tag or other detectable molecule or a cell
compartmentalization domain. The different elements can be
different types of a particular regulatory element, for example,
constitutive or inducible promoters or tissue specific promoters,
or can be different types of elements including, for example,
transcriptional and translational regulatory elements, epitope
tags, and the like. Such nucleic acid molecules can be
topoisomerase-activated, and can contain 5' overhanging sequences
that facilitate operably covalently linking the elements in a
predetermined orientation, particularly such that a polypeptide
such as a selectable marker is expressible in vitro or in one or
more cell types.
[0482] Such kits also may contain primers, including first and
second primers, such that a primer pair comprising a first and
second primer can be selected and used to amplify a desired
covalently linked ds recombinant nucleic acid molecule generated
using components of the kit. For example, the primers can include
first primers that are complementary to elements that generally are
positioned at the 5' end of a generated ds recombinant nucleic acid
molecule, for example, a portion of a nucleic acid molecule
comprising a promoter element, and second primers that are
complementary to elements that generally are positioned at the 3'
end of a generated ds recombinant nucleic acid molecule, for
example, a portion of a nucleic acid molecule comprising a
transcription termination site or encoding an epitope tag.
Depending on the elements selected from the kit for generating a
covalently linked ds recombinant nucleic acid molecule, the
appropriate first and second primers can be selected and used to
amplify a full length functional construct.
[0483] In another embodiment, a kit of the invention contains a
plurality of different elements, each of which can be
topoisomerase-activated at one or both ends, and each of which can
contain a 5' overhanging sequence. The 5' overhanging sequences can
be unique to a particular element, or can be common to plurality of
related elements, for example, to a plurality of different promoter
element. Preferably, the 5' overhanging sequences of elements are
designed such that one or more elements can be operably covalently
linked to provide a useful function, for example, an element
comprising a Kozak sequence and an element comprising a translation
start site can have complementary 5' overhangs such that the
elements can be operably covalently linked according to a method of
the invention.
[0484] The plurality of elements in the kit can comprise any
elements, including transcription or translation regulatory
elements; elements required for replication of a nucleotide
sequence in a bacterial, insect, yeast, or mammalian host cell;
elements comprising recognition sequences for site specific nucleic
acid binding proteins such as restriction endonucleases or
recombinases; elements encoding expressible products such as
epitope tags or drug resistance genes; and the like. As such, a kit
of the invention provides a convenient source of different elements
that can be selected depending, for example, on the particular
cells that a construct generated according to a method of the
invention is to be introduced into or expressed in. The kit also
can contain PCR primers, including first and second primers, which
can be combined as described above to amplify a covalently linked
ds recombinant nucleic acid molecule generated using the elements
of the kit. Optionally, the kit further contains one or more
topoisomerases (e.g., one or more site-specific topoisomerases)
and/or one or more recombinases (or recombination proteins) in an
amount useful for covalently linking a first nucleic acid molecule
comprising a topoisomerase recognition site to a second (or other)
nucleic acid molecule, which can be topoisomerase-activated nucleic
acid molecules or can be nucleotide sequences that comprise a
topoisomerase recognition site.
[0485] In still another embodiment, a kit of the invention contains
a first nucleic acid molecule, which encodes a selectable marker,
and contains a topoisomerase recognition site at each end; a first
and second PCR primer pair, which can produce a first and second
amplification products that can be covalently linked to the first
nucleic acid molecule in a predetermined orientation according to a
method of the invention. Such a generated construct can be
introduced into a cell and can incorporate into the genome of the
cell by homologous recombination in a site specific manner, where
it can be stably maintained and can express a heterologous
polypeptide in the cell or can knock-out a target gene function. A
target gene to be knocked-out, for example, can be any gene for
which at least part of the sequence is known or can be readily
determined and the function of which it is desired to disrupt, for
example, an oncogene, a gene involved in apoptosis, a gene encoding
a serine/threonine or a tyrosine kinase, or any other gene.
[0486] The first PCR primer pair in a kit of the invention includes
a first primer that comprises, in an orientation from 5' to 3', a
nucleotide sequence complementary to a 5' overhanging sequence of a
nucleic acid molecule to which it is to be covalently linked (for
example, an end of the nucleic acid molecule encoding the
selectable marker), a nucleotide sequence complementary to a
topoisomerase recognition site, such that PCR introduces a
functional recognition site in the opposite strand (see primer
sequences in FIG. 9D), and/or to a recombination site, and a
nucleotide sequence complementary to a 3' sequence of the target
DNA sequence. The first PCR primer pair also includes a second
primer that comprises a nucleotide sequence of the target DNA
sequence upstream of the 3' sequence to which the first primer is
complementary.
[0487] The second PCR primer pair of a kit of the invention
includes a first primer that comprises, from 5' to 3', a nucleotide
sequence complementary to a 5' overhanging sequence of a nucleic
acid molecule to which it is to be covalently linked, a nucleotide
sequence complementary to a topoisomerase recognition site, such
that PCR introduces a functional recognition site in the opposite
strand (see primer sequences in FIG. 9D), and optionally, a
nucleotide sequence complementary to a recombination site, and a
nucleotide sequence of a 5' sequence of the target DNA sequence,
wherein the 5' sequence of the target gene is downstream of the 3'
sequence of the target DNA sequence to which the first primer of
the first primer pair is complementary. The second PCR primer pair
also includes a second primer that comprises a nucleotide sequence
complementary to a 3' sequence of the target gene that is
downstream of the 5' sequence of the target DNA sequence contained
in the first primer.
[0488] In another embodiment, a kit of the invention contains a
first nucleic acid molecule, which encodes a transcription
activation domain and comprises a topoisomerase recognition site,
or cleavage product thereof, at or near a 3' terminus; and a second
nucleic acid molecule, which encodes a DNA binding domain and
comprises a topoisomerase recognition site and optionally a
recombination site, or cleavage product thereof, at or near a 3'
terminus. Upon cleavage by the site specific topoisomerase, the
first or second nucleic acid molecule can have a 5' overhang, or
both sequences can have 5' overhangs, which are the same or are
different from each other. Where the nucleic acid molecules have a
5' overhang, the overhang generally is complementary to a nucleic
acid molecule to which first or second nucleic acid molecule is to
be covalently linked according to a method of the invention.
[0489] The kit also can contain one or a pair of adapters, linkers
or the like, which comprise a topoisomerase recognition site and,
optionally, a recombination site, or cleavage product thereof, at
one or both 3' termini, and, optionally, a hydroxyl group at the
same terminus/termini. Such adapters, linkers, or the like are
selected such that they contain a 5' overhang that is complementary
to one or the other of the two nucleic acid molecules described
above and part of the kit. Similarly, the kit also can contain one
or a pair of adapters, linkers or the like, which comprise a
topoisomerase recognition site and, optionally, a recombination
site, or cleavage product thereof, at one or both 5' termini, and,
optionally, a hydroxyl group at the same terminus/termini. Such
adapters, linkers, or the like are selected such that they contain
a 3' overhang that is complementary to one or the other of the two
nucleic acid molecules described above and part of the kit. In
addition, the kit can contain one or a pair of adapters, linkers or
the like, which comprise a topoisomerase recognition site, or
cleavage product thereof, at or near one or both 5' and/or 3'
termini, and, optionally, a hydroxyl group at the same
terminus/termini. Such adapters, linkers, or the like are selected
such that they contain a 5' and/or a 3' overhang that is
complementary to one or the other of the two nucleic acid molecules
described above and part of the kit. Such adapters, linkers, or the
like can be joined to the ends of nucleic acid molecules that are
to covalently linked to one or the other of the first or second
nucleic acid molecules provided with the kit, thus facilitating the
construction of chimeric polynucleotides encoding the bait and prey
polypeptides useful in a two hybrid assay. Such a kit also can
contain a PCR primer or primer pair, which can be used to prepare
an amplified plurality of nucleotide sequences comprising a
topoisomerase recognition site, or cleavage product thereof.
Additional kits according to this aspect of the invention may
optionally comprise one or more additional components such as one
or more topoisomerases, one or more recombination proteins, one or
more vectors, one or more polypeptides having polymerase activity,
and one or more host cells.
[0490] It will be understood by one of ordinary skill in the
relevant arts that other suitable modifications and adaptations to
the methods and applications described herein are readily apparent
from the description of the invention contained herein in view of
information known to the ordinarily skilled artisan, and may be
made without departing from the scope of the invention or any
embodiment thereof. Having now described the present invention in
detail, the same will be more clearly understood by reference to
the following examples, which are included herewith for purposes of
illustration only and are not intended to be limiting of the
invention.
EXAMPLES
Example 1
Construction of Covalently Linked Double Stranded Recombinant
Nucleic Acid Molecules Using Topoisomerase
[0491] This example demonstrates that topoisomerase can be used to
produce covalently linked double stranded (ds) recombinant nucleic
acid molecules.
A. Methods
[0492] Except where indicated, studies were performed using the
following methods. PCR was performed in 50 .mu.l reactions,
including 10 ng plasmid (template), 100 ng each primer, 2.5 Units
Taq DNA polymerase (Sigma), 5 .mu.l 10.times.PCR buffer, and 4
.mu.l of dNTPs (200 .mu.M each). An initial denaturation was
performed by incubating the reaction at 94.degree. C. for 4 min;
followed by 30 cycles of PCR using 94.degree. C. (45 sec) for
denaturation, 55.degree. C. (45 sec) for primer annealing and
72.degree. C. (1 min per kb of target sequence) for extension.
After cycling, the reactions were incubated at 72.degree. C. (10
min), and then placed at 4.degree. C.
[0493] Topoisomerase joining reactions were performed in 5 .mu.l,
including 50-100 ng each amplified element (PCR-generated or
synthetic), 0.5 .mu.l 500 mM Tris (pH 7.5), and 0.5 .mu.g
topoisomerase. Reactions were incubated at room temperature for 5
min, then 1-2 .mu.l of the Topo-linked product was used for linear
fragment generation.
[0494] Linear fragment generation by PCR was performed in 50 .mu.l
reactions, including 1-2 .mu.l of the Topo-linked product
(template), 100 ng each primer, 2.5 U Taq DNA polymerase (Sigma), 5
.mu.l 10.times.PCR buffer, and 4 .mu.l dNTPs (200 .mu.M each). PCR
was performed as described above.
[0495] The resultant linear fragment was purified using a SNAP
Miniprep Kit (Invitrogen Corporation, Carlsbad, Calif.) as
described by the manufacturer. Essentially, 100 .mu.l PCR product
was mixed with 300 .mu.l Binding Buffer; 750 .mu.l isopropanol, and
the mixture was applied to a SNAP Miniprep Column/Collection Tube
and centrifuged at 7,000 rpm for 30 sec. The column was washed with
700 .mu.l Wash Buffer, centrifuged at 7,000 rpm for 30 sec; then
washed with 900 .mu.l 1.times. Final Wash and centrifuged at 7,000
rpm for 30 sec. The column was then centrifuged at 7,000 rpm for an
additional 30 sec to remove all remaining liquid. Water (30 to 50
.mu.l) was added and the column was centrifuged at 7,000 rpm for 30
sec to elute the purified DNA. DNA concentration was determined by
spectrophotometry.
B. Generation of Topoisomerase Linked Linear Nucleic Acid
Molecules
[0496] PCR primers were designed to examine the directional
addition of elements to the coding sequence of green fluorescent
protein (GFP; see FIG. 9A-C). The CMV promoter (approximately 700
bp) and BGH polyadenylation signal sequence (approximately 380 bp)
were amplified from a pCMV/myc/nuc plasmid template, and the GFP
element (approximately 700 bp) was amplified from a pcDNA3.1/GFP
plasmid template (Invitrogen Corporation, Carlsbad, Calif.) using
the primers indicated in FIG. 9D. The resultant amplification
products were joined using topoisomerase as described above, and a
portion of the ligation reaction was used as template for PCR with
primers F6945 (SEQ ID NO: 11) and F6948 (SEQ ID NO: 15) to amplify
the entire construct (CMV+GFP+BGH; approximately 1,700 bp). In
addition, 5 .mu.l of the ligation mixture was treated with
proteinase K for 30 min at 37.degree. C. to remove any bound
topoisomerase, and then subjected to electrophoresis on a 3-8%
NuPAGE Tris-acetate gel to examine the ligated products.
[0497] Only a small amount of ligation product of the correct size
(1.7 kb) was observed when the recombinant nucleic acid molecules
were generated using elements having palindromic overhanging
sequence (FIG. 9A or 9B), whereas significant quantities of the
desired product were generated using elements having
non-palindromic overhangs (FIG. 9C). These results demonstrate that
the efficiency of generating ds recombinant nucleic acid molecule
covalently linked in both strands containing nucleotide sequences
operatively linked in a predetermined orientation is related to the
nature of the overhang sequence. In particular, the selection of
overhanging sequences that lack palindromic regions result in the
efficient generation of a desired ds recombinant nucleic acid
molecule covalently linked in both strands, whereas the presence of
palindromic sequences in the overhangs allows the formation of
ligation products other than the intended product, thus decreasing
the efficiency of generating a desired product.
Example 2
Functional Characterization of Topoisomerase-generated ds
Recombinant Nucleic Acid Molecules
[0498] This example demonstrates that a method of the invention
provides a means to generate functional ds recombinant nucleic acid
molecules covalently linked in both strands.
A. Expression of Sense and Antisense mRNA from a Topo-Ligated
Construct
[0499] The ability to create a ds recombinant nucleic acid molecule
containing functional upstream and downstream elements flanking a
gene of interest was examined using two synthetic elements
containing either a T7 or a T3 promoter sequence. The elements were
made by annealing pairs of synthetic oligonucleotides. The T7
linker was generated by mixing equal molar amounts of T7top (F9304;
SEQ ID NO: 20) and T7bottom (F9305; SEQ ID NO: 21) oligonucleotides
(FIG. 9D). The T3 linker was generated by mixing equal molar
amounts of T3top (F9661; SEQ ID NO: 23) and T3bottom (F9662; SEQ ID
NO: 24) oligonucleotides (FIG. 9D). The mixtures were heated in
boiling water for 5 min, then allowed to cool to room temperature.
Both elements were designed to contain a topoisomerase recognition
site at one end.
[0500] The GFP gene was amplified with GFP primers F8418 (SEQ ID
NO: 17) and F8420 (SEQ ID NO: 18, FIG. 9D; see, also, FIG. 9C).
Unpurified GFP PCR product (2 .mu.l) was mixed with 50 ng of T7
linker and 50 ng of T3 linker, topoisomerase was added, and the
topo-joining reaction was allowed to proceed at room temperature
for 5 min. Two .mu.l of the joining reaction was used as template
for a 50 .mu.l PCR reaction with primers for the T7 and T3
sequences.
[0501] After amplification, a 4 .mu.l aliquot of the PCR reaction
was used as template for in vitro transcription. The reaction was
performed using a Promega RiboProbe In Vitro Transcription Systems
kit according to the manufacturer's instruction. The reaction was
allowed to proceed for 60 min at 37.degree. C. with T7 or T3 RNA
polymerase (final volume, 20 .mu.l). Aliquots of the in vitro
transcription reactions were digested with RNase or DNase, then
undigested and digested samples were subjected to electrophoresis
in a 2% TBE gel. A predominant band of the predicted size (either
sense or antisense orientation) was observed in the undigested
samples. No decrease in the product band was noted in samples
treated with DNase. The product bands disappeared when samples were
treated with RNase indicating the product was RNA. These results
demonstrate that topoisomerase can be used according to a method of
the invention to generate a ds recombinant nucleic acid molecule
covalently linked in both strands in a predetermined orientation,
and that an RNA transcript can be expressed from such a nucleic
acid molecule.
B. Expression of a Translation Product from a Topo-Ligated
Construct
[0502] The ability of topoisomerase ligated polynucleotide to
support coupled in vitro transcription/translation was examined. A
ds recombinant nucleic acid molecule was generated according to a
method of the invention by linking an element containing a T7
promoter (plus a Kozak sequence) to lacZ PCR products of 1 kb, 2
kb, or 3 kb. Two 2 .mu.l of the generated products were used as
template for PCR amplification reactions (primers, SEQ ID NOS:
25-28; FIG. 9D). Unpurified aliquots of the amplification reactions
(3 .mu.l) were used as templates for coupled
transcription/translation with a TNT T7 Quick for PCR DNA Kit
according to the manufacturer's instructions (Promega).
[0503] Two .mu.l aliquots from each reaction were separated by
electrophoresis on a Tris-glycine gel (Novex), then visualized by
autoradiography, which revealed protein products that migrated at
the expected sizes. These results demonstrate that a method of the
invention can be used to produce a ds recombinant nucleic acid
molecule covalently linked in both strands useful as a template for
expressing a polypeptide by a coupled in vitro
transcription/translation reaction.
C. Generation of Topo-Ligated Constructs for Performing a Two
Hybrid Assay
[0504] Two hybrid assays provide a powerful method for detecting
protein-protein interactions in vivo. These assays are based on the
fact that many eukaryotic transcriptional activators consist of two
physically and functionally separable domains, including a DNA
binding domain, which binds to a specific DNA sequence, and a
transcriptional activation domain, which interacts with the basal
transcriptional machinery. The association of a transactivation
domain with a DNA binding domain can promote the assembly of a
functional RNA polymerase II complex, thereby allowing
transcriptional activation, for example, of a detectable reporter
gene (Field and Song, Nature 340:245-246, 1989). Where a first
protein, X, is fused to a DNA binding domain, for example, a GAL4
binding domain, and a second protein, Y, which can be the same or
different from X, is fused into a transactivation domain, for
example, a VP16 domain, an interaction of proteins X and Y can be
identified by detecting transcription of a reporter gene having a
GAL4 promoter.
[0505] The ability of a method of the invention to generate linear
constructs for expressing fusion proteins for performing a
mammalian two-hybrid assay was examined. PCR was used to generate
GAL4 (F10779 and F12667 primers; SEQ ID NOS: 1 and 3,
respectively), VP16 (F10779 and F12668 primers; SEQ ID NOS: 1 and
5, respectively), p53 (F12669 and F12505 primers; SEQ ID NOS: 8 and
4, respectively), T antigen (F12670 and F12505 primers; SEQ ID NOS:
9 and 4, respectively), and SV40pA (F12016 and F561 primers; SEQ ID
NOS: 6 and 7, respectively) elements containing topoisomerase sites
at the appropriate ends. Topoisomerase was used to create the
covalently linked, double stranded constructs GAL4+p53+SV40pA and
VP16+T antigen+SV40pA, and the resultant ligation products were
used as templates for PCR amplification.
[0506] Purified GAL4+p53+SV40pA and VP16+T antigen+SV40pA PCR
constructs were co-transfected with a lacZ reporter gene
(pGene/lacZ plasmid; Invitrogen Corporation, Carlsbad, Calif.) Into
CHO cells (6 well plate, 1.times.10.sup.5 cells/well). In parallel
studies, the use of plasmid vectors containing the expression
constructs was examined, as was the use of PCR reaction mixtures
containing the unpurified constructs. Control reactions were
performed using GAL4+pA and VP16+pA without inserts (negative
controls) or p53+VP16 (positive control). Cells were lysed 48 hr
after transfection and reporter gene activity was measured using a
beta-galactosidase assay kit.
[0507] A high level of reporter gene activity was detected with the
positive control (FIG. 10, sample 3) and in the sample
co-transfected with the reporter gene and the linear
GAI4+p53+SV40pA and VP16+T antigen+SV40pA constructs (FIG. 10,
sample 4). Low level activity (but greater than that of the
negative controls; samples 5, 6, 8 and 9) was detected when the
plasmid version of the constructs was used (FIG. 10, sample 1). Low
level activity was also observed in the sample co-transfected with
the unpurified, PCR-generated prey and bait constructs (sample 7).
These results demonstrate that a method of the invention can be
used to prepare constructs useful for performing a two hybrid
assay.
Example 3
Production and Use of Directionally Topo-Charged Gateway Vectors
Introduction
[0508] As a combination of Topoisomerase and GATEWAY.TM.
recombinational cloning technologies, directionally Topo-charged
Gateway vectors were developed. These tools facilitate easy entry
into the Gateway system by alleviating the necessity of adding attB
sites (25 base pairs) to either side of a PCR amplified ORF prior
to recombination into a Donor vector. Instead, a four base tag
recognition sequence (CACC) is added to the 5' end of the ORF and
PCR products are then directionally TOPO-cloned to create an Entry
or a Gateway compatible expression vector (See FIG. 29).
[0509] In the present Example, three Topo-Gateway vectors and one
Destination vector were created in all. Two topo entry vectors have
been produced: (1) pENTR/D-TOPO.RTM. (FIG. 22), which allows ORFs
directionally cloned between attL sites to be transferred to any of
the N-terminal fusion prokaryotic and all of the eukaryotic DEST
vectors; and (2) pENTR/SD/D-TOPO.RTM. (FIG. 23), which allows ORFs
to be directionally top( ) cloned downstream of a prokaryotic
ribosome binding site Shine-Dalgarno). Genes cloned in this manner
can be transferred to prokaryotic DEST vectors without N-terminal
tags and expressed in bacteria yielding proteins with native
N-termini.
[0510] One directional Topo Gateway mammalian expression vector has
also been constructed, pcDNA/GW-DT (FIG. 19). This vector allows
directional cloning of an ORF into a pcDNA 3.1 derivative. ORFs
cloned into this vector are expressed in mammalian cells under the
control of the CMV promoter. Cloned ORFs are flanked by attB sites
in the vector, allowing them to be moved around in the Gateway
system via BP and LR Clonase reactions. This vector also encodes a
C-terminal V5 tag, the TK poly adenylation signal, and the neomycin
(G418) resistance marker for selection of stable clones in
mammalian cell lines. Finally, a Gateway Destination vector was
constructed from pcDNA/GW-DT by transferring the ccdB and
chloramphenicol resistance cassettes.
[0511] These Topo Gateway Entry and Expression vectors improve the
ease of entry into the Gateway system by allowing the researcher to
directly clone a PCR amplified gene without the necessity of adding
attB sites to the primers and performing a BP clonase reaction.
Materials and Methods
[0512] Construction of pcDNA/GW-DT. pcDNA/GW-DT was constructed by
first replacing the multiple cloning site in pcDNA3.1 attB (an
early version with the BGH polyadenylation signal). This was done
by digesting the parent vector with BsrG I (which cuts within each
att site flanking the MCS) and inserting a double stranded
oligonucleotide encoding the new MCS (FIG. 18). Once the proper
insertion was confirmed, the V5/His tag and BGH polyadenylation
signal were replaced with a V5 tag followed by three stop codons
(TAG, TGA, TAA) and the thymidine kinase (TK) polyadenylation
signal from Herpes Simplex Virus. This was accomplished by
digesting the vector with AscI and AvrII, purification of the
vector fragment, and inserting two fragments encoding the new
sequences in a triple ligation (see FIG. 19).
[0513] Construction of pcDNA-DEST 40. pcDNA-DEST 40 was created
from pcDNA/GW-DT via a BP clonase reaction with pDONR221. pDONR221
was combined with pcDNAGW-DT(sc) and BP clonase (Invitrogen
Corporation; Carlsbad, Calif.) In the appropriate buffer. The
reaction was incubated according to the standard protocol and
transformants selected for on Kanamycin plates. The product, a
pcDNA destination vector containing attP sites flanking the ccdB,
ccdA, and chloramphenicol resistance genes was selected on
ampicillin/chloramphenicol containing media. In one alternative of
this aspect of the invention, the chloramphenicol resistance gene
in the cassette can be replaced by a spectinomycin resistance gene
(see Hollingshead et al., Plasmid 13(1):17-30 (1985), NCBI
accession no. X02340 M10241), and the Destination Vector can be
selected on ampicillin/spectinomycin-containing media. It has
recently been found that the use of spectinomycin selection instead
of chloramphenicol selection results in an increase in the number
of colonies obtained on selection plates, indicating that use of
the spectinomycin resistance gene may lead to an increased
efficiency of cloning from that observed using cassettes containing
the chloramphenicol resistance gene.
[0514] Construction of pENTR/D-TOPO.TM. (sc). pDONOR221 was
modified by adding an adaptation sequence cassette between the attP
sites by BP recombination with pcDNA/GW-DT(sc) creating
pENTR/D-TOPO.RTM. (sc) (FIG. 22). pDONR221 was combined with
pcDNA/GW-DT (sc) and BP clonase in the appropriate buffer. The
reaction was incubated according to the standard protocol except
that DH10BsbcC cells were used for transformation and propagation
of pENTR/D-TOPO.RTM. (sc). This cell line carries a mutation that
allows maintenance of plasmids that carry hairpin structures (e.g.
attL sites) that are in close proximity. This plasmid did not
support growth of Top 10 cells in selective media.
[0515] Creation of pENTR/D-TOPO.RTM. and pENTR/SD/D-TOPO.RTM.. The
vector pENTR/D-TOPO.RTM. (sc) was directionally topo charged by
sequential digestion with Not I, Asc I, and Xho I followed by
ligation with the directional topo adapters Topo-D71, -D72, -D75
and -D76 for pENTR/SD/D-TOPO.RTM. or Topo D-73, -D74, -D75, and
-D76 for pENTR/D-TOPO.RTM. overnight at 15.degree. C. (see FIG.
26). The adapted vectors were separated from free oligonucleotides
by isopropanol precipitation at room temperature. The purified,
adapted vector was topo charged by addition of the common annealing
oligo Topo D-70, T4 Kinase, and recombinant vaccinia topoisomerase
I. After incubation at 37.degree. C. for 15 minutes, charged vector
was purified either by agarose gel electrophoresis (NB JC-12,
2001-035, pg. 3) or chromatography on a 25 Q MacroPrep column
(BioRad) (NB2000-0342, pg. 45). Directional topo cloning efficiency
was assayed by incubation of 1 ng purified vector with 5 ng
directional (CACC) 750 bp test insert for 5 minutes at room
temperature. Top 10 chemically competent cells were then
transformed with 2 ul of the cloning reaction and grown out on LB
plates containing Kanamycin as antibiotic selection.
[0516] Topo-Gateway cloning and gene expression. To test the
ability of these vectors to support Topo cloning, Gateway cloning
and protein production, the gene encoding human HLA class I
(accession No. D32129) was amplified by PCR with primers that
incorporated the four base CACC tag at its 5' end immediately
upstream of the ATG start codon. This PCR product was cloned into
both pENTR/D-TOPO.RTM. and pENTR/SD/D-TOPO.RTM.. Ten clones from
each HLA reaction were used in colony directional PCR reactions
(d-PCR). In this study, clones were amplified with a T7 primer
(binds 5' to the attL 1 site) and 129 reverse primer (specific for
the 3' end of HLA).
[0517] In addition to the HLA gene, the gene for chloramphenicol
acetyl transferase (CAT) was similarly amplified and cloned into
the two entry vectors. After miniprep and digestion analysis,
single clones from each reaction were isolated and sequenced using
the M13 Forward and M13 Reverse primers. All entry clones were
confirmed by sequencing and recombined by L/R Clonase reaction with
pcDNA/GW DEST 40 (pENTR-D-TOPO.RTM. clones) or pET DEST 42
(pENTR/SD/D-TOPO.RTM. clones). Positive clones were confirmed by
digestion with NcoI (site appears at the 5' end of directionally
adapted ORFs, caCCATGG), and NotI (data not shown). The resulting
pcDNA-DEST 40 (HLA and CAT) and pcDNA/GW-DT (HLA and CAT)
constructs were then used to transfect COS cells. Cells were
transfected using Lipofectamine 2000, 8 .mu.g DNA and Optimem
buffer. Reactions were applied to the cells for 5 hours then the
media changed. After an overnight incubation at 37.degree. C., the
cells were harvested, lysed and run on a 4-20% Tris-Glycine gel
using standard procedures. After electrophoresis, proteins were
transferred to nitrocellulose membranes, blocked, and probed with
V5-HRP antibody and ECL detection.
[0518] One positive clone from each pET DEST 42 reaction was used
to transform BL21(DE3) cells and grown overnight in LB/Amp. The
culture was then diluted 1:25 in the same medium and allowed to
grow to O.D. (600 nm)=0.5 at which time expression of recombinant
protein was induced by addition of IPTG to a final concentration of
1 mM. After the cultures were allowed to grow 3 hours at 37.degree.
C., cells were harvested by centrifugation. Aliquots of cell
pellets were boiled in NuPage denaturing sample buffer, run on
4-12% NuPage polyacrylamide gels, and stained using SafeStain.TM.
(Invitrogen Corporation, Carlsbad, Calif.). As a positive control
for expression of test genes in the pET DEST 42 vector, the HLA and
CAT genes were directly topo cloned into pET100 CAT and HLA (dTopo,
no attB sites). These constructs were used to transfect BL21(DE3)
E. coli cells, grown to log phase and induced with IPTG as
described above.
Results and Discussion
[0519] Directional cloning efficiency of HLA and CAT clones in
pENTR-dTopo and pENTR/SD-dTopo. Directional PCR reactions were
designed to ensure that the HLA ORF cloned into pENTR/D-TOPO.RTM.''
and pENTR/SD/D-TOPO.RTM. were in the correct orientation. Ten
colonies were picked from each of the Topo cloning transformations
and put directly into PCR reactions as described in "Materials and
Methods." Eight of ten pENTR/SD-HLA clones tested were correctly
oriented while nine of ten pENTR-HLA clones were correct. These
tests were done with gel purified vector which had approximately
10-15% no insert background (data not shown).
[0520] Alternatively, restriction analysis of the CAT clones was
done. Clones were isolated and the DNA digested with NcoI and AscI.
One of the two NcoI sites in a correctly oriented CAT clone appears
at the 5' end of each ORF as part of the Kozac directional
adaptation sequence and the first two codons of the CAT gene
(caCCATGG). AscI is present in the vector at the 3' end of the ORF.
A correctly oriented clone will have two NcoI sites (one at the 5'
end and one internal) and will yield 500 bp and 150 bp fragments
after a double digest with Asc I. The CAT ORF encodes at its 3' end
the sequence, CGCC, which is a one base pair mismatch to the
optimum tag sequence. This close homology caused the CAT PCR
product to directionally clone with only 50% efficiency (four of
eight clones, data not shown).
[0521] Sequencing of Entry Clones. Each of the Entry clones chosen
for recombination into DEST vectors and subsequent expression were
sequenced from both ends to confirm that the adapters and ORFs
ligated correctly. M13 forward and reverse primers were used and
the reactions were sent to ResGen for sequencing on an ABI 3700
capillary sequencer. From these reactions a minimum of 600 bases of
readable sequence were obtained. It is clear that there is some
loss of signal as the reaction proceeds through the attL sites but
significant signal remains after this point using this procedure
(data not shown).
[0522] Expression of HLA and CAT in COS cells. Expression from
pcDNA/GW/D-TOPO.RTM. and pcDNA-DEST 40 was tested by transfection
of COS cells with HLA and CAT as the test gene in these constructs.
Harvested lysates were probed for VS-tagged recombinant protein by
Western blot using the V5 antibody. Data shown in FIG. 27 indicates
that both the HLA and CAT genes express in these vectors whether
the genes were cloned directly via Topo cloning (FIG. 27, lanes 3
and 6) or after LR clonase transfer from pENTR/D-TOPO.RTM. (FIG.
27, lanes 2 and 5).
[0523] Bacterial expression of HLA and CAT. The CAT and HLA genes
cloned into pENTR/SD/D-TOPO.RTM. were transferred via LR Clonase
reaction to pDEST-42 (pET, C-terminal V5/His). The results shown in
FIG. 28 suggest that the CAT gene expressed in bacteria whether it
is flanked by attB sites or not (FIG. 28, compare lanes 6 and 7).
The finding that the CAT gene expresses well in E. coli after being
transferred to a pET DEST vector from pENTR/SD/D-TOPO.RTM.
validates the utility of this system for cloning and expressing
ORFs using the Topo-Gateway system.
[0524] Interestingly, HLA cloned into pDEST 42 (flanked by attB
sites) failed to express in BL21(DE3) cells in two independent
studies (FIG. 28, lanes 3 and 4). As seen above, the HLA gene from
the same Entry clone expressed well in COS cells when recombined
into a mammalian DEST vector. Further, the fact that the pET system
was unable to support expression of the HLA gene when it was
flanked by attB sites suggests that there can be gene specific
variations on expression using the Gateway system at least in
bacteria. One factor that may be involved in this result is that
HLA expressed from the control vector (pET 100 d-Topo) ran
anomalously in the gel (30 kDa instead of the predicted 41 kDa).
This human protein may not express well in bacteria in any case and
the expression problem may be exacerbated by addition of attB
sites.
[0525] In conclusion, we have described the construction and
testing of two new Topo Gateway Entry vectors, one new Topo Gateway
Expression vector and a new DEST vector that followed from that. In
all, these new tools that combine the ease and efficiency of Topo
cloning and the versatility of the Gateway system permit the
cloning and expression of large numbers of genes in many different
contexts with a minimum of expense and effort.
Example 4
Alternative Methods of Topoisomerase Cloning
[0526] In one preferred alternative embodiment of the present
invention, a TOPO SSS vector is made by first obtaining a
commercially available cloning vector. One such vector is
pUni/V5-His version A (Invitrogen Corporation, Carlsbad, Calif.), a
circular supercoiled vector that contains uniquely designed
elements. These elements include a BGH polyadenylation sequence to
increase mRNA stability in eukaryotic hosts, a T7 transcription
termination region, an R6 Kg DNA replication origin and a kanamycin
resistance gene and promoter for antibiotic resistance selection.
Additionally, pUniN5-His version A contains a multiple cloning
site, which is a synthetic DNA sequence encoding a series of
restriction endonuclease recognition sites. These sites are
engineered for cloning of DNA into a vector at a specific position.
Also within the vector's multiple cloning site is a loxP site
inserted 5' to the endonuclease recognition sites thereby
facilitating Cre recombinase-mediated fusion into a variety of
other expression vectors, (Echo.TM. Cloning System, Invitrogen
Corporation, Carlsbad, Calif.). An optional C-terminal V5 epitope
tag is present for easy detection of expressed fusion proteins
using an Anti-V5 Antibody. An optional C-terminus polyhistidine
(6.times.His) tag is also present to enable rapid purification and
detection of expressed proteins. A bacterial ribosomal binding site
downstream from the loxP site makes transcription initiation in E.
coli possible. Though this combination of elements is specific for
pUni/V5-His version A cloning vector, many similar cloning and
expression vectors are commercially available or may be assembled
from sequences and by methods well known in the art. pUni/V5-His
version A is a 2.2 kb double stranded plasmid.
[0527] Construction of a topoisomerase I charged cloning vector
from pUniN5-His version A is accomplished by endonuclease digestion
of the vector, followed by complementary annealing of synthetic
oligonucleotides and site-specific cleavage of the heteroduplex by
Vaccinia topoisomerase I. Sad and EcoRI are two of the many
restriction endonuclease sites present within the multiple cloning
site of pUni/V5-His version A. Digestion of pUni/V5-His version A
with the corresponding restriction enzymes, Sad and EcoRI will
leave cohesive ends on the vector (5'-AGCT-3' and 5'-AATT-3').
These enzymes are readily available from numerous vendors including
New England Biolabs (Beverly, Mass., Catalogue Nos. R0156S, Sad and
RO101S, EcoRI). The digested pUni/V5-His version A is easily
separated from the digested fragments using isopropanol
precipitation. These and other methods for digesting and isolating
DNA are well known to those of ordinary skill in the art (Sambrook,
J., Fritsch, E. F., and T. Maniatis. (1989) Molecular Cloning, A
Laboratory Manual. Second edition. Cold Spring Harbor Laboratory
Press. pp 5.28-5.32.)
[0528] The purified, digested vector is then incubated with two
specific oligonucleotide adapters and T4 DNA ligase. The adapters
are oligonucleotide duplexes containing ends that are compatible
with the Sad and EcoRI ends of the vector. One of skill in the art
will readily appreciate that other adapter oligonucleotides with
appropriate sequences can be made for other vectors having
different restriction sites. Following incubation with T4 DNA
ligase, the vector containing the ligated adapters is purified
using isopropanol. The adapter duplex that results from the
annealing of TOPO D1 and TOPO D2 has a single-stranded Eco R1
overhang at one end and a 12-nucleotide single-stranded overhang at
the other end.
[0529] The first adapter oligonucleotide, (TOPO D1), has
complementation to the EcoRI cohesive end, 3'-TTAA-5'. Furthermore,
TOPO D1 has an additional 24-bp including the topoisomerase
consensus pentapyridine element 5'-CCCTT located 16-bp upstream of
the 3' end. The remaining sequence and size of TOPO D1 adapter
oligo is variable, and may be modified to fit a researcher's
particular needs. According to one such aspect of this preferred
embodiment of the invention, 5'-AATTGATCCCTTCACCGACATAGTACAG-3 (SEQ
ID NO:33) is the full sequence of the adapter used.
[0530] The second adapter oligonucleotide, (TOPO D2), must have
full complementation to TOPO. D1. TOPO D2 complements directly 5'
of the EcoRI cohesive flap, extending the bottom strand of the
linearized vector. Additionally, TOPO D2 contains the sequence
3'-GTGG, which is the necessary SSS for directional cloning. In
this embodiment, the SSS was chosen to complement the Kozak
sequence known to help expression of ORFs in eukaryotic cells by
increasing the efficiency of ribosome binding on the mRNA, however,
sequence and length are highly variable to meet the specific needs
of individual users. The complete sequence of TOPO D2 is
3-CTAGGGAAGTGG-5 (SEQ ID NO:34). Similar to above, the adapter
duplex that results from the annealing of oligonucleotides TOPO D4
and TOPO D5 has a single-stranded Sad overhang at one end, and a 12
nucleotide single-stranded overhang at the other end.
[0531] The third adapter oligonucleotide (TOPO D5), has
complementation to the Sad cohesive end, 3'-TCGA-5'. Similar to
TOPO D1, TOPO D5 has additional bases creating a single stranded
overhang. The length and sequence can vary based on the needs of
the user. In the current embodiment TOPO D5's sequence is
5'-AAGGGCGAGCT-3' (SEQ ID NO:35).
[0532] The fourth adapter oligonucleotide (TOPO D4), has full
complementation to TOPO D5, and complements directly 5' of the SacI
cohesive flap extending the top strand of the linearized vector.
TOPO D4 also contains the topoisomerase consensus sequence
5'-CCCTT. The remaining sequence and size of TOPO D4 adapter oligo
is variable and may be modified to fit particular needs. In the
current embodiment, the sequence of TOPO D4 is
3'-GACATGATACAGTTCCCGC-5' (SEQ ID NO:36), which includes an
additional 12 bp single stranded overhang.
[0533] These adapter oligonucleotides can be chemically synthesized
using any of numerous techniques, including the phosphoramadite
method, (Caruthers, M. H., Barone, A. D., Beaucage, S. L., Dodds,
D. R., Fisher, E. F., McBride, L. J., Matteucci, M., Stabinsky, Z.,
and Tang, J. Y., (1987) Chemical Synthesis of
Deoxyoligonucleotides, Methods Enzymol. 154: 287-313). This and
other methods for the chemical synthesis of oligos are well known
to those of ordinary skill in the art.
[0534] Complementary annealing of the purified digested vector and
the adapter oligonucleotides is done by incubation of the DNA in
the presence of T4 DNA ligase. Typical ligation reactions are
performed by incubation of a cloning vector with suitable DNA
fragments in the presence of ligase and an appropriate reaction
buffer. Buffers for ligation reactions should contain ATP to
provide energy to for the reaction, as well as, reducing reagents
like dithiothreitol and pH stabilizers like Tris-HCl. The ratio of
concentrations for the cloning vector and the DNA fragments are
dependent on each individual reaction, and formulae for their
determination are abundant in the literature, (See e.g. Protocols
and Applications Guide (1991), Promega Corporation, Madison, Wis.,
p. 45). T4 Ligase will catalyze the formation of a phosphodiester
bond between adjacent 5'-phosphates and 3'-hydroxyl termini during
the incubation. Cohesive end ligation can generally be accomplished
in 30 minutes at 12-15.degree. C., while blunt end ligation
requires 4-16 hours at room temperature, (Ausubel, F. M., Brent,
R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A.,
Struhl, K. (1992) Second Edition; Short Protocols in Molecular
Biology, John Wiley & Sons, Inc., New York, N.Y., pp.
3.14-3.37), however parameter range varies for each study. In the
current embodiment, purified, digested pUni/V5-His version A and
the adapter oligos were incubated in the presence of T4 ligase and
a suitable buffer for sixteen hours at 12.5.degree. C. The
resulting linearized and adapted vector comprises the purified
cloning vector attached to the adapter oligonucleotides through
base pair complementation and T4 ligase-catalyzed, phosphodiester
bonds.
[0535] Efficient modification of the adapted vector with
topoisomerase requires the addition of an annealing oligo to
generate double, stranded DNA on TOPO D1's and TOPO D4's single
stranded overhangs. Vaccinia topoisomerase I initially binds
non-covalently to double stranded DNA. The enzyme then diffuses
along the duplex until locating and covalently attaching to the
consensus pentapyrimidine sequence 5'-CCCTT, forming the
topoisomerase adapted complex (See Shuman et al., U.S. Pat. No.
5,766,891). Modification of the adapted vector takes place in the
absence of DNA ligase to prevent the formation of phosphodiester
bonds between the adapted vector and the annealing oligo, since
phosphodiester bonds in the non-scissile strand will prevent the
dissociation of the leaving group upon cleavage.
[0536] The annealing oligonucleotide (TOPO D3), must have
complementation to the single stranded DNA overhangs of TOPO D1 and
TOPO D4. In the current embodiment the overhangs both share the
following sequence, 5'-GACATAGTACAG-3' (SEQ ID NO:37). Therefore,
TOPO D3 has the following sequence, 3-CTGTATCATGTCAAC-5 (SEQ ID
NO:38), which comprises full complementation to the adapter oligos'
single stranded overhang and an additional 3 bp overhang,
3'-AAC-5'.
[0537] Incubation of the adapted vector with the annealing oligo in
the presence of topoisomerase will create double stranded DNA to
which topoisomerase can non-covalently bind. Bound topoisomerase
will search the double stranded DNA by a facilitated diffusion
mechanism, until the 5'-CCCTT recognition motif is located.
Cleavage of the phosphodiester backbone of the scissile strand 3'
of the motif is catalyzed via a nucleophilic attack on the 3'
phosphorus atom of the preferred oligonucleotide cleavage sequence
5-CCCTT, resulting in covalent attachment of the DNA to the enzyme
by a 3'-phosphotyrosyl linkage, (See Shuman, S., Kane, E. M.,
Morham, S. G. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9793-9796).
Cleavage of the scissile strand creates a double stranded leaving
group comprising the 3' end adapter oligo, downstream from the
5'-CCCTT motif, and the annealing oligo TOPO D3. Although the
leaving group can religate to the topoisomerase-modified end of the
vector via 5' hydroxyl-mediated attack of the phosphotyrosyl
linkage, this reaction is disfavored when the leaving group is no
longer covalently attached to the vector. The addition of T4
polynucleotide kinase and ATP to the cleavage/religation reaction
further shifts the equilibrium toward the accumulation of trapped
topoisomerase since the kinase can phosphorylate the 5' hydroxyl of
the leaving group to prevent the rejoining from taking place,
(Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman,
J. G., Smith, J. A., Struhl, K. (1992) Second Edition; Short
Protocols in Molecular Biology, John Wiley & Sons, Inc., New
York, N.Y., pp. 3.14-3.30). The resulting linearized vector
comprises a blunt end from the TOPO D4/D3 leaving group and a SSS
bearing end from the TOPO D1/D3 leaving group. Both of the
linearized cloning vector's ends are charged with topoisomerase,
enabling fast, efficient and directional topoisomerase mediated
insertion of an acceptor molecule.
[0538] Although the above example details the modification of
pUni/V5-His version A to form the topoisomerase-modified
directional cloning vector, a person of ordinary skill in the art
will appreciate how to apply these methods to any plasmid, cosmid,
virus, or other DNA. It should also be noted that this example
demonstrates a vector containing a 5' single-stranded overhang
comprising the sequence 5'-GGTG-3', however the design of adapter
duplexes and annealing oligonucleotides would allow one of skill in
the art to custom design overhangs of any sequence or length at one
or both ends of a given vector.
[0539] Specifically, any plasmid, cosmid, virus or other DNA can be
modified to possess a SSS of any convenient sequence and length.
These are the basic steps: the vector is first subjected to a
treatment that is known to linearize the DNA. Common procedures
include, but are not limited to, restriction digestion and
treatment with topoisomerase II. Following linearization, a custom
SSS is added. In the above example, complementary oligonucleotides
are added to the sticky ends of a restriction digestion giving the
desired SSS, however SSS forming oligonucleotides can be added by
T4 blunt end ligation, as well. The SSS sequence is exposed by a
topoisomerase I mediated, single strand nicking. In turn, this SSS
can be used to directionally insert a PCR product comprising one or
more complimentary SSS.
[0540] Likewise, topoisomerase modification can be applied to any
double-stranded plasmid, cosmid, virus or other piece of DNA.
Methods for the attachment of topoisomerase I to double stranded
DNA are well known in the art, (See Shuman et al., U.S. Pat. No.
5,766,891). The strategic placement of topoisomerase on to a piece
of double stranded DNA is determined by the incorporation of a
topoisomerase I consensus sequence, (See Shuman et al., U.S. Pat.
No. 5,766,891). The topoisomerase I will bind the double stranded
DNA, nick the scissile strand thus revealing the predetermined
single-stranded overhang sequence, and ligate the incoming PCR
product in the correct, SSS mediated orientation.
Example 5
Production of Custom Topoisomerase I-Adapted Vectors
[0541] As an example of the application of this aspect of the
current invention to another plasmid, pCR 2.1 (Invitrogen
Corporation; Carlsbad, Calif.) was modified to create a
topoisomerase I adapted vector with a custom single stranded
sequence.
[0542] Plasmid pCR 2.1 is 3.9 kb T/A cloning vector. Within the
sequence of this vector are many uniquely designed elements. These
elements include an f1 origin, a ColE1 origin, a kanamycin
resistance gene, an ampicillin resistance gene, a LacZ-alpha
fragment and a multiple cloning sequence located within the
LacZ-alpha fragment allowing for blue-white selection of
recombinant plasmids. The multiple cloning sequence of pCR 2.1
contains; numerous restriction sites, including but not limited to,
HindIII, SpeI and EcoRI; M13 forward and reverse primers and a T7
RNA polymerase promoter.
[0543] Construction of the topoisomerase I charged vector
possessing a custom single stranded sequence consists of
endonuclease digestion followed by complementary annealing of
synthetic oligonucleotides and the site specific cleavage of the
heteroduplex by Vaccinia topoisomerase I. Digestion of pCR 2.1 with
the restriction enzymes HindIII, SpeI and EcoRI leaves HindIII and
EcoRI cohesive ends on the vector. The dissociated fragment of pCR
2.1 downstream from the HindIII cleavage site is further cleaved
with SpeI in order to reduce its size. By reducing the size of the
fragment, the digested vector is easily purified away from the
smaller digested pieces by isopropanol precipitation. These enzymes
are readily available from numerous vendors including New England
Biolabs, (Beverly, Mass., Catalogue Nos.; RO104S, HindIII; RO133S,
SpeI; RO101S, EcoRI). Methods for the digestion and the isolation
of DNA are well known to those skilled in the art, (Sambrook, J.,
Fritsch, E. F., and T. Maniatis. (1989) Molecular Cloning, A
Laboratory Manual. Second edition. Cold Spring Harbor Laboratory
Press. pp. 5.28-5.32.)
[0544] The purified digested vector is incubated with four adapter
oligonucleotides and T4 DNA ligase. These adapter oligonucleotides
are designed to have complementation to either the HindIII cohesive
end, the EcoRI cohesive end, or to each other. Following incubation
with T4 DNA ligase the adapted vector is purified using
isopropanol.
[0545] The first adapter oligonucleotide, (TOPO H), has
complementation to the HindIII cohesive end, 3'-TCGA-5'.
Furthermore, TOPO H has an additional 24 bp including the
topoisomerase consensus pentapyrimidine element 5'-CCCTT located
19-bp upstream of the 3' end. The remaining sequence and size of
TOPO H adapter oligo is variable, and may be modified to fit a
researcher's particular needs. In the current embodiment
5'-AGCTCGCCCTTATTCCGATAGTG-3' (SEQ ID NO:39) is the full sequence
of the adapter used.
[0546] The second adapter oligonucleotide (TOPO 16), must have full
complementation to TOPO H. TOPO 16 complements directly 5' of the
HindIII cohesive end, extending the bottom strand of the linearized
vector. Additionally, TOPO 16 contains the sequence 3'-TAAG, which
is the chosen single stranded sequence for directional cloning. The
complete sequence of TOPO 16 is 3'-GCGGGAATAAG-5' (SEQ ID
NO:40).
[0547] The third adapter oligonucleotide (TOPO 1), has
complementation to the EcoRI cohesive end, 3'-TTAA-5'. Similar to
TOPO H, TOPO 1 has additional bases containing the topoisomerase I
consensus sequence CCCTT located 12 bp upstream of the 3' end. The
length and sequence of TOPO 1 can vary based on the needs of the
user. In the current embodiment TOPO 1's sequence is
5'-AATTCGCCCTTATTCCGATAGTG-3' (SEQ ID NO:41).
[0548] The fourth adapter oligonucleotide (TOPO 2), has full
complementation to TOPO 1, and complements directly 5' of the EcoRI
cohesive end extending the top strand of the linearized vector. In
the current embodiment, the sequence of TOPO 2 is
3'-GCGGGAA-5'.
[0549] Complementary annealing of the purified digested vector and
the adapter oligonucleotides is done by incubation of the DNA in
the presence of T4 DNA ligase. T4 Ligase will catalyze the
formation of a phosphodiester bond between adjacent 5'-phosphates
and 3'-hydroxyl termini during the incubation. In the current
embodiment, purified, digested pCR 2.1 and the adapter oligos were
incubated in the presence of T4 ligase and a suitable buffer for
sixteen hours at 12.5.degree. C. The resulting linearized and
adapted vector comprises the purified cloning vector attached to
the adapter oligonucleotides through base pair complementation and
T4 ligase-catalyzed, phosphodiester bonds. Ligation techniques are
abundant in the literature, (see Ausubel, F. M., et al, (1992)
Second Edition; Short Protocols in Molecular Biology, John Wiley
& Sons, Inc., New York, N.Y., pp. 3.14-3.37)
[0550] Charging of the adapted vector with topoisomerase requires
the addition of annealing oligonucleotides to generate double
stranded DNA on TOPO H's and TOPO1's single stranded overhangs.
Charging of the adapted vector takes place in the absence of DNA
ligase to prevent the formation of phosphodiester bonds between the
adapted vector and the annealing oligo, since phosphodiester bonds
in the non-scissile strand will prevent the dissociation of the
leaving group upon cleavage.
[0551] The annealing oligonucleotide (TOPO 17), must have
complementation to the single stranded DNA overhang of TOPO H. In
the current embodiment the overhang has the following sequence,
5'-CGATAGTG 3'. Therefore, TOPO 17 has the following sequence,
3'-GCTATCAC 5', which comprises full complementation to the adapter
oligo's single stranded overhang.
[0552] The annealing oligonucleotide (TOPO 3), must have
complementation to the single stranded DNA overhang of TOPO 1. In
the current embodiment the overhang has the following sequence,
3'-GTGATAGCCTTA-5' (SEQ ID NO:42). Therefore, TOPO 3 has the
following sequence, 5'-CAACACTATCGGAAT-3' (SEQ ID NO:43), which
comprises full complementation to the adapter oligo's single
stranded overhang and an additional 3 bp overhang, 5'-CAA-3'.
[0553] Incubation of the adapted vector with the annealing oligo in
the presence of topoisomerase will create double stranded DNA to
which topoisomerase can non-covalently bind. Bound topoisomerase
will search the double stranded DNA by a facilitated diffusion
mechanism, until the 5'-CCCTT recognition motif is located.
Cleavage of the phosphodiester backbone of the scissile strand 3'
of the motif will result in the covalent attachment of the DNA to
the enzyme by a 3'-phosphotyrosyl linkage, (See Shuman, S., et al
(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9793-9796). Cleavage of
the scissile strand creates a double stranded leaving group
comprising the 3' end the adapter oligos, downstream from the
5'-CCCTT motif, and the complementary annealing oligonucleotide.
The leaving group can religate to the topoisomerase adapted vector
through its 5' hydroxyl's attack of the phosphotyrosyl linkage,
also catalyzed by topoisomerase. Addition of T4 polynucleotide
kinase to the equilibrium reaction prevents the back reaction via
the kinase-mediated phosphorylation of the leaving group's 5'
hydroxyl, (Ausubel, F. M., et al (1992) Second Edition; Short
Protocols in Molecular Biology, John Wiley & Sons, Inc., New
York, N.Y., pp. 3.14-3.30). The resulting linearized vector
comprises a blunt end from the TOPO 1/3 leaving group and a single
stranded sequence end from the TOPO H/17 leaving group. Both of the
linearized cloning vector's ends are charged with topoisomerase,
enabling fast, efficient and directional topoisomerase mediated
insertion of an acceptor molecule.
Example 6
Directional Cloning Using Topoisomerase
[0554] This aspect of the invention also provides a method for
directional cloning of DNA. In such methods, the TOPO SSS vector
constructed from pUni/V5-His version A was used for the directional
insertion of ORFs from the GeneStorm Expression Ready Clones
(Invitrogen Corporation, Carlsbad, Calif.). The modified pUni
vector was selected for the cloning of these ORF's because the
single strand added to the vector has homology to the Kozak
sequence known to enhance ORF expression. Note, however, that, as
before, any plasmid, cosmid, virus or other DNA could be modified
to possess the necessary single stranded sequence. Likewise, any
DNA fragment could be modified to possess a homologous sequence to
any vector SSS. As a point of interest, the sequence of the SSS can
effect directional cloning efficiencies. For example, SSSs with low
GC content will have lower annealing stability, also SSSs that have
high complementation to both ends of a DNA fragment to be cloned
will loose the capability to direct these DNA inserts. Thus the
sequence of a SSS should be carefully designed to avoid these and
similar problems.
[0555] This aspect of the present invention is particularly useful
in the directional insertion of PCR products into vectors
constructed according to the present invention. In the PCR
amplification of the desired insert, the PCR primers are designed
so as to complement identified sequences of the insert(s) that are
to be directionally cloned into the TOPO SSS vector. The primer
designed to bind upstream of the DNA's coding strand is modified
with an additional vector SSS complementation sequence on its 5'
end. The resulting PCR product will possess a complementary
sequence allowing SSS mediated directional insertion into the TOPO
SSS cloning vector and subsequent expression of the product.
[0556] One such embodiment comprises introducing to a donor duplex
DNA substrate a SSS site by PCR amplifying the donor duplex DNA
molecule with the 5' oligonucleotide primer containing the SSS. PCR
amplification of a region of DNA is achieved by designing
oligonucleotide primers that complement a known area outside of the
desired region. In a preferred embodiment the primer that has
homology to the coding strand of the double stranded region of DNA
will possess an additional sequence of nucleotides complementary to
the SSS of the TOPO SSS cloning vector.
[0557] Using the current invention in a high throughput format, we
selected 82 known ORFs from the GeneStorm expression system
(Invitrogen Corporation, Carlsbad, Calif.) for directional cloning
into the TOPO SSS vector, however, any sequence of DNA may be
selected as desired by individual users. For each of these ORFs,
primers are designed with homology to the coding and the non-coding
strands. To clone PCR products in a directional fashion into the
modified pUni/V5-His version A TOPO SSS vector as described in
Example 4, one primer of a given pair was modified to contain the
nucleotide sequence complementary to the SSS contained within the
vector. In the current example, the coding primer contained the
added sequence 5'-CACC-3', which complements the `SSS`, 3'-GTGG-5',
of the TOPO SSS cloning vector. PCR amplification of the above ORFs
with their respective primers will produce double stranded DNA
fragments, which possess the SSS at their 5' end. We used Pfu
polymerase in our PCR amplification, but it is well-known that PCR
reactions can be performed with either a non-thermophillic
polymerase such as Pfu or with a thermophillic polymerase like Taq
followed by a blunting step to remove the non-template nucleotide
these enzymes leave at the end of PCR products.
[0558] In the present example, 0.1 .mu.g of each primer was
combined with 0.05 .mu.g of DNA containing an ORF in a PCR reaction
mix totaling 50 .mu.l total volume. Besides the primers and vector,
the reaction mix also contained water, PCR buffer salts, 10 mM
dNTPs and 1.25 units of Pfu polymerase. Thermal cycling
temperatures were as follows: an initial 94.degree. C.
denaturation; followed by 25 repetitions of 94.degree. C.
denaturation, 55.degree. C. primer annealing, and 72.degree. C.
elongation, each at one minute; and ended with a 72.degree. C.,
fifteen minute elongation. These parameters will vary with each DNA
fragment to be amplified, and can be optimized for fragments of
varying lengths and composition using methods well known to those
of ordinary skill in the art (Ausubel, F. M., Brent, R., Kingston,
R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K.
(1992) Second Edition; Short Protocols in Molecular Biology, John
Wiley & Sons, Inc., New York, N.Y., pp. 15.3-15.4). Techniques
for the conversion of 3' overhangs to blunt end termini will also
be familiar to those of ordinary skill in the art (Protocols and
Applications Guide (1991), Promega Corporation, Madison, Wis., pp.
43-44).
[0559] Incubation of the PCR amplified donor duplex DNA containing
the SSS complementary sequence with the modified pUni/V5-His
version A TOPO SSS vector results in the directional cloning of the
donor DNA. For example, the eighty-two ORFs from the GeneStorm
clone collection (Invitrogen Corporation, Carlsbad, Calif.) were
amplified using SSS adapted primers. Amplification of the 82
GeneStorm ORFs with the described modified primer pairs resulted in
PCR products that had the SSS complementary sequence at their 5'
end. This ORF PCR product is combined with 10 ng of TOPO SSS
cloning vector in either sterile water or a salt solution. The
reaction is mixed gently and incubated for 5 minutes at room
temperature (22-23.degree. C.). After five minutes, we placed the
reaction on ice then proceeded to the OneShot.RTM. Chemical
Transformation or Electroporation (Invitrogen Corporation,
Carlsbad, Calif., Catalogue Nos. C4040-10 and C4040-50,
respectively) (Invitrogen TOPO Cloning Protocol. Invitrogen
Corporation Carlsbad, Calif.). Topoisomerase had joined the
adjacent strands of the vector and the product by catalyzing a
rejoining reaction (FIG. 29). DNA fragments constructed with the
SSS at their 5' ends were thus correctly inserted into TOPO SSS
cloning vectors with a high efficiency.
[0560] Directional insertion of DNA fragments containing 5' SSS
occurs with greater than 90% efficiency as shown by sequencing
multiple colonies of transformed host cells. In the current
example, the TOPO SSS cloning vectors containing the GeneStorm ORFs
were incubated with transformation competent E. coli host cells. In
74 of the transformation reactions, the directional cloning of the
ORFs into the TOPO SSS cloning vector occurred in at least seven of
the eight colonies picked, and 59 of these cloning reactions were
directional in all eight colonies picked. The overall directional
cloning score was 609 of 656, thus, directional insertion was
present in over 93% of the clones picked (see Table 5).
TABLE-US-00005 TABLE 5 Directional Cloning of ORFs using a TOPO SSS
Cloning Vector Positive colonies. dPCR reactions Clones tested 8/8
59 7/8 15 6/8 2 5/8 1 4/8 3 3/8 2
Example 7
Directional Cloning of a Reporter Gene
[0561] In a similar example, using the above described modified
pCR2.1 TOPO SSS vector, a PCR-generated ORF encoding the gene
encoding the reporter molecule Green Fluorescent Protein (GFP) was
directionally cloned in frame with the lacZ a fragment present in
the vector. The primers used to amplify the GFP gene contained the
requisite SSS complementation sequence 5'-ATTC-3', and the known
sequence for translation initiating methionine, 5'-ATG-3'. Using
the necessary cloning steps noted above, the PCR amplified GFP was
inserted into the vector and transformed cells were grown on solid
Agar plates. Glowing colonies represented a correctly inserted PCR
product (see Table 6).
TABLE-US-00006 TABLE 6 In-frame and Directional Insertion of GFP
Into Modified pCR2.1 TOPO SSS Cloning Vector. Percentage 5'
sequence of Correct Total White of PCR Product Inserts Colonies
5'-ATTCATG-3' 86% 457 (homologous) 5'-CAAGATG-3' 35% 118
(non-homologous) 5'-ATTCGGATG-3' 0% 268 (frame shift) VECTOR ONLY
0% 31
[0562] These data represent a substantial improvement over the
current state of the art in cloning, and furthermore present an
invention in cloning that is highly compatible with high throughput
techniques. Given directional cloning efficiencies greater than
90%, a user need only screen two colonies for each cloned DNA
fragment. Thus, on a 96-well plate, 48 separate clones can be
screened for directional insertion, 400% more than current cloning
techniques. Use of this invention will streamline many
high-throughput-gene-expression operations, and allow them to be
run at a fraction of their current costs.
Example 8
Directional Topoisomerase Cloning of Blunt-End PCR Products into
Entry Vectors Overview
[0563] In additional embodiments, the compositions, kits and
methods of the invention combine a highly efficient, 5-minute
cloning strategy ("TOPO.RTM. Cloning;" Invitrogen Corporation,
Carlsbad, Calif.) to directionally clone blunt-end PCR products
into vectors for entry into the recombinational cloning system of
the invention (e.g., the GATEWAY.TM. System available from
Invitrogen Corporation, Carlsbad, Calif.). Using this cloning
strategy of the invention, blunt-end PCR products clone
directionally at greater than 90% efficiency, with no ligase,
post-PCR procedures, or restriction enzymes required.
[0564] For optimal expression of a PCR product after recombination
with the GATEWAY.TM. destination vector of interest, any suitable
expression vector may be used. Examples include, but are not
limited to, the pENTR Directional TOPO.RTM. vectors available
commercially (Invitrogen Corporation; Carlsbad, Calif.), which have
a number of benefits including the following:
[0565] Vector Benefits [0566] pENTR/D-TOPO.RTM. For efficient
expression of a gene of interest after recombination with a
GATEWAY.TM. destination vector [0567] pENTR/SD/D-TOPO.RTM. Contains
a T7 gene 10 translational enhancer and a ribosome binding site for
optimal expression of native protein after recombination with a
prokaryotic GATEWAY.TM. destination vector [0568] Also suitable for
efficient expression of a gene of interest in other host cell
systems (e.g., mammalian, insect, yeast) after recombination with a
suitable GATEWAY.TM. destination vector
[0569] These pENTR/D-TOPO.RTM. and pENTR/SD/D-TOPO.RTM. vectors are
designed to facilitate rapid, directional TOPO.RTM. Cloning of
blunt-end PCR products for entry into the GATEWAY.TM. System.
Features of these vectors include: [0570] attL1 and attL2 sites for
site-specific recombination of the entry clone with a GATEWAY.TM.
destination vector; [0571] Directional TOPO.RTM. Cloning site for
rapid and efficient directional cloning of blunt-end PCR products;
[0572] rmB transcription termination sequences to prevent basal
expression of the PCR product of interest in E. coli; [0573]
Kanamycin resistance gene for selection in E. coli; [0574] pUC
origin for high-copy replication and maintenance of the plasmid in
E. coli; and [0575] T7 gene 10 translation enhancer and ribosome
binding site for efficient translation of the PCR product in
prokaryotic systems (pENTR/SD/D-TOPO.RTM. only).
[0576] Using these pENTR Directional TOPO.RTM. vectors in
conjunction with the GATEWAY.TM. recombinational cloning system of
the invention, genes of interest contained in blunt-end PCR
products may be readily expressed by following several simple
steps:
[0577] 1. the blunt-end PCR product is cloned (using topoisomerase
in the "TOPO.RTM. Cloning" procedures described herein) into one of
the pENTR TOPO.RTM. vectors described above, to generate an entry
clone;
[0578] 2. an expression construct is generated by performing a
recombination reaction between this entry clone and a GATEWAY.TM.
destination vector of choice (such as those described elsewhere
herein); and
[0579] 3. the expression construct is introduced into an
appropriate host cell (e.g., a bacterial, mammalian, yeast, insect,
or other appropriate host cell, the choice depending on the
specific destination vector chosen for production of the expression
construct above), and the recombinant protein encoded by the gene
of interest on the PCR product (and now contained on the expression
construct) is expressed using expression conditions appropriate for
the particular host cell system.
Directional TOPO.RTM. Cloning
[0580] Topoisomerase I from Vaccinia virus binds to duplex DNA at
specific sites (CCCTT) and cleaves the phosphodiester backbone in
one strand (Shuman, 1991). The energy from the broken
phosphodiester backbone is conserved by formation of a covalent
bond between the 3' phosphate of the cleaved strand and a tyrosyl
residue (Tyr-274) of topoisomerase I. The phospho-tyrosyl bond
between the DNA and enzyme can subsequently be attacked by the 5'
hydroxyl of the original cleaved strand, reversing the reaction and
releasing topoisomerase (Shuman, 1994). TOPO.RTM. Cloning exploits
this reaction to efficiently clone PCR products.
[0581] Directional joining of double-stranded DNA using
TOPO.RTM.-charged oligonucleotides occurs by adding a 3'
single-stranded end (overhang) to the incoming DNA (Cheng and
Shuman, 2000). This single-stranded overhang is identical to the 5'
end of the TOPO.RTM.-charged DNA fragment. By the present
invention, this approach has been modified by adding a 4 nucleotide
overhang sequence to the TOPO.RTM.-charged DNA and adapting it to a
"whole vector" format.
[0582] In this system, PCR products are directionally cloned by
adding four bases to the forward primer (CACC). The overhang in the
cloning vector (GTGG) invades the 5' end of the PCR product,
anneals to the added bases, and stabilizes the PCR product in the
correct orientation. Inserts can be cloned in the correct
orientation with efficiencies equal to or greater than 90%.
Methods
[0583] Designing PCR Primers. The design of the PCR primers to
amplify a gene of interest is critical for expression. Depending on
the pENTR TOPO.RTM. vector being used, several considerations must
be kept in mind during design of PCR primers, including: [0584] the
sequences required to facilitate directional cloning; [0585] the
sequences required for proper translation initiation of the PCR
product; and [0586] whether or not the PCR product is to be fused
in frame with an N- or C-terminal tag after recombination of the
entry clone with a GATEWAY.TM. destination vector.
[0587] Guidelines to Design the Forward PCR Primer. When designing
the forward PCR primer, the following points must be
considered.
[0588] To enable directional cloning, the forward PCR primer MUST
contain the sequence, CACC, at the 5' end of the primer. The four
nucleotides, CACC, base pair with the overhang sequence, GTGG, in
each pENTR TOPO.RTM. vector.
[0589] If the PCR product is to be expressed in mammalian cells
(following recombination of the entry clone with a GATEWAY.TM.
destination vector), the sequence of interest must include a Kozak
translation initiation sequence with an ATG initiation codon for
proper initiation of translation (Kozak, 1987; Kozak, 1991; Kozak,
1990). An example of a Kozak consensus sequence is (G/A)NNATGG.
Other sequences are possible, but the G or A at position -3 and the
G at position +4 are the most critical for function (shown in
bold). The ATG initiation codon is shown underlined. Note: If the
sequence of interest does not contain an initiation codon within
the context of a Kozak sequence, the forward PCR primer may be
designed so as to contain a Kozak sequence at the 5' end of the
primer (see below).
[0590] If the PCR product is to be expressed in prokaryotic cells
without an N-terminal fusion tag (following recombination of the
entry clone with a GATEWAY.TM.-destination vector), the PCR product
should be TOPO.RTM. Cloned into a pENTR/SD/D-TOPO.RTM. entry
vector. As noted above, pENTR/SD/D-TOPO.RTM. contains a T7 gene 10
translational enhancer and a ribosome binding site (RBS) to enable
efficient translation of the PCR product in E. coli. To ensure
optimal spacing for proper translation, the forward PCR primer
should be designed such that that the ATG initiation codon of the
PCR product directly follows the CACC necessary for directional
cloning (see below).
[0591] Example of Forward Primer Design. Below is the DNA sequence
of the N-terminus of a theoretical protein and the proposed
sequence for a corresponding forward PCR primer. The ATG initiation
codon is underlined.
TABLE-US-00007 DNA sequences:, (SEQ ID NO. 122) 5'-ATG GGA TCT GAT
AAA Proposed Forward PCR primer: (SEQ ID NO. 123) 5'-CACC ATG GGA
TCT GAT AAA.
[0592] If the forward PCR primer is designed as noted above, then
(a) the ATG initiation codon falls within the context of a Kozak
sequence (see boxed sequence), allowing proper translation
initiation of the PCR product in mammalian cells (note that the
first three base pairs of the PCR product following the 5' CACC
overhang will constitute a functional codon); and (b) the ATG
initiation codon is properly spaced from the RBS (in
pENTR/SD/D-TOPO.RTM. only), allowing proper translation of the PCR
product in prokaryotic cells.
[0593] Guidelines to Design the Reverse primer. When designing your
reverse PCR primer, consider the following points below. See FIGS.
26 and 27 for diagrams of the TOPO.RTM. Cloning sites for
pENTR/D-TOPO.RTM. and pENTR/SD/D-TOPO.RTM., respectively.
[0594] To ensure that the PCR product clones directionally with
high efficiency, the reverse PCR primer MUST NOT be complementary
to the overhang sequence GTGG at the 5' end. A one base pair
mismatch can reduce the directional cloning efficiency from 90% to
50%, increasing the likelihood that the ORF will be cloned in the
opposite orientation (see "example A" below). We have not observed
evidence of PCR products cloning in the opposite orientation from a
two base pair mismatch.
[0595] If the PCR product is to be fused in frame with a C-terminal
tag (following recombination of the entry clone with a
GATEWAY.TM.-destination vector), then the reverse PCR primer should
be designed so as to remove the native stop codon in the gene of
interest (see "example B" below).
[0596] If the PCR product is NOT to be fused in frame with a
C-terminal tag (following recombination of the entry clone with a
GATEWAY.TM.-destination vector), then the native sequence
containing the stop codon should be included in the reverse primer,
or it should be ensured that the stop codon is upstream from the
reverse PCR primer binding site (see "example B" below).
[0597] Example A of Reverse Primer Design. Below is the sequence of
the C-terminus of a theoretical protein. The protein should be
fused in frame with a C-terminal tag (following recombination of
the entry clone with a GATEWAY.TM.-destination vector). The stop
codon is underlined.
TABLE-US-00008 DNA sequence: (SEQ ID NO. 46) AAG TCG GAG CAC TCG
ACG ACG GTG TAG-3'.
One solution is to design the reverse PCR primer to start with the
codon just upstream of the stop codon, but the last two codons
contain GTGG (underlined below), which is identical to the 4 bp
overhang sequence. As a result, the reverse primer will be
complementary to the 4 bp overhang sequence, increasing the
probability that the PCR product will clone in the opposite
orientation. This situation should be avoided.
TABLE-US-00009 DNA sequence: (SEQ ID NO. 46) AAG TCG GAG CAC TCG
ACG ACG GTG TAG-3'. Proposed Reverse PCR primer sequence: (SEQ ID
NO. 47) TG AGC TGC TG C CAC AAA-5'.
[0598] Another solution is to design the reverse primer so that it
hybridizes just downstream of the stop codon, but still includes
the C-terminus of the ORF. Note that the stop codon will need to be
replaced with a codon for an innocuous amino acid such as glycine,
alanine, or lysine.
[0599] Example B of Reverse Primer Design. Below is the sequence
for the C-terminus of a theoretical protein. The stop codon is
underlined.
TABLE-US-00010 (SEQ ID NO. 48) GCG GTT AAG TCG GAG CAC TCG ACG ACT
GCA TAG-3'.
To fuse the ORF in frame with a C-terminal tag (supplied by the
destination vector after recombination), remove the stop codon by
starting with nucleotides homologous to the last codon (TGC) and
continue upstream. The reverse primer will be:
TABLE-US-00011 (SEQ ID NO. 49) 5'-TGC AGT CGT CGA GTG CTC CGA
CTT-3'.
This will amplify the C-terminus without the stop codon and allow
the ORF to be joined in frame with a C-terminal tag. If it is not
desirable to join the ORF in frame with a C-terminal tag, the
reverse primer should simply be designed to include the stop
codon:
TABLE-US-00012 (SEQ ID NO. 50) 5'-CTA TGC AGT CGT CGA GTG CTC CGA
CTT-3'.
Important: It must be remembered that the pENTR TOPO.RTM. vectors
accept blunt-end PCR products. 5' phosphates should not be added to
the primers for PCR, as this will prevent ligation into the pENTR
TOPO.RTM. vectors. In addition, it is recommended that the
oligonucleotides be gel-purified prior to use, especially if they
are long (>30 nucleotides).
Producing Blunt-End PCR Products
[0600] Once a PCR strategy has been chosen and primers synthesized
according to the guidance presented above, the blunt-end PCR
product can be produced. Any thermostable, proofreading polymerase
may be used for this purpose, including ThermalAce.TM.,
PLATINUM.RTM., Pfr, Pfu, or Vent.RTM. for PCR. To produce blunt-end
PCR products, the instructions and recommendations of the
manufacturer of the polymerase should be followed. It is important
to optimize PCR conditions to produce a single, discrete PCR
product. Gel purification of PCR fragments, according to methods
outlined below, is also recommended.
Producing PCR Products
[0601] To produce amplification products via PCR, 25 .mu.l or 50
.mu.l PCR reaction mixtures are set up using the following
guidelines:
[0602] Follow the manufacturer's instructions for the DNA
polymerase that is being used.
[0603] Use the cycling parameters suitable for the primers and
template.
[0604] Use a 7 to 30 minute final extension to ensure that all PCR
products are completely extended.
[0605] After cycling, the tube should be placed on ice or stored at
-20.degree. C. for up to 2 weeks.
Checking the PCR Product
[0606] To verify quality and quantity of the PCR product, 5 .mu.l
to 10 .mu.l should be removed from each PCR reaction and analyzed
by agarose gel electrophoresis for the following:
[0607] The presence of a single, discrete band of the correct size.
If there is not a single, discrete band, consult the manufacturer's
recommendations for optimizing PCR reactions with the chosen
polymerase. Alternatively, the desired product may be gel purified
(see below).
[0608] Estimate the concentration of the PCR product. For TOPO.RTM.
Cloning, a 5:1 molar ratio of PCR product to TOPO.RTM. vector is
recommended to obtain the highest cloning efficiency. For example,
20 ng of a 500 bp PCR product, or 10 ng of a 1000 bp PCR product,
may be used in a TOPO.RTM. Cloning reaction. The concentration of
the PCR product may need to be adjusted before proceeding to
TOPO.RTM. Cloning. Note: If ThermalAce.TM. polymerase is being used
to produce the blunt-end PCR product, it should be noted that
ThermalAce.TM. can generate higher yields than other proofreading
polymerases. When generating PCR products in the 0.5 to 1.0 kb
range, we generally dilute the PCR reaction 1:5 in 1.times.
ThermalAce.TM. buffer before performing the TOPO.RTM. Cloning
reaction. For PCR products larger than 1.0 kb, dilution may not be
required. Setting Up the TOPO.RTM. Cloning Reaction
Introduction
[0609] Once you have produced the desired PCR product, you are
ready to TOPO.RTM. Clone it into the pENTR TOPO.RTM. vector and
transform the recombinant vector into TOP10 E. coli. It is
important to have everything you need set up and ready to use to
ensure that you obtain the best possible results. We suggest that
you read the sections entitled Setting Up the TOPO.RTM. Cloning
Reaction and Transforming OneShot.RTM. TOP 10 Competent Cells
before beginning. If this is the first time you have TOPO.RTM.
Cloned, perform the control reactions described below in parallel
with your samples.
[0610] If you are TOPO.RTM. Cloning in HTP format (see below), you
may transform TOP10 E. coli using Bulk TOP10 cells (500 reaction
kits) or MultiShot.TM. TOP10 cells (480 reaction kits). Depending
on which kit you are using, see the TOPO.RTM. Cloning and
transformation protocols below.
[0611] Note: Recent studies demonstrate that including salt (200 mM
NaCl, 10 mM MgCl.sub.2) in the TOPO.RTM. Cloning reaction may
result in an increase in the number of transformants. From these
results, we recommend adding salt to the TOPO.RTM. Cloning
reaction. A stock salt solution is provided in the kit for this
purpose. Please note that the amount of salt added to the TOPO.RTM.
Cloning reaction varies depending on whether you plan to transform
chemically competent cells or electrocompetent cells. For this
reason two different TOPO.RTM. Cloning reactions are provided to
help you obtain the best possible results.
Transforming Chemically Competent E. coli
[0612] For TOPO.RTM. Cloning and transformation into chemically
competent E. coli, adding sodium chloride and magnesium chloride to
a final concentration of 200 mM NaCl, 10 mM MgCl.sub.2 in the
TOPO.RTM. (Cloning reaction increases the number of colonies over
time. A Salt Solution (1.2 M NaCl, 0.06 M MgCl.sub.2) is provided
to adjust the TOPO.RTM. Cloning reaction to the recommended
concentration of NaCl and MgCl.sub.2.
Transforming Electrocompetent E. coli
[0613] For TOPO.RTM. Cloning and transformation of electrocompetent
E. coli, salt may also be included in the TOPO.RTM. Cloning
reaction, but the amount of salt must be reduced to 50 mM NaCl, 2.5
mM MgCl.sub.2 to prevent arcing when electroporating. Dilute the
Salt Solution 4-fold with water to prepare a 300 mM NaCl, 15 mM
MgCl.sub.2 solution for convenient addition to the TOPO.RTM.
Cloning reaction.
Setting Up the TOPO.RTM. Cloning Reaction
[0614] The table below describes how to set up your TOPO.RTM.
Cloning reaction (6 .mu.l) for eventual transformation into either
chemically competent One Shot! TOP0 E. coli or electrocompetent E.
coli. Additional information on optimizing the TOPO.RTM. Cloning
reaction for your needs can be found below. If you generated your
PCR product using ThermalAce-polymerase, please note that you may
need to dilute your PCR reaction before proceeding.
[0615] Note: The blue color of the TOPO.RTM. vector solution is
normal and is used to visualize the solution.
TABLE-US-00013 TABLE 7 Setting Up a TOPO .RTM. Cloning Reaction
Mixture. Chemically Competent Electrocompetent Reagents* E. coli E.
coli Fresh PCR product 0.5-4 .mu.l 0.5-4 .mu.l Salt Solution 1
.mu.l -- Dilute Salt Solution (1:4) -- 1 .mu.l Sterile Water Add to
final Add to final volume of 5 .mu.l volume of 5 .mu.l TOPO .RTM.
vector 1 .mu.l 1 .mu.l *Store all reagents at -20.degree. C. when
finished. Salt solutions and water can be stored at room
temperature or 4.degree. C.
Performing the TOPO.RTM. Cloning Reaction
[0616] Mix reaction gently and incubate for 5 minutes at room
temperature (22-23.degree. C.).
[0617] Note: For most applications, 5 minutes will yield plenty of
colonies for analysis. Depending on your needs, the length of the
TOP011 Cloning reaction can be varied from 30 seconds to 30
minutes. For routine subcloning of PCR products, 30 seconds may be
sufficient. For large PCR products (>1 kb) or if you are
TOPO.RTM. Cloning a pool of PCR products, increasing the reaction
time may yield more colonies.
[0618] Place the reaction on ice and proceed to Transforming One
Shot? TOP10 Competent Cells. Note: You may store the TOPO7 Cloning
reaction at -20.degree. C. overnight.
Transforming One Shot.RTM. TOP10 Competent Cells
Introduction
[0619] Once you have performed the TOPO.RTM. Cloning reaction, you
will transform your pENTR TOPO.RTM. construct into competent E.
coli. One Shots TOP10 Chemically Competent E. coli are included
with the 20 reaction kit to facilitate transformation, however, you
may also transform electrocompetent cells (see page x for ordering
information). Protocols to transform chemically competent or
electrocompetent E. coli are provided in this section.
Materials Supplied by the User
[0620] In addition to general microbiological supplies (i.e.
plates, spreaders), you will need the following reagents and
equipment.
[0621] (a) 42.degree. C. water bath (or electroporator with
cuvettes, optional)
[0622] (b) LB plates containing 50 .mu.g/mlkanamycin (two for each
transformation)
[0623] (c) 37.degree. C. shaking and non-shaking incubator
There is no blue-white screening for the presence of inserts. Most
transformants will contain recombinant plasmids with the PCR
product of interest cloned in the correct orientation. Sequencing
primers are included in the kit to sequence across an insert in the
multiple cloning site to confirm orientation and reading frame.
Preparing for Transformation
[0624] For each transformation, you will need one vial of competent
cells and two selective plates.
[0625] Equilibrate a water bath to 42.degree. C. (for chemical
transformation) or set up your electroporator if you are using
electrocompetent E. coli.
[0626] For electroporation, dilute a small portion of the Salt
Solution 4-fold to prepare Dilute Salt Solution (e.g. add 5 .mu.l
of the Salt Solution to 150 sterile water).
[0627] Warm SOC medium to room temperature.
[0628] Warm LB plates containing 50 .mu.g/ml kanamycin at
37.degree. C. for 30 minutes.
[0629] Thaw on ice 1 vial of One Shot.RTM. TOP 10 cells for each
transformation.
[0630] Important: Please note that directional TOPO.RTM. Cloning
generally yields 5 to 10-fold fewer colonies than traditional
bidirectional TOPO TA Cloning.RTM.. When directionally TOPO.RTM.
Cloning a 750 bp test insert, we generally obtain 1800-3000
colonies using the protocol described herein. Although fewer total
colonies are obtained, greater than 90% of the colonies will
contain plasmid with your PCR insert in the correct
orientation.
One Shot.RTM. TOP10 Chemical Transformation Protocol
[0631] 1. Add 2 .mu.l of the TOPO.RTM. Cloning reaction from
Performing the TOPO.RTM. Cloning Reaction (above) into a vial of
One Shot TOP10 Chemically Competent E. coli and mix gently. Do not
mix by pipetting up and down.
[0632] 2. Incubate on ice for 5 to 30 minutes.
[0633] Note: Longer incubations on ice seem to have a minimal
effect on transformation efficiency. The length of the incubation
is at the user's discretion.
[0634] 3. Heat-shock the cells for 30 seconds at 42.degree. C.
without shaking
[0635] 4. Immediately transfer the tubes to ice.
[0636] 5. Add 250 .mu.l of room temperature SOC medium.
[0637] 6. Cap the tube tightly and shake the tube horizontally (200
rpm) at 37.degree. C. for 30 minutes.
[0638] 7. Spread 50-200 .mu.l from each transformation on a
prewarmed selective plate and incubate overnight at 37.degree. C.
We recommend that you plate two different volumes to ensure that at
least one plate will have well-spaced colonies.
[0639] 8. An efficient TOPO7 Cloning reaction may produce several
hundred colonies. Pick .about.5 colonies for analysis (see
Analyzing Transformants, below).
Transformation by Electroporation
[0640] Use ONLY electrocompetent cells for electroporation to avoid
arcing. Do not use the One Shot.RTM. TOP10 chemically competent
cells for electroporation.
[0641] 1. Add 2 .mu.l of the TOPO.RTM. Cloning reaction from
Performing the TOPO.RTM. Cloning Reaction (above) into a 0.1 cm
cuvette containing 50 .mu.l of electrocompetent E coli and mix
gently. Do not mix by pipetting up and down. Avoid formation of
bubbles.
[0642] 2. Electroporate your samples using your own protocol and
your electroporator.
[0643] Note: If you have problems with arcing, see below.
[0644] 3. Immediately add 250 .mu.l of room temperature SOC
medium.
[0645] 4. Transfer the solution to a 15 ml snap-cap tube (i.e.
Falcon) and shake for at least 1 hour at 37.degree. C. to allow
expression of the kanamycin resistance gene.
[0646] 5. Spread 20-100 .mu.l from each transformation on a
prewarmed selective plate and incubate overnight at 37.degree. C.
To ensure even spreading of small volumes, add 20 .mu.l of SOC. We
recommend that you plate two different volumes to ensure that at
least one plate will have well-spaced colonies.
[0647] 6. An efficient TOPO7 Cloning reaction may produce several
hundred colonies. Pick .about.5 colonies for analysis (see
Analyzing Transformants, below).
[0648] Addition of the Dilute Salt Solution in the TOPO.RTM.
Cloning Reaction brings the final concentration of NaCl and
MgCl.sub.2 in the TOPO.RTM. Cloning Reaction to 50 mM and 2.5 mM,
respectively. To prevent arcing of your samples during
electroporation, the volume of cells should be between 50 and 80
.mu.l (0.1 cm cuvettes) or 100 to 200 .mu.l (0.2 cm curettes).
[0649] If you experience arcing during transformation, try one of
the following suggestions:
[0650] Reduce the voltage normally used to charge your
electroporator by 10%
[0651] Reduce the pulse length by reducing the load resistance to
100 ohms
[0652] Ethanol precipitate the TOPO.RTM. Cloning reaction and
resuspend in water prior to electroporation.
High-Throughput Applications
[0653] The 480 and 500 reaction pENTR and pENTR/SD Directional
TOPO.RTM. Cloning Kits are specifically designed to allow
production of GATEWAY.TM. entry clones for use in high-throughput
(HTP) applications. In these kits, the pENTR TOPO.RTM. vector is
provided in bulk and chemically competent TOP10 E. coli are
provided in a choice of two formats:
[0654] Cells are provided in bulk aliquots of 5 ml to allow simple
transfer of the cells from a sterile trough into a 96-well plate
containing the TOPO7 Cloning reaction (Catalog nos. K2400-500 and
K2420-500).
[0655] Cells are provided pre-aliquoted in 96-well plates (in
12-well stripwells) to allow addition of the TOPO7 Cloning reaction
to the cells (Invitrogen Corporation, Carlsbad, Calif.; Catalog
nos. K2400-480 and K2420-480).
HTP TOPO.RTM. Cloning and Transformation with Bulk Cells
Description
[0656] In this protocol, the TOPO.RTM. Cloning reaction is set up
in a 96-well U bottom, polystyrene plate (Costar, Catalog no. 3366,
330 .mu.l/well) and the TOP10 competent cells are placed in a
trough for dispensing.
[0657] Before Starting
[0658] Chill a 96-well metal heating block (VWR, Catalog no.
13259-260) on ice until the block is cold.
[0659] Bring a vial of SOC to room temperature.
[0660] Preheat a heat block or thermocycler containing a 96-well
metal block to 42.degree. C.
[0661] Note: You can also use a water bath, but be careful not to
contaminate the cells. [0662] Thaw 1 tube (5 ml) of TOP10
chemically competent E. coli on ice (30-60 minutes). [0663] Warm LB
agar plates containing 50 .mu.g/ml kanamycin to 37.degree. C. If
you plan to include a pUC19 control to test the transformation
efficiency of the cells, you will need LB agar plates containing
50-100 .mu.g/ml ampicillin. Controls: For your convenience a 50
.mu.l aliquot of competent cells is provided to perform a test
TOPO.RTM. Cloning and transformation reaction. In addition, you can
include the pUC19 plasmid as an internal control (see Procedure
below).
[0664] Procedure
[0665] 1. Set up the 6 .mu.l TOPO.RTM. Cloning reaction in each
well as follows. If you include pUC19 as a control, leave 2-3 wells
empty.
PCR product 1 .mu.l
Salt Solution 1 .mu.l
Sterile Water 3 .mu.l
[0666] pENTR TOPO.RTM. vector 1 .mu.l
Final Volume 6 .mu.l
[0667] 2. Incubate 5-10 minutes at room temperature.
[0668] 3. Place the 96-well plate on the cooling block for 5
minutes.
[0669] 4. If you are including pUC19, add 1 .mu.l (10 .mu.g) of the
plasmid to 2-3 empty wells.
[0670] 5. Pour thawed TOP10 E. coli into a sterile trough and
immediately dispense 45 .mu.l/well. Gently pipet up and down 1-2
times to mix.
[0671] 6. Cover the plate with Parafilm.RTM. and incubate it on the
chilled block for 20 minutes.
[0672] 7. Transfer the plate to either the pre-warmed heat block or
the thermocycler and heat-shock the cells at 42.degree. C. for 30
seconds.
[0673] 8. Transfer the plate back to the cooling block and press
down to ensure the plate is in complete contact with the cooling
block. Incubate for 1 minute.
[0674] 9. Remove the Parafilm.RTM. and add 150 .mu.L/well of
SOC.
[0675] 10. Re-cover the plate and incubate the plate at 37.degree.
C. for 1 hour. Note: Gentle shaking (125 RPM) is optional.
[0676] 11. Plate 50 .mu.L from each well onto LB agar plates
containing 50 .mu.g/ml kanamycil. For the pUC19 controls, plate 10
.mu.l of the transformation mixture plus 20 .mu.l of SOC on LB
plates containing 100 .mu.g/ml ampicillin. Incubate overnight at
37.degree. C.
[0677] 12. The next day, select 5-10 colonies and process as
desired.
[0678] Too Many Colonies
[0679] If you obtain too many colonies, reduce the amount of
bacterial culture plated and/or dilute the transformation with
additional SOC.
[0680] HTP TOPO Cloning and Transformation with MultiShot.TM.
Cells
[0681] Description
[0682] In this protocol, the TOPO.RTM. Cloning reaction is set up
in a 96-well plate and 2 .mu.l are transferred to each well of a
96-well MultiShot.TM. plate containing 15 .mu.l of chemically
competent TOP 10 E. coli per well.
[0683] Before Starting [0684] Chill two 96-well metal heating
blocks (VWR, Catalog no. 13259-260) on ice until the blocks are
cold. [0685] Bring a vial of SOC to room temperature. [0686] Warm
LB agar plates containing 50 .mu.g/ml kanamycin to 37.degree. C. If
you plan to include a pUC19 control to test the transformation
efficiency of the cells, you will need LB agar plates containing
50-100 .mu.g/ml ampicillin. [0687] Pre-heat a heat block or
thermocycler containing a 96-well metal block to 42.degree. C.
[0688] Note: You can also use a water bath, but be careful not to
contaminate the cells. [0689] If you are using a thermocycler,
program the machine to hold the temperature at 42.degree. C.
[0690] Controls: A test plate containing 1 row (12 wells) of TOP10
cells is included to perform test TOPO.RTM. Cloning reactions and
transformations. In addition, you can include the pUC19 plasmid as
an internal control (see Procedure below).
[0691] Procedure
[0692] 1. In a 96-well plate, set up the following 6 .mu.l
TOPO.RTM. Cloning reaction in each well.
PCR product 1 .mu.l
Salt Solution 1 .mu.l
Sterile Water 3 .mu.l
[0693] pENTR TOPO.RTM. vector 1 .mu.l
Final Volume 6 .mu.l
[0694] 2. Incubate 5-10 minutes at room temperature.
[0695] 3. Place the 96-well plate on one of the cooling blocks for
5 minutes.
[0696] 4. Remove a 96-well MultiShot.TM. plate of chemically
competent TOP10 E. coli from the freezer and place it in the second
cooling block. Cells should thaw within 30 seconds.
[0697] 5. Carefully remove the aluminum foil seal.
[0698] 6. Use a multi-channel pipet to add 2 .mu.l of each
TOPO.RTM. Cloning reaction (.about.3.3 ng) to each well of the
96-well plate containing cells. Keep the volume around 2 .mu.l for
uniform results. For the pUC19 control, add 1 .mu.l (10 .mu.g) of
the DNA.
[0699] 7. Cover the cells with the supplied plastic lid and
incubate the cells and DNA in the chilled block for 20 minutes.
[0700] 8. Transfer the cell plate to either the pre-warmed heat
block or thermocycler and heat-shock for 30 seconds at 42.degree.
C.
[0701] 9. Transfer the cell plate back to a cooling block, press
the plate into the block and allow the plate to cool for 1
minute.
[0702] 10. Remove the plastic lid and add 90 .mu.l SOC to each
well.
[0703] 11. Cover the plate with the lid and incubate the plate at
37.degree. C. for 1 hour. Note: Gentle shaking (125 RPM) is
optional.
[0704] 12. Plate 100 .mu.l from each well onto LB agar plates
containing 50 .mu.g/ml kanamycin. For the pUC19 controls, plate 10
.mu.l of the transformation mixture plus 20 .mu.l of SOC on LB
plates containing 100 .mu.g/ml ampicillin. Incubate overnight at
37.degree. C.
[0705] NOTE: If you obtain too many colonies, you can reduce the
amount of cells plated or dilute the TOPO.RTM. Cloning reactions
with sterile water or TE buffer prior to adding the reaction to the
cells.
[0706] Analyzing Transformants
[0707] Analyzing Positive Clones
[0708] 1. Pick 5 colonies and culture them overnight in LB or SOB
medium containing 50-100 .mu.g/ml kanamycin.
[0709] 2. Isolate plasmid DNA using your method of choice. If you
need ultra-pure plasmid DNA for automated or manual sequencing, we
recommend using the S.N.A.P.J MidiPrep Kit (Catalog no.
K1910-01).
[0710] 3. Analyze the plasmids by restriction analysis to confirm
the presence and correct orientation of the insert. Use a
restriction enzyme or a combination of enzymes that cut once in the
vector and once in the insert.
[0711] Sequencing
[0712] You may sequence your construct to confirm that your gene is
cloned in the correct orientation. The M13 Forward (-20) and M13
Reverse primers are included in the kit to help you sequence your
insert. The M13 Forward (-20) and M13 Reverse primers are also
available separately from Invitrogen Corporation, Carlsbad,
Calif.
[0713] Important: If you download the sequence for
pENTR/D-TOPO.RTM. or pENTR/SD/D-TOPO.RTM. from the Invitrogen
Corporation Web site (see description for FIG. 22), note that the
overhang sequence (GTGG) will be shown already hybridized to CACC.
No DNA sequence analysis program allows us to show the overhang
without the complementary sequence.
[0714] Analyzing Transformants by PCR
[0715] You may analyze positive transformants using PCR. For PCR
primers, use a combination of the M13 Forward (-20) primer or the
M13 Reverse primer and a primer that hybridizes within your insert.
You will have to determine the amplification conditions. If you are
using this technique for the first time, we recommend performing
restriction analysis in parallel. Artifacts may be obtained because
of mispriming or contaminating template.
[0716] The protocol below is provided for your convenience. Other
protocols are suitable.
[0717] 1. Prepare a PCR cocktail consisting of PCR buffer, dNTPs,
primers, and Taq polymerase. Use a 20 .mu.l reaction volume.
Multiply by the number of colonies to be analyzed (e.g. 5).
[0718] 2. Pick 5 colonies and resuspend them individually in 20
.mu.l of the PCR cocktail (remember to make a patch plate to
preserve the colonies for further analysis).
[0719] 3. Incubate reaction for 10 minutes at 94.degree. C. to lyse
cells and inactivate nucleases.
[0720] 4. Amplify for 20 to 30 cycles.
[0721] 5. For the final extension, incubate at 72.degree. C. for 10
minutes. Store at -4.degree. C.
[0722] 6. Visualize by agarose gel electrophoresis.
[0723] Important: If you have problems obtaining transformants or
the correct insert, perform the control reactions described herein.
These reactions will help you troubleshoot your experiment.
[0724] Long-Term Storage
[0725] Once you have identified the correct clone, be sure to
purify the colony and make a glycerol stock for long term storage.
We recommend that you store a stock of plasmid DNA at -20.degree.
C.
[0726] 1. Streak the original colony out for single colony on LB
plates containing 50 .mu.g/ml kanamycin.
[0727] 2. Isolate a single colony and inoculate into 1-2 ml of LB
containing 50 .mu.g/ml kanamycin.
[0728] 3. Grow until culture reaches stationary phase.
[0729] 4. Mix 0.85 ml of culture with 0.15 ml of sterile glycerol
and transfer to a cryovial.
[0730] 5. Store at -80.degree. C.
[0731] Recombining the Entry Construct with a Destination
Vector
[0732] Once you have obtained your entry clone, you may recombine
the pENTR TOPO.RTM. construct with any GATEWAY.TM. destination
vector of choice to generate an expression clone. This "LR"
recombination reaction is mediated by LR CLONASE.TM., a cocktail of
recombination proteins. LR CLONASE.TM. Enzyme Mix is available from
Invitrogen Corporation (Carlsbad, Calif.). In certain such methods,
for example, TOPO-adapted vectors are incubated with one or more
nucleic acid segments (e.g., one or more PCR products) at room
temperature (e.g., about 20-20.degree. C.) for about 5-30 (and
preferably about 10) minutes; the reaction is then heat-treated by
incubation at about 80.degree. C. for about 20 minutes, and the
reaction mixture then used in a standard LR reaction according to
manufacturer's instructions (Invitrogen Corporation, Carlsbad,
Calif.), except the incubation time for the LR reaction is
increased to about 3 hours.
[0733] Optimizing the TOPO.RTM. Cloning Reaction
[0734] Speeding up the Cloning Process. The high efficiency of
TOPO.RTM. Cloning allows you to streamline the cloning process. If
you routinely clone PCR products and wish to speed up the process,
consider the following: [0735] Incubate the TOPO.RTM. Cloning
reaction for only 30 seconds instead of 5 minutes.
[0736] You may not obtain the highest number of colonies, but with
the high efficiency of TOPO.RTM. Cloning, most of the transformants
will contain your insert. [0737] After adding 3 .mu.l of the
TOPO.RTM. Cloning reaction to chemically competent cells, incubate
on ice for only 5 minutes.
[0738] Increasing the incubation time to 30 minutes does not
significantly improve transformation efficiency.
[0739] Obtaining More Transformants. If you are TOPO.RTM. Cloning
large PCR products, toxic genes, or cloning a pool of PCR products,
you may need more transformants to obtain the clones you want. To
increase the number of colonies: [0740] Incubate the
salt-supplemented TOPO.RTM. Cloning reaction for 20 to 30 minutes
instead of 5 minutes.
[0741] Increasing the incubation time of the salt-supplemented
TOPO.RTM. Cloning reaction allows more molecules to ligate,
increasing the transformation efficiency. Addition of salt appears
to prevent topoisomerase I from rebinding and nicking the DNA after
it has ligated the PCR product and dissociated from the DNA. [0742]
Titrate the amount of PCR product used in the TOPO7 Cloning
reaction for maximum colony output.
[0743] Cloning Dilute PCR Products
[0744] To clone dilute PCR products, you may: [0745] Increase the
amount of the PCR product [0746] Incubate the TOPO.RTM. Cloning
reaction for 20 to 30 minutes [0747] Concentrate the PCR
product
[0748] Performing the Control Reactions
[0749] Introduction
[0750] We recommend performing the following control TOPO.RTM.
Cloning reactions the first time you use the 20 reaction kit to
help you evaluate your results. Performing the control reactions
involves producing a control PCR product using the reagents
included in the kit and using this product directly in a TOPO.RTM.
Cloning reaction.
[0751] Before Starting
[0752] For each transformation, prepare two LB plates containing 50
.mu.g/ml kanamycin.
[0753] Producing the Control PCR Product
[0754] Use your thermostable, proofreading polymerase and the
appropriate buffer to amplify the control PCR product. Follow the
manufacturer's recommendations for the polymerase you are
using.
[0755] 1. To produce the 750 bp control PCR product, set up the
following 50 .mu.l PCR: Control DNA Template (100 ng) 1 .mu.l
10.times.PCR Buffer (appropriate for enzyme) 5 .mu.l dNTP Mix 0.5
.mu.l Control PCR Primers (0.1 .mu.g/.mu.l each) 1 .mu.l
Sterile Water 41.5 .mu.l
[0756] Thermostable polymerase (1-2.5 units/.mu.l) 1 .mu.l
Total Volume 50 .mu.l
[0757] 2. Overlay with 70 .mu.l (1 drop) of mineral oil.
[0758] 3. Amplify using the following cycling parameters:
TABLE-US-00014 Stop Time Temperture Cycles Initial Denaturation 2
minutes 94.degree. 1X Denaturation 1 minutes 94.degree. Annealing 1
minutes 55.degree. Extension 1 minutes 72.degree. 25X Final
Extension 7 minutes 72.degree. 1X
[0759] 4. Remove 10 .mu.l from the reaction and analyze by agarose
gel electrophoresis. A discrete 750 bp band should be visible.
Proceed to the Control TOPO7 Cloning Reactions.
[0760] Control TOPO.RTM. Cloning Reactions
[0761] Using the control PCR product produced on the previous page
and the pENTR is TOPO.RTM. vector, set up two 6 .mu.l TOPO.RTM.
Cloning reactions as described below.
[0762] 1. Set up control TOPO.RTM. Cloning reactions: 14 Reagent
"Vector Only" "Vector+PCR Insert" Sterile Water 4 .mu.l 3 .mu.l
Salt Solution or Dilute 1 .mu.l 1 .mu.l Salt Solution Control PCR
Product--1 .mu.l pENTR TOPO.RTM. 1 .mu.l 1 .mu.l vector
TABLE-US-00015 Reagent "Vector Only" "Vector + PCR Insert" Sterile
Water 4 .mu.l 3 .mu.l Salt Solution or Dilute Salt 1 .mu.l 1 .mu.l
Solution Control PCR Product -- 1 .mu.l pENTER TOPO vector 1 .mu.l
1 .mu.l
[0763] 2. Incubate at room temperature for 5 minutes and place on
ice.
[0764] 3. Transform 3 .mu.l of each reaction into separate vials of
One Shot.RTM. TOP10 cells.
[0765] 4. Spread 100-200 .mu.l of each transformation mix onto LB
plates containing 50 .mu.g/ml kanamycin. Be sure to plate two
different volumes to ensure that at least one plate has well-spaced
colonies.
[0766] 5. Incubate overnight at 37.degree. C.
[0767] Analysis of Results
[0768] Hundreds of colonies from the vector+PCR insert reaction
should be produced. To analyze the transformations, isolate plasmid
DNA and digest with the appropriate restriction enzymes. Greater
than 90% of the colonies should contain the 750 bp insert in the
correct orientation. Relatively few colonies should be produced in
the vector-only reaction.
[0769] Transformation Control
[0770] pUC19 plasmid is included to check the transformation
efficiency of the One Shot.RTM. TOP10 competent cells. Transform
one vial of One Shot.RTM. TOP10 cells with 10 .mu.g of pUC19 using
the protocol described above. Plate 10 .mu.l of the transformation
mixture plus 20 .mu.l of SOC on LB plates containing 100 .mu.g/ml
ampicillin. Transformation efficiency should be -1.times.10.sup.9
cfu/.mu.g DNA.
[0771] Factors Affecting Cloning Efficiency
[0772] Please note that lower cloning efficiencies will result from
the following variables. Most of these are easily corrected, but if
you are cloning large inserts, you may not obtain the expected 90%
directional cloning efficiency.
TABLE-US-00016 Variable Solution Low efficiency of Forward primner
should contain CACC at directional cloning 5' end. Reverse primer
is complementary to the overhang at the 5' end. Re-design primer to
avoid base pairing to the overhang. pH > 9 in PCR Check the pH
of the PCR amplification amplification reaction reaction and adjust
with 1M Tris-HCl, pH 8. Incomplete extension Be sure to include a
final extension step of during PCR 7 to 30 minutes during PCR.
Longest PCR products will need a longer extension time. Cloning
large inserts Increase amount of insert or gel-purify as (>1 kh)
described on pages 25-26. Excess (or overly dilute) Reduce (or
concentrate) the amount of PCR PCR product product. PCR cloning
artifacts TOPO .RTM. Cloning is very efficient for small ("false
positives") fragments (<100 bp) present in certain PCR
reactions. Gel-purify your PCR product or optimize your PCR.
[0773] Gel Purifying PCR Products
[0774] Introduction
[0775] Smearing, multiple banding, primer-dimer artifacts, or large
PCR products (>3 kb) may necessitate gel purification. If you
wish to purify your PCR product, be extremely careful to remove all
sources of nuclease contamination. There are many protocols to
isolate DNA fragments or remove oligonucleotides. Please refer to
Current Protocols in Molecular Biology, Unit 2.6 (Ausubel et al.,
1994) for the most common protocols. Three simple protocols are
provided below.
[0776] Note: cloning efficiency may decrease with purification of
the PCR product (e.g. PCR product too dilute). You may wish to
optimize your PCR to produce a single band (see Producing Blunt-End
PCR Products, herein).
[0777] Using the S.N.A.P..TM. Gel Purification Kit
[0778] The S.N.A.P..TM. Gel Purification Kit available from
Invitrogen Corporation, Carlsbad, Calif. (Catalog no. K1999-25)
allows you to rapidly purify PCR products from regular agarose
gels.
[0779] 1. Electrophorese amplification reaction on a 1 to 5%
regular TAE agarose gel. (Note: Do not use TBE to prepare agarose
gels. Borate interferes with the sodium iodide step, below.)
[0780] 2. Cut out the gel slice containing the PCR product and melt
it at 65.degree. C. in 2 volumes of the 6 M sodium iodide
solution.
[0781] 3. Add 1.5 volumes Binding Buffer.
[0782] 4. Load solution (no more than 1 ml at a time) from Step 3
onto a S.N.A.P..TM. column. Centrifuge 1 minute at 3000.times.g in
a microcentrifuge and discard the supernatant.
[0783] 5. If you have solution remaining from Step 3, repeat Step
4.
[0784] 6. Add 900 .mu.l of the Final Wash Buffer.
[0785] 7. Centrifuge 1 minute at full speed in a microcentrifuge
and discard tile flowthrough.
[0786] 8. Repeat Step 7.
[0787] 9. Elute the purified PCR product in 40 .mu.l of TE or
sterile water. Use 4 .mu.l for the TOPO.RTM. Cloning reaction and
proceed as described above.
[0788] Quick S.N.A.P..TM. Method
[0789] An even easier method is to simply cut out the gel slice
containing your PCR product, place it on top of the S.N.A.P..TM.
column bed, and centrifuge at full speed for 10 seconds. Use 1-2
.mu.l of the flow-through in the TOPO.RTM. Cloning reaction. Be
sure to make the gel slice as small as possible for best
results.
[0790] Low-Melt Agarose Method
[0791] If you prefer to use low-melt agarose, use the procedure
below. Please note that gel purification will result in a dilution
of your PCR product and a potential loss of cloning efficiency.
[0792] 1. Electrophorese as much as possible of your PCR reaction
on a low-melt agarose gel (0.8 to 1.2%) in TAE buffer.
[0793] 2. Visualize the band of interest and excise the band.
[0794] 3. Place the gel slice in a microcentrifuge tube and
incubate the tube at 65.degree. C. until the gel slice melts.
[0795] 4. Place the tube at 37.degree. C. to keep the agarose
melted.
[0796] 5. Add 4 .mu.l of the melted agarose containing your PCR
product to the TOPO.RTM. Cloning reaction as described above.
[0797] 6. Incubate the TOPO.RTM. Cloning reaction at 37.degree. C.
for 5 to 10 minutes. This is to keep the agarose melted.
[0798] 7. Transform 2 to 4 .mu.l directly into OneShot.RTM. TOP10
cells using the method on page 13.
[0799] Note: the cloning efficiency may decrease with purification
of the PCR product. You may wish to optimize your PCR to produce a
single band.
Example 9
Optimization of Reaction Conditions for TOPO Joining Reactions
Using GATEWAY.TM. Vectors
[0800] To use TOPO Cloning procedures in conjunction with GATEWAY
vectors, the optimal conditions for the combined reactions were
investigated. In carrying out these studies, several questions were
addressed.
[0801] Sufficiency of Template for BP Reaction, and Inhibition of
BP Reaction by TOPO Reaction Components
[0802] To address these issues, TOPO Tools was used as described
elsewhere herein to generate attB1+CAT+attB2 templates. Secondary
PCR was then performed to generate sufficient template for testing
studies, and BP reactions were performed using the products. The
following reaction conditions were used for each step of the
process:
TABLE-US-00017 TOPO Joining Reaction: BP Reaction: X ng of PCR
product (see below) 2 .mu.l salt-free buffer 1 .mu.l topoisomerase
1 .mu.l TOPO Joining Product 0.5 .mu.l of 500 mM Tris 0.5 .mu.l of
pDONR222 (300 ng/.mu.l) 1 .mu.l of 40 mM NaCl 2 .mu.l of BP Clonase
(Invitrogen Corporation, Carlsbad, CA) 37.degree. C. for 15 min
room temp for 25 min .fwdarw. Proteinase Transformation (chemical)
K treatment
[0803] Following BP reactions, mixtures were chemically transformed
into chemically competent E. coli cells (e.g., TOP10; Invitrogen
Corporation Carlsbad, Calif.) and cells were plated to determine
recombination efficiency.
[0804] Results
TABLE-US-00018 1 2 3 4 5 6 Colonies 149 270 514 0 0 0 Template 0.8
ng 1.6 ng 4 ng 1.6 ng 4 ng 0 ng Used TOPO No No No Yes Yes No
Joining?
[0805] These results demonstrate that TOPO Tools generates
sufficient template for the subsequent BP reaction. In addition,
these results demonstrate that TOPO joining inhibits the subsequent
BP reaction.
[0806] Effect of Presence of attB1 and attB2 Adapters on BP
Reactions
[0807] In this portion of the studies, the effects of the presence
of excess attB1 and attB2 adapters in the reaction mixtures on the
subsequent BP reaction were examined. To address this issue,
different amounts of attB1 and attB2 adapters were added to
templates (attB1+CAT+attB2, 20 ng), and BP reactions were performed
under standard conditions (60 minutes at room temperature).
Following BP reactions, mixtures were chemically transformed into
chemically competent E. coli cells (e.g., TOP10; Invitrogen
Corporation, Carlsbad, Calif.) and cells were plated to determine
recombination efficiency.
[0808] Results:
TABLE-US-00019 1 2 3 4 5 6 Adaptor amount (ng) 20 10 5 2.5 1 0 No.
of colonies formed 270 475 760 590 340 460
[0809] These results demonstrate that the presence of an excess of
attB1 and attB2 adapters has no significant effect on the
transformation efficiencies observed, indicating that the BP
reaction is not significantly influenced by the presence of attB1
and attB2 adapters in the reaction mixture.
[0810] Removal of Inhibitors from TOPO Joining Reactions
[0811] To address the optimal methods for removing inhibitors from
TOPO Joining reactions prior to use of the products in BP
reactions, various treatment methods were assessed. TOPO Joining
reactions were performed using the following reaction mixtures,
incubated at room temperature for 5 minutes:
TABLE-US-00020 attB1 + attB2 (20 ng/.mu.l each) 2 .mu.l CAT (100
ng/.mu.l) 1.7 .mu.l attB1 + CAT + attB2 product (10 ng/.mu.l) 1
.mu.l 500 mM Tris 0.5 .mu.l Topoisomerase (1 .mu.g/.mu.l) 1
.mu.l
[0812] Following TOPO Joining reactions, seven different samples of
the reaction mixtures were treated under one of the following
conditions prior to carrying out BP reactions:
[0813] (1) add 1 .mu.l of 0.6% SDS+3 mM EDTA to one reaction,
37.degree. C. for 15 min;
[0814] (2) add 4 .mu.l of 0.6% SDS+3 mM EDTA to four reactions,
37.degree. C. for 15 min, then SNAP purify into 20 .mu.l of
water;
[0815] (3) add 4 .mu.l of 0.6% SDS+3 mM EDTA+1 .mu.l of proteinase
K (2 .mu.g/.mu.l) to 4 reactions, 37.degree. C. for 15 minutes,
then SNAP purify into 20 .mu.l of water;
[0816] (4) add 0.8 .mu.l of 2.5 M NaCl to one reaction, 37.degree.
C. for 17 minutes;
[0817] (5) add 3.2 .mu.l of 2.5 M NaCl to four reactions,
37.degree. C. for 15 min, then SNAP purify into 20 .mu.l of
water;
[0818] (6) add 3.2 .mu.l of 2.5 M NaCl and 1 .mu.l of 2 .mu.g/.mu.l
proteinase K to 4 reactions, 37.degree. C. for 15 min, then SNAP
purify into 20 .mu.l of water (positive control; 0.8 ng template
used);
[0819] (7) (negative control; no template used).
[0820] BP reactions were performed using salt-free buffer for 60
min at room temperature. For unpurified mixtures, 1 .mu.l of TOPO
Joining reaction mixture was used per 10 .mu.l of BP reaction. For
purified mixtures, 5.5 .mu.l of TOPO Joining reaction mixture was
used per 10 .mu.l of BP reaction. Following BP reactions, mixtures
were chemically transformed into chemically competent E. coli cells
(e.g., TOP10; Invitrogen Corporation, Carlsbad, Calif.) and cells
were plated to determine recombination efficiency.
[0821] Results
TABLE-US-00021 1 2 3 4 5 6 7 8 Treatment SDS SDS SDS NaCl NaCl NaCl
(+) (-) Proteinase K - - + - - + Purification - + + - + + No. of
Colonies 6 515 400 0 550 657 179 0
[0822] These results demonstrate that: (1) purification is not
necessary to carry out the BP reaction efficiently; (2) treatment
of reaction mixtures with proteinase K is not required following
TOPO Joining reactions for maximum efficiency of subsequent BP
reactions; and (3) SDS treatment and NaCl treatment of reaction
mixtures give the same transformation efficiencies (and therefore
have the same effects upon the BP reaction).
[0823] Optimization of BP Reaction Temperature
[0824] To determine the optimum reaction temperature for carrying
out BP reactions following TOPO Joining, attB1+CAT+attB2 PCR
product was used as the template for BP reactions conducted under
various temperatures. Following BP reactions, mixtures were
chemically transformed into chemically competent E. coli cells
(e.g., TOP10; Invitrogen Corporation, Carlsbad, Calif.) and cells
were plated to determine recombination efficiency.
[0825] Results
TABLE-US-00022 BP Reaction Temperature Room 42.degree. C.
37.degree. C. Temp 14.degree. C. No. of Colonies (+Template) 3 337
588 195 No. of Colonies (no Template) 0 4 0 0
[0826] These results demonstrate that room temperature (about
20-25.degree. C.) Is the optimal reaction temperature for carrying
out BP reactions.
[0827] Optimization of Molar Ratio of attB1:insert:attB2
[0828] To determine the optimal molar ratio for attB1, insert and
attB2 templates in the BP reaction, these templates were mixed in
various molar ratios and BP reactions carried out under optimal
conditions described above. Following BP reactions, mixtures were
chemically transformed into chemically competent E. coli cells
(e.g., TOP10; invitrogen Corporation, Carlsbad, Calif.) and plated
to determine recombination efficiency.
Results
TABLE-US-00023 [0829] Ratio of attB1:insert:attB2 2:1:2 1.5:1:1.5
1:1:1 1:2:1 0 (control) No. of Colonies 81 93 165 154 9
[0830] These results demonstrate that a ratio of attB1:insert:attB2
at 1:1:1 is optimal for carrying out BP reactions.
[0831] Determination of Effect of Salt on BP Reaction
[0832] To determine whether the presence of salt in the BP reaction
solution influences the recombination efficiency, BP reactions were
carried out in salt-free buffers, or in standard BP reaction
buffers containing salt.
[0833] Results
TABLE-US-00024 Buffer Salt - + +template 108 109 -template (neg.
control) 1 0
[0834] These results demonstrate that the presence or absence of
salt in the reaction buffer during the BP reaction has no impact
upon the recombination efficiency.
[0835] Determination of Optimal Number of TOPO Joining
Reactions
[0836] In the next series of studies, the question of whether one
TOPO Joining reaction is sufficient to provide optimal
recombination efficiency for BP reactions after purification was
examined. A single TOPO Joining reaction was carried out using the
following reaction mixture:
TABLE-US-00025 24 attB1 and attB2 (20 ng/.mu.l each) 0.5 .mu.l CAT
(100 ng/.mu.l) 1.7 .mu.l 500 mM Tris 0.5 .mu.l Topoisomerase (1
.mu.g/.mu.l) 1 .mu.l dH.sub.2O sufficient to bring final volume to
5 .mu.l
[0837] The reaction mixture was incubated at 37.degree. C. for 15
minutes, then 1 .mu.l of 0.6% SDS+3 mM EDTA was added; the mixture
was incubated at 37.degree. C. for 15 minutes, and then purified
using a SNAP column (see above) into 20 .mu.l of water. A BP
reaction was then carried out using the product of this TOPO
Joining reaction as follows:
TABLE-US-00026 standard BP reaction buffer 2 .mu.l pDONR222 (300
ng/.mu.l) 0.5 .mu.l TOPO Joining product (from above) 5.5 .mu.l BP
Clonase 2 .mu.l
[0838] The reaction mixture was incubated at room temperature for
60 minutes, then 1 .mu.l of 2 .mu.g/.mu.l proteinase K was added;
the mixture was incubated at 37.degree. C. for 15 minutes, and then
at 75.degree. C. for 15 minutes. 4 .mu.l of this reaction mixture
was then used for chemical transformation into chemically competent
E. coli cells (e.g., TOP10; Invitrogen Corporation, Carlsbad,
Calif.) and cells were then plated to determine recombination
efficiency.
[0839] Results (No. of Colonies Formed):
TABLE-US-00027 +Templates -Template (neg. control) 188 0
[0840] These results demonstrate that one TOPO Joining reaction
provides sufficient template to carry out an efficient BP
reaction.
[0841] Optimization of Purification Methods
[0842] Studies were also conducted to determine whether the SNAP
purification column (Invitrogen Corporation, Carlsbad, Calif.) or
the CONCERT purification system (Invitrogen Corporation, Carlsbad,
Calif.) differed in providing optimal purified template for
carrying out BP reactions after TOPO Joining TOPO Joining reactions
and BP reactions were conducted as described above, except that
some samples were purified using SNAP columns, and other samples
were purified using the CONCERT plasmid purification system after
conducting the TOPO Joining reaction. Purified samples were then
carried through a standard BP reaction, and reaction mixtures were
then used either for transformation via chemical transformation or
electroporation. Following transformation, cells were plated to
determine recombination efficiency.
[0843] Results (No. of Colonies Formed)
TABLE-US-00028 Transformation Method SNAP Concert No template (neg.
control) Chemical 188 254 0 Electroporation 8220 11,460 672
[0844] These results demonstrate that both SNAP and CONCERT
purification systems work well to provide purified template for BP
reactions after TOPO Joining reactions.
[0845] Optimal Conditions
[0846] Based on the results of the above studies taken together, it
was determined that the optimal conditions for combination TOPO
Joining-Gateway reactions are as follows:
[0847] (1) TOPO Joining Reaction
[0848] (a) attB 1/insert at 1:1 molar ratio, in 5 .mu.l reaction
volume
[0849] (b) incubate at 37.degree. C. for 15 minutes
[0850] (c) add 1 .mu.l of 0.6% SDS+3 mM EDTA; incubate at
37.degree. C. for 15 minutes
[0851] (d) purify with SNAP column or CONCERT system into 20 .mu.l
of dH.sub.2O
[0852] (2) BP Reaction
[0853] (a) prepare reaction mixture: 28 (i) purified TOPO Joining
product 5.5 .mu.l; (ii) standard BP reaction buffer 2 .mu.l; (iii)
pDONR222 (30 ng/.mu.l) 0.5 .mu.l; (iv) BP Clonase 2 .mu.l;
[0854] (b) incubate reaction mixture at room temperature for 60
minutes;
[0855] (c) add 1 .mu.l of 2 .mu.g/.mu.l proteinase K;
[0856] (d) incubate at 37.degree. C. for 15 minutes;
[0857] (e) incubate at 75.degree. C. for 15 minutes;
[0858] (3) Transformation
[0859] (a) use 2-4 .mu.l of reaction mixture from BP reaction, and
carry out either chemical transformation or electroporation.
[0860] To demonstrate the efficacy of these optimized conditions,
studies were conducted using CAT and lacZ inserts of various sizes
subjected to TOPO Joining and subsequent BP reactions, followed by
transformation and plating.
[0861] Results
[0862] Chemical Transformation
TABLE-US-00029 lacZ lacZ lacZ lacZ Insert CAT (1 kb) (1.5 kb) (2
kb) (3.2 kb) none No. of Colonies 188 180 182 177 71 3 Right-sized
Clone 10/10 18/18 16/16 17/18 18/18 --
[0863] Electrical Transformation
TABLE-US-00030 lacZ lacZ lacZ lacZ Insert CAT (1 kb) (1.5 kb) (2
kb) (3.2 kb) none No. of Colonies 8222 7335 7320 7500 6150 510
[0864] These results, taken together, demonstrate that the
conditions described above are optimal for combination TOPO
Joining-Gateway reactions on inserts of various sizes.
Example 10
Construction of a Mammalian Expression Cassette Without Secondary
PCR Methods
[0865] Preparation of Elements and Gene of Interest
[0866] The following primer sets (see Table 8 below) and templates
were used for PCR amplification of elements and gene of
interest:
[0867] (A) Primer set: Sequence #1 and #2; template: pcDNA 4/TetO.
PCR product: 5' element.
[0868] (B) Primer set: Sequence #3 and #4; template: pcDNA 3.2N5.
PCR product: 3' element.
[0869] (C) Primer set: Sequence #5 and #6; template: pcDNA 3.1/CAT.
PCR product: CAT insert.
TABLE-US-00031 TABLE 8 Primers Used for Construction of Expression
Cassette. SEQ ID NO: 51 GTTGACATTGATTATTGACTAG SEQ ID NO: 52
GTTCCGAAGGGTTAACGCTAGAGTCCGGAGGC SEQ ID NO: 53
GACTCAAAGGGAAGGTAAGCCTATCCCTAAGG SEQ ID NO: 54 GCGCAGATCTGCTATGGCAG
SEQ ID NO: 55 CGGAACAAGGGACCATGGAGAAAAAAATCACT GGATA SEQ ID NO: 56
TGAGTCAAGGGCGCCCCGCCCTGCTGCCACTC ATCG SEQ ID NO: 57
GGGGACAAGTTTGTACAAAAAAGCAGGCTTCC CTTCGGAAC SEQ ID NO: 58
GTTCCGAAGGGAAGCCTGCTTTTTTGTACAAA CTTGTCCCC SEQ ID NO: 59
GAGTCAAAGGGACCCAGCTTTCTTGTACAAAG TGGTCCCC SEQ ID NO: 60
GGGGACCACTTTGTACAAGAAAGCTGGGTCCC TTTGAGTC SEQ ID NO: 61
CACGACGTTGTAAAACGACG SEQ ID NO: 62 ATGTAATAGGAGTCACTATAGG
[0870] Platinum Taq DNA polymerase High Fidelity (Invitrogen
Corporation; Carlsbad, Calif.) was used for PCR. The PCR conditions
were as follows:
TABLE-US-00032 Components Volume Final Concentration dH.sub.2O 35.5
.mu.l 10 mM dNTP mixture (2.5 mM each) 4 .mu.l 0.2 mM each 10 X
High Fidelity PCR Buffer 5 .mu.l 1X 50 mM MgSO.sub.4 2 .mu.l 2 mM
Primer 1 (100 ng/.mu.l) 1 .mu.l Primer 2 (100 ng/.mu.l) 1 .mu.l
Template (10 ng/.mu.l) 1 .mu.l Platinum Taq High Fidelity (5
U/.mu.l) 0.5 .mu.l .mu.l
[0871] 94.degree. C.: 4 min (1 cycle)
[0872] 94.degree. C. 30 sec->55.degree. C. 30 sec->68.degree.
C. 1 min (30 cycles)
[0873] 68.degree. C. 10 min (1 cycle)
[0874] 4.degree. C. (to completion)
[0875] The following conditions were used to purify PCR generated
fragments:
[0876] Reagent: SNAP MiniPrep kit (Invitrogen Corporation,
Carlsbad, Calif.).
[0877] Steps
[0878] (1) Mix 50 .mu.l PCR product with 150 .mu.l Binding Buffer.
Mix well.
[0879] (2) Add 350 .mu.l of Isopropanol. Mix well.
[0880] (3) Load the sample onto a SNAP MiniPrep Column.
[0881] (4) Centrifuge at 14000 rpm for 1 min. Discard the column
flow through.
[0882] (5) Add 500 .mu.l of Wash Buffer and centrifuge at 14000 rpm
for 1 min. Discard the column flow through.
[0883] (6) Add 700 .mu.l of 1.times. Final Wash Buffer and
centrifuge at 14000 rpm for 1 min. Discard the column flow
through.
[0884] (7) Dry the column by centrifuge at 14000 rpm for 1 min.
[0885] (8) Transfer the column to a new centrifuge tube. Add 50
.mu.l of dH.sub.2O to the column. Incubate at room temperature for
2-5 min. Centrifuge at 14000 rpm for 1 min. Collect the flow
through.
[0886] (9) DNA concentration measurement by UV absorbance at 260
nm.
[0887] TOPO Joining Reaction
[0888] For production of expression cassettes with secondary PCR,
the following joining conditions were used: 33 5' element (700 bp)
75 ng 3' element (350 bp) 35 ng 500 mM Tris (pH7.5) 0.5 .mu.l
Topoisomerase (1 .mu.g/.mu.l) 0.5 .mu.l CAT insert (700 bp) 150 ng
dH2O enough to bring final volume to 5 .mu.l
[0889] The reaction was performed at room temperature for 5-15 min.
Half volume of the reaction was used as template for the second
round PCR with primer set sequence #1 and sequence #4. PCR
conditions were the same as above except that the extension time
was 2 min. After PCR, DNA was purified as mentioned above. Purified
DNA was used for transfection.
[0890] For production of expression cassette without secondary PCR,
the following joining conditions were used:
TABLE-US-00033 5' element (700 bp) 510 ng 3' element (350 bp) 230
ng 500 mM Tris (pH 7.5) 1.5 .mu.l Topoisomerase (1 .mu.g/.mu.l) 3
.mu.l CAT insert (700 bp) 450 ng dH.sub.2O enough to bring final
volume to 15 .mu.l
[0891] The reaction was performed at 37.degree. C. for 15 min.
Proteinase K was added to a final concentration of 50 .mu.g/ml and
the mixture was incubated at 37.degree. C. for 10 min. The treated
DNA was ready for transfection.
[0892] Gene Expression Study
[0893] Three cell lines (suspension TRex-CHO, adherent TRex-CHO and
adherent TRex-293 cell lines) were used as model cell lines to test
these expression cassettes. Standard cell culture methods were
used. Twenty-four well cell culture plates were used. Lipofectamine
2000 was used as transfection reagent. Twenty-four hours after
transfection, tetracycline was added at a final concentration of 1
.mu.g/ml. For control studies, no tetracycline was added. Cells
were incubated for another 24 hours before lysis. Western blot was
used for transfer of proteins and anti-V5 or anti-CAT antibody was
used for detection.
[0894] Results and Discussion
[0895] The purpose of this study was to demonstrate that expression
cassettes could be generated without secondary PCR. In this study,
we compared the expression data generated from an expression
cassette produced using a secondary PCR step to that obtained using
an expression cassette produced without a secondary PCR step. For
the expression cassette produced with secondary PCR, about 1.2
.mu.g/well of DNA was used for transfection into 24-well plate
format. For the expression cassette without secondary PCR, the
product from one joining reaction was used (about 1.2 .mu.g/well).
The detection data showed that functional expression cassettes can
be produced using the methods of the present invention, without
using a secondary PCR step (FIG. 30).
Example 11
Generation of Gateway Compatible Cassettes with Topo Tools
Methods
[0896] Preparation of Adaptors
[0897] Equal amounts of sequence #7 and sequence #8 (see Table 8,
above) were mixed in 40 mM NaCl and the mixture was denatured at
95.degree. C. for 5 min and slowly cooled to room temperature to
form the attB1adaptor. Equal amounts of sequence #9 and sequence
#10 (see Table 8, above) were mixed in 40 mM NaCl and the mixture
was denatured at 95.degree. C. for 5 min and slowly cooled to room
temperature to form the attB2 adaptor.
[0898] TOPO Joining
[0899] CAT insert was generated as in example 10. The joining
conditions were as optimized above (see Examples 9 and 10): 35
TABLE-US-00034 attB1 adaptor (40 bp) 10 ng attB2 adaptor (40 bp) 10
ng 500 mM Tris (pH 7.5) 0.5 .mu.l Topoisomerase (1 .mu.g/.mu.l) 1
.mu.l CAT insert (700 bp) 170 ng dH.sub.2O sufficient to bring
final volume to 5 .mu.l
[0900] The reaction was performed at 37.degree. C. for 15 min. SDS
and EDTA were added to a final concentration of 0.1% and 0.5 mM
respectively. The mixture was incubated at 37.degree. C. for 15
min.
[0901] Purification
[0902] Water (15 P.mu.l) was added to the treated mixture. DNA was
purified with SNAP MiniPrep kit (Invitrogen Corporation, Carlsbad,
Calif.).
[0903] Steps
[0904] (1) Mix the treated product with 60 .mu.l Binding Buffer.
Mix well.
[0905] (2) Add 140 .mu.l of Isopropanol. Mix well.
[0906] (3) Load the sample onto a SNAP MiniPrep Column.
[0907] (4) Centrifuge at 14000 rpm for 1 min. Discard the column
flow through.
[0908] (5) Add 500 .mu.l of Wash Buffer and centrifuge at 14000 rpm
for 1 min. Discard the column flow through.
[0909] (6) Add 700 .mu.l of 1.times. Final Wash Buffer and
centrifuge at 14000 rpm for 1 min. Discard the column flow
through.
[0910] (7) Dry the column by centrifuge at 14000 rpm for 1 min.
[0911] (8) Transfer the column to a new centrifuge tube. Add 20
.mu.l of dH.sub.2O to the column. Incubate at room temperature for
2-5 min. Centrifuge at 14000 rpm for 1 min. Collect the flow
through.
[0912] BP Reaction
TABLE-US-00035 BP reaction buffer 2 .mu.l Purified product 5.5
.mu.l pDONR 222 (300 ng/.mu.l) 0.5 .mu.l BP clonase 2 .mu.l
[0913] The reaction mixture was incubated at room temperature for
60 min then 1 .mu.l of Proteinase K (2 .mu.g/.mu.l) was added. The
mixture was incubated at 37.degree. C. for 15 min followed by 15
min at 75.degree. C. to inactive the enzyme.
[0914] Transformation
[0915] The treated mixture was transformed into TOP10 competent
cells (chemical) or eletroporated into ElectroMax competent cells.
Cells were plated onto LP-Kanamycin plates and incubated at
37.degree. C. overnight. The number of colonies was counted. To
make sure that insert was present in these colonies, we designed
primer sets (sequence #11 and #12) to do colony PCR. If insert was
present, the PCR product would have produced a band of about 700
bp; if no insert was present, however, the PCR product band would
be about 2.2 kb in size.
[0916] Results and Discussion
[0917] In this study, we wanted to demonstrate that PCR products
produced with TOPO Tools sticky ends can be directly joined to
attB1 and attB2 adaptors. The joined product can be directly used
in the BP recombination reaction to create GATEWAY.TM. entry clones
(Table 9).
TABLE-US-00036 TABLE 9 Colonies Generated from BP Reaction.
Transformation Type attB1-Cat-attB2 Vector only Chemical 188 0
Electroporation 8220 672
[0918] To further confirm the insert was present in these colonies,
we picked 18 positive colonies and 2 negative colonies to do PCR.
PCR results showed that right-sized product was present in all 18
colonies checked (FIG. 31).
[0919] The present invention has been described in some detail by
way of illustration and example for purposes of clarity of
understanding, it will be obvious to one of ordinary skill in the
art that the same can be performed by modifying or changing the
invention within a wide and equivalent range of conditions,
formulations and other parameters without affecting the scope of
the invention or any specific embodiment thereof, and that such
modifications or changes are intended to be encompassed within the
scope of the appended claims.
[0920] The following commonly owned, co-pending U.S. patent
applications are incorporated herein by reference in their
entireties: U.S. Provisional Appl. No. 60/254,510, filed Dec. 8,
2000; U.S. application Ser. No. 09/732,914, filed Dec. 11, 2000;
U.S. Provisional Appl. No. 60/291,972, filed May 21, 2001; U.S.
Provisional Appl. No. 60/318,902, filed Sep. 14, 2001; and U.S.
Provisional Appl. No. 60/326,092, filed Sep. 28, 2001.
[0921] All publications, patents and patent applications mentioned
in this specification are indicative of the level of skill of those
skilled in the art to which this invention pertains, and are herein
incorporated by reference to the same extent as if each individual
publication, patent or patent application was specifically and
individually indicated to be incorporated by reference.
Example 12
[0922] In vitro transcription with the T7 bacteriophage promoter
and RNA polymerase is commonly used to generate RNAs for downstream
studies such as probing of Northern blots, RNase protection assays,
and RNA interference. In order to produce the template molecule,
the sequence of interest is usually cloned into a vector downstream
of a T7 promoter sequence or PCR amplified with primers including
20-30 nt T7 promoters at their 5' ends. The first method requires
subcloning, growth, isolation, and sometimes sequencing of the
recombinant plasmid. The second is rapid but requires 40-50 nt
primers designed and synthesized in advance of transcription.
[0923] T7 TOPO linkers provide a way to quickly and easily add a T7
promoter to an existing PCR product without the need to order new
primers and without subcloning. A TOPO-charged linker containing
the T7 promoter sequence is joined to a Taq-generated PCR product
in a 15 minute reaction. A secondary amplification with a
linker-specific primer and one of the original gene-specific
primers produces ample template for as many T7 transcription
reactions as needed and determines the orientation of the RNA that
will be produced.
[0924] Here we test conditions for purification of a T7 TOPO
linker, and we show that it is capable of efficient ligation to
actin and GFP PCR products and can direct T7 transcription from
secondarily amplified templates at levels comparable to primary
amplification products with a T7 promoter in one primer.
[0925] Materials and Methods
[0926] The following materials may be used to prepare T7 promoter
linkers and attach them to a PCR product (e.g., the actin ORF), and
then isolate a construct having the T7 promoter attached to the PCR
product in the desired orientation such that an RNA molecule can be
transcribed with a T7 polymerase that corresponds to the sense or
the antisense or both strands of the PCR fragment (see FIG. 42). T7
TOPO linkers are prepared ase described below. A T7 secondary
amplification primer having a sequence that anneals to all or a
portion of the T7 promoter such that extension of the primer is in
the direction of the attached PCR product. Various buffer,
nucleotide and/or salt solutions may be mployed in the reactions
described, for example, salt solution (available from Invitrogen
Corporation, Carlsbad Calif., catalog number 46-0205), 10.times.PCR
buffer (available from Invitrogen Corporation, Carlsbad Calif.,
catalog number 46-0121), 10 mM dNTPs (available from Invitrogen
Corporation, Carlsbad Calif., catalog number 46-0344).
[0927] As an example of the use of the methods of the present
invention, a PCR fragment containing all or portions of the actin
gene is prepared using forward and reverse actin control primers
described below to amplify a fragment from an actin control
template. After attachment of the T7 promoter to the fragment, in
vitro transcription was performed using a commercially available T7
transcription kit.
[0928] Construction and Purification of a T7 Promoter Linker
[0929] The following oligos were synthesized and gel-purified:
TABLE-US-00037 T7topG (SEQ ID NO: 124)
5'-pGACTCGTAATACGACTCACTATAGGGCCCTTATTCCGATAGTG-3' T7botG (SEQ ID
NO: 125) pAGGGCCCTATAGTGAGTCGTATTACGAGTCAAAAAAAAAAAA TOPO-5 (SEQ ID
NO: 126) pCAACACTATCGGAATA
[0930] A total of 50 .mu.g oligos were annealed in a 1:1:3 molar
ratio (T7topG:T7botG:TOPO-5) in 1.times.PNK buffer (New England
Biolabs) and 200 mM NaCl by incubation in a thermal cycler for 5
min at 95.degree. C., 5 min at 65.degree. C., 5 min at 37.degree.
C., and 5 min at 25.degree. C. 10 .mu.g of annealed oligos were
charged in a 400 .mu.l reaction consisting of 1.times.PNK buffer
(New England Biolabs) with 1 mM ATP, 20 U of polynucleotide kinase
(New England Biolabs), and 50 .mu.g Vaccinia topoisomerase I for 15
min at 37.degree. C. This results in the attachment of the
topoisomerase to the 3' terminus of the T7 promoter linker that is
to be attached to the PCR product.
[0931] The topoisomerase-charged promoter linker was purified using
an Akta-FPLC and Unicom software ver. 4.00 (Amersham). 360 .mu.l of
the linking reaction was loaded onto a 1 ml HiTrap SP Sepharose HP
pre-packed column (Amersham) pre-equilibrated in buffer A (50 mM
Tris-HCl pH 7.0), washed at 0.5 ml/min with 11 ml buffer A
(collected in 1 ml fractions), and eluted with a 5 ml, 0-100%
buffer B (50 mM Tris-HCl pH 7.0, 1M NaCl) gradient followed by 3 ml
of 100% buffer B. The eluate was collected in 0.2 ml fractions.
Representative chromatograms of the FPLC purification are shown in
FIG. 43.
[0932] The peak fractions were identified by treating 4 .mu.l of
"load" fraction (unpurified linking reaction), 10 .mu.l of each
flow-through fraction, and 16 .mu.l of each eluate fraction with 5
.mu.g proteinase K for 30 min prior to loading on a 10%
polyacrylamide Novex TBE gel and electrophoresing for 45 min at
200V. The gel was stained for 30 min in 0.2 .mu.g/ml ethidium
bromide and destained by washing 10 min in ddH.sub.2O. Linker
concentration can be estimated by running low DNA mass ladder on
the same gel and comparing band intensities (see FIG. 44A).
[0933] The location of free topoisomerase among the fractions was
determined by loading the same fraction volumes as above but
without proteinase K treatment onto Novex 4-12% Tris-Bis NUPAGE
gels. 0.3 .mu.g of three topoisomerase were run for comparison. The
gels were electrophoresed for 35 min at 200V in MES buffer and
stained with Coomassie R-250 (see FIG. 44B).
[0934] Three peak fractions (#33-35) of T7 TOPO linker were pooled,
diluted with 2 volumes of storage buffer (60% glycerol, 67 .mu.g/ml
BSA, 50 mM Tris-HCl pH 7.4, 0.3 mM EDTA, 1.3 mM DTT, 0.07% Triton-X
100), and stored at -20.degree. C.
[0935] Plasmid Templates
[0936] pcDNA5/FRT/TO/GFP was from Invitrogen Corporation.
[0937] pBAD/TOPO-actin-as was created by TOPO cloning a blunt PCR
product amplified with an actin forward, actinF, primer having the
sequence 5'-GCTCACCATGGATGATGATATCGC-3' (SEQ ID NO:127) and an
actin reverse, actinR, primer having the sequence
5'-GGAGGAGCAATGATCTTGATCTTC-3' (SEQ ID NO:128) from the HeLa cDNA
PCR control template (available from Invitrogen Corporation,
Carlsbad Calif., catalog number 46-0324) into pBAD/TOPO in the
antisense orientation.
[0938] pUC 19/actin (FIG. 46A) was created by cloning of a
BamHI-HindIII digested PCR product amplified from the HeLa cDNA
template with BamHI-actinF primer having the sequence
5'-CACGGATCCGCTCACCATGGATGATGATAT-CGC-3' (SEQ ID NO:129) and
actinR-HindIII primer having the sequence
5'-CACAAGCTTGGAGGAGCAATGATCTTGATCTTC (SEQ ID NO:130) into
BamHI-HindIII digested pUC19.
[0939] PCR
[0940] 50 .mu.l reactions were used for both primary and secondary
amplifications using 10 .mu.mol each primer, 0.2 mM dNTPs,
1.times.PCR buffer (from 10.times. stock, Invitrogen Corporation,
Carlsbad Calif.), and 2.5 U Platinum Taq DNA polymerase or
Recombinant Taq DNA polymerase. Primary reactions were performed
using 1 ng of pBAD/TOPO-actin-as, pUC19/actin, or pcDNA5/FRT/TO/GFP
plasmids as templates and actinF+actinR or GFPstart
(5'-ATGGCTAGCAAAGGAGAAGAACTTT-3' (SEQ ID NO:131))+GFPstop2
(5'-TTATTTGTAGAGCTCATCCATGCCA-3' (SEQ ID NO:132)) primers. For
transcription control templates, the GFP and actin forward primers
were paired with reverse primers appended with a 5' T7 promoter
sequence (5'-GATGACTCGTAATACGACTCACTATAGGG-3' (SEQ ID NO:133)).
Secondary reactions were the same as the primary reactions except 1
.mu.l of T7 TOPO linking reaction was used as template and either
actinF or GFPstart primers were combined with the linker-specific
primer T7ampl (5'-GATGACTCGTAATACGACTCA-CTA-3' (SEQ ID
NO:134)).
[0941] GFP primary and secondary amplifications were incubated for
2 min at 94.degree. C. followed by 30 cycles of 94.degree. C. for
15s, 57.degree. C. for 30s, and 72.degree. C. for 45s. Actin
primary and secondary amplifications were incubated for 2 min at
94.degree. C. followed by 30 cycles of 94.degree. C. for 15s,
58.degree. C. for 30s, and 72.degree. C. for 1 min. All
amplifications included a final extension step of 7 min at
72.degree. C.
[0942] PCR product concentrations were estimated by running on 1.2%
agarose-TAE gels with Low DNA Mass ladder and comparing band
intensities.
[0943] T7 TOPO Linking Reactions
[0944] Linking was performed by combining 1 .mu.l of pooled T7 TOPO
linker eluate fractions in storage buffer with 1 .mu.l of primary
GFP or actin PCR reaction, 3 .mu.l ddH.sub.2O, and 1 .mu.l salt
solution (1.2M NaCl, 60 mM MgCl.sub.2) and incubating at 37.degree.
C. for 15 min. Reactions were checked by running 8 .mu.l from a
double reaction on a 6% polyacrylamide Novex TBE gel for 1 hr at
200V, staining in 0.2 .mu.g/ml ethidium bromide, and destaining for
10 min in ddH.sub.2O. For the negative control, 1.times.TOPO
storage buffer (2 vol. storage buffer, above, +1 vol. buffer A) was
substituted for the pooled TOPO linker eluate in vitro
transcription
[0945] 1 .mu.l-1.5 .mu.l of secondary PCR reaction was mixed with 2
.mu.l 75 mM NTPs (Amersham), 4.5 .mu.l ddH.sub.2O, 1 .mu.l
10.times. transcription buffer (400 mM Tris-HCl pH 8.0, 100 mM DTT,
20 mM spermidine, 100 mM MgCl.sub.2), and 1.5 .mu.l enzyme mix (4
parts 50 U/.mu.l T7 RNA polymerase, 1 part 40 U/.mu.l RNaseOUT, 1
part 0.6 U/.mu.l yeast inorganic pyrophosphatase). Reactions were
incubated for 1 hr at 37.degree. C. followed by addition of 0.51
.mu.l DNase I (50 U/.mu.l) and continued incubation for 15 min. 0.5
.mu.l of each reaction was then mixed with 4.5 .mu.l ddH.sub.2O and
5 .mu.l of Gel Loading Buffer II (Ambion), denatured for 5 min at
95.degree. C., cooled on ice for 5 min, and run on a 1.2%
agarose-TAE gel for 45 min at 100V. The gel was stained for 30 min
in 1 .mu.g/ml ethidium bromide and destained for 10 min in
ddH.sub.2O.
[0946] Results
[0947] T7 TOPO Linker Purification by FPLC
[0948] The T7 TOPO linker is produced by annealing three oligos,
charging with Vaccinia topoisomerase I, and purifying over SP
Sepharose as described in the Materials & Methods. The final
product is a double-stranded oligo covalently bound to
topoisomerase (FIG. 42A). The linker self-joins to Taq-generated
PCR products in a 15 min reaction, forming a template for secondary
PCR and subsequent transcription (FIG. 42B).
[0949] FIG. 43A shows a chromatogram from the FPLC purification.
The peak in UV absorbance (254 nm) in flow-through fractions 2 and
3 corresponds to unbound linker, a small cleavage product, and ATP.
The small peak corresponding to TOPO-bound linker appears in
fractions 30-33 and is magnified in FIG. 43B. Gel electrophoresis
(FIG. 44A) reveals that fractions digested with proteinase K can be
visualized as discrete bands (compare undigested load with load).
The digested topoisomerase/linker covalent complex runs at a lower
molecular weight than annealed oligos alone due to cleavage of the
oligo duplex by topoisomerase. Little covalent complex is evident
in the flow-through fractions (F-T 2 through 5). The lanes
corresponding to elution fractions 29-40 demonstrate that the peak
elution position of the linker is offset from the UV absorbance
trace on the chromatogram in FIG. 43A by approximately 3 fractions
(0.6 ml) to fractions 33-36.
[0950] Undigested fractions run on protein gels and stained with
Coomassie reveal that free topoisomerase elutes in fractions 37-42
(FIG. 44B, compare to free topoisomerase control lane). T7 TOPO
linker can be joined to actin and GFP PCR products
[0951] T7 TOPO linker from peak fractions #33-35 was pooled and
tested for the ability to join with actin (pBAD/TOPO-actin-as
template) and GFP (pcDNA5/FRT/TO/GFP template) test PCR products
(see FIG. 45A and Materials & Methods). A brief incubation with
the linker causes a portion of each Platinum Taq-generated PCR
product to shift into a more slowly migrating band during agarose
gel electrophoresis (FIG. 45B). This band presumably represents a
single copy of the T7 linker joined to one end of the PCR
product.
[0952] Secondary PCR reactions using the T7 linker specific primer
T7amp1 and the appropriate gene-specific forward primer (actinF or
GFPstart) produce strong bands when the linker reaction is used as
the template but not when control reactions lacking linker are used
(FIG. 45C). Relatively weak bands are seen when only the T7ampl
primer is used in the secondary amplification, possibly created
from a small amount of template carrying T7 linkers on both ends.
Background bands are also evident in some negative control lanes,
in which the mock linking reactions (no T7 TOPO linker) were used
as templates for the secondary amplification.
[0953] T7 TOPO Linkers can be Used to Generate Competent Templates
for Transcription
[0954] Approximately 40-50 ng of actin and GFP secondary PCR
products (1.0 .mu.l) (see FIG. 45C) or T7-actin (1.0 .mu.l) and
T7-GFP (1.5 .mu.l) primary PCR products (see FIG. 45A) were used as
templates in 10 .mu.l transcription reactions as described in the
Materials & Methods. The secondary amplification products from
both the actin and GFP linking reactions, but not from the
corresponding negative controls, are competent templates for
transcription by T7 RNA polymerase (FIG. 45D).
[0955] Transcription reactions using these secondary PCR reactions
as templates produce equivalent or greater amounts of RNA to those
using primary PCR reactions with the T7 promoter sequence added to
the 5' end of one of the primers, another common method of
generating transcription templates (FIG. 45D). Thus the amplified
product of the T7 TOPO linking reaction is a fully competent
template. pUC19/actin can also function as an actin template for
use with the T7 TOPO linker
[0956] Similar results were obtained using Recombinant Taq DNA
Polymerase and a pUC19/actin template (FIG. 46). Success with a
non-Platinum polymerase shows that use of the linkers does not
require automatic hot start.
[0957] The amount of T7 TOPO linker in the peak fraction (#34) was
estimated to be 0.1 ng/.mu.l in the final storage buffer. The
linker concentration can be roughly quantitated by running Low DNA
Mass Ladder on a gel along with the proteinase K digested peak
fractions as in FIG. 44A.
[0958] The success of PCR reactions, both primary and secondary,
can be estimated by rough quantitation of the products on agarose
gels as in FIG. 45A. 20-60 ng/.mu.l should be acceptable for the
primary reaction, as the PCR products are in excess in the linking
reactions. For the secondary reaction, at least 25 ng/.mu.l should
be produced as significantly lower levels will influence the yield
of the transcription reaction.
[0959] Transcription reaction yields should be judged according to
the criteria developed for the forthcoming transcription kit.
[0960] Having now fully described the present invention in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious to one of ordinary skill in
the art that the same can be performed by modifying or changing the
invention within a wide and equivalent range of conditions,
formulations and other parameters without affecting the scope of
the invention or any specific embodiment thereof, and that such
modifications or changes are intended to be encompassed within the
scope of the appended claims.
[0961] All publications, patents and patent applications mentioned
in this specification are indicative of the level of skill of those
skilled in the art to which this invention pertains, and are herein
incorporated by reference to the same extent as if each individual
publication, patent or patent application was specifically and
individually indicated to be incorporated by reference.
Sequence CWU 1
1
148127DNAArtificialPrimer 1tatgtatcat acacatacga tttaggt
27220DNAArtificialPrimer 2accgcctctc cccgcgcgtt
20334DNAArtificialPrimer 3gttccgaagg gggcgataca gtcaactgtc tttg
34436DNAArtificialPrimer 4ttggccaagg gtatctagaa gcttctgcag acgcgt
36534DNAArtificialPrimer 5gttccgaagg gccaccgtac tcgtcaattc caag
34636DNAArtificialPrimer 6ggccaaaagg gaacttgttt attgcagctt ataatg
36722DNAArtificialPrimer 7ctctgacttg agcgtcgatt tt
22832DNAArtificialPrimer 8cggaacaagg ggaattccct gtcaccgaga cc
32934DNAArtificialPrimer 9cggaacaagg ggaattcccg gggatctgga attc
341029DNAArtificialPrimer 10tcgaaagggt cgaggtcgac ctgcagctg
291126DNAArtificialPrimer 11aattcacatt gattattgag tagtta
261230DNAArtificialPrimer 12tcgaaagggt aatggccagc aaaggagaag
301327DNAArtificialPrimer 13ggccaagggt ttgtagagct catccat
271429DNAArtificialPrimer 14ggccaagggt ctgaatgggg ccgcatagt
291520DNAArtificialPrimer 15aagccataga gcccgggcca
201631DNAArtificialPrimer 16gttccgaagg gtcgaggtcg acctgcagct g
311730DNAArtificialPrimer 17cggaacaagg gatggccagc aaaggagaag
301831DNAArtificialPrimer 18taggccaagg gtttgtagag ctcatccatg c
311929DNAArtificialPrimer 19ggcctaaagg gtgaatgggg ccgcatagt
292050DNAArtificialPrimer 20gaaggagtaa tacgactcac tatagggagc
caccatgggc ccttcggaac 502150DNAArtificialPrimer 21gttccgaagg
gcccatggtg gctccctata gtgagtcgta ttactccttc
502221DNAArtificialPrimer 22gaaggagtaa tacgactcac t
212338DNAArtificialPrimer 23ggcctaaagg gtccctttag tgagggttaa
ttgcgcgc 382438DNAArtificialPrimer 24gcgcgcaatt aaccctcact
aaagggaccc tttaggcc 382534DNAArtificialPrimer 25cggaacaagg
gatgatagat cccgtcgttt taca 342632DNAArtificialPrimer 26taggccaagg
ggaccatttt caatccgcac ct 322732DNAArtificialPrimer 27taggccaagg
ggaggcactt caccgcttgc ca 322833DNAArtificialPrimer 28taggccaagg
gtttgacacc agaccaactg gta 332912DNAArtificialPrimer 29gcccttattc cc
123012DNAArtificialPrimer 30tcgcccttat tc 123112DNAArtificialPrimer
31tgtcgccctt at 123212DNAArtificialPrimer 32gtgtcgccct ta
123328DNAArtificialPrimer 33aattgatccc ttcaccgaca tagtacag
283412DNAArtificialPrimer 34ggtgaaggga tc 123511DNAArtificialPrimer
35aagggcgagc t 113619DNAArtificialPrimer 36cgcccttgac atagtacag
193712DNAArtificialPrimer 37gacatagtac ag 123815DNAArtificialPrimer
38caactgtact atgtc 153923DNAArtificialPrimer 39agctcgccct
tattccgata gtg 234011DNAArtificialPrimer 40gaataagggc g
114123DNAArtificialPrimer 41aattcgccct tattccgata gtg
234212DNAArtificialPrimer 42attccgatag tg 124315DNAArtificialPrimer
43caacactatc ggaat 154414DNAArtificialPrimer 44atggatctga taaa
144514DNAArtificialPrimer 45accgatctga taaa
144627DNAArtificialPrimer 46aagtcggagc actcgacgac ggtgtag
274717DNAArtificialPrimer 47aaacaccgtc gtcgagt
174833DNAArtificialPrimer 48gcggttaagt cggagcactc gacgactgca tag
334924DNAArtificialPrimer 49tgcagtcgtc gagtgctccg actt
245027DNAArtificialPrimer 50ctatgcagtc gtcgagtgct ccgactt
275122DNAArtificialPrimer 51gttgacattg attattgact ag
225232DNAArtificialPrimer 52gttccgaagg gttaacgcta gagtccggag gc
325332DNAArtificialPrimer 53gactcaaagg gaaggtaagc ctatccctaa gg
325420DNAArtificialPrimer 54gcgcagatct gctatggcag
205537DNAArtificialPrimer 55cggaacaagg gaccatggag aaaaaaatca
ctggata 375636DNAArtificialoligonucleotide primer 56tgagtcaagg
gcgccccgcc ctgctgccac tcatcg 365741DNAArtificialPrimer 57ggggacaagt
ttgtacaaaa aagcaggctt cccttcggaa c 415841DNAArtificialPrimer
58gttccgaagg gaagcctgct tttttgtaca aacttgtccc c
415940DNAArtificialPrimer 59gactcaaagg gacccagctt tcttgtacaa
agtggtcccc 406040DNAArtificialPrimer 60ggggaccact ttgtacaaga
aagctgggtc cctttgagtc 406120DNAArtificialPrimer 61cacgacgttg
taaaacgacg 206222DNAArtificialPrimer 62atgtaatacg actcactata gg
226311DNAArtificialPrimer 63cggaacaagg g 116411DNAArtificialPrimer
64taggccaagg g 116516DNAArtificialPrimer 65cccttcggaa caaggg
166616DNAArtificialPrimer 66cccttggcca taaggg
166775DNAArtificialPrimer 67ttgtacaaaa aagcaggctc cgcggccgcc
gtactcgaga aagggcgcgc cgacccagct 60ttcttgtaca aagtg
756810PRTArtificialPrimer 68Leu Tyr Lys Lys Ala Gly Ser Ala Ala Ala
1 5 10 6911PRTArtificialPrimer 69Gly Arg Ala Asp Pro Ala Phe Leu
Tyr Lys Val 1 5 10 702591DNAArtificialPlasmid 70ctttcctgcg
ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 60taccgctcgc
cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
120gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
gcagctggca 180cgacaggttt cccgactgga aagcgggcag tgagcgcaac
gcaattaata cgcgtaccgc 240tagccaggaa gagtttgtag aaacgcaaaa
aggccatccg tcaggatggc cttctgctta 300gtttgatgcc tggcagttta
tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 360acaacgttca
aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
420caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat
ttgatgcctg 480gcagttccct actctcgcgt taacgctagc atggatgttt
tcccagtcac gacgttgtaa 540aacgacggcc agtcttaagc tcgggcccca
aataatgatt ttattttgac tgatagtgac 600ctgttcgttg caacaaattg
atgagcaatg cttttttata atgccaactt tgtacaaaaa 660agcaggctcc
gcggccgccc cttcaccatg nnnnnnnnna agggtgggcg cgccgaccca
720gctttcttgt acaaagttgg cattataaga aagcattgct tatcaatttg
ttgcaacgaa 780caggtcacta tcagtcaaaa taaaatcatt atttgccatc
cagctgatat cccctatagt 840gagtcgtatt acatggtcat agctgtttcc
tggcagctct ggcccgtgtc tcaaaatctc 900tgatgttaca ttgcacaaga
taaaaatata tcatcatgaa caataaaact gtctgcttac 960ataaacagta
atacaagggg tgttatgagc catattcaac gggaaacgtc gaggccgcga
1020ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga
taatgtcggg 1080caatcaggtg cgacaatcta tcgcttgtat gggaagcccg
atgcgccaga gttgtttctg 1140aaacatggca aaggtagcgt tgccaatgat
gttacagatg agatggtcag actaaactgg 1200ctgacggaat ttatgcctct
tccgaccatc aagcatttta tccgtactcc tgatgatgca 1260tggttactca
ccactgcgat ccccggaaaa acagcattcc aggtattaga agaatatcct
1320gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt
gcattcgatt 1380cctgtttgta attgtccttt taacagcgat cgcgtatttc
gtctcgctca ggcgcaatca 1440cgaatgaata acggtttggt tgatgcgagt
gattttgatg acgagcgtaa tggctggcct 1500gttgaacaag tctggaaaga
aatgcataaa cttttgccat tctcaccgga ttcagtcgtc 1560actcatggtg
atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt
1620attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat
cctatggaac 1680tgcctcggtg agttttctcc ttcattacag aaacggcttt
ttcaaaaata tggtattgat 1740aatcctgata tgaataaatt gcagtttcat
ttgatgctcg atgagttttt ctaatcagaa 1800ttggttaatt ggttgtaaca
ctggcagagc attacgctga cttgacggga cggcgcaagc 1860tcatgaccaa
aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc agaccccgta
1920gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
ctgcttgcaa 1980acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
atcaagagct accaactctt 2040tttccgaagg taactggctt cagcagagcg
cagataccaa atactgtcct tctagtgtag 2100ccgtagttag gccaccactt
caagaactct gtagcaccgc ctacatacct cgctctgcta 2160atcctgttac
cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
2220agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
gtgcacacag 2280cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
tacagcgtga gcattgagaa 2340agcgccacgc ttcccgaagg gagaaaggcg
gacaggtatc cggtaagcgg cagggtcgga 2400acaggagagc gcacgaggga
gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 2460gggtttcgcc
acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
2520ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
ctggcctttt 2580gctcacatgt t 2591712607DNAArtificialPlasmid
71ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
60taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
120gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
gcagctggca 180cgacaggttt cccgactgga aagcgggcag tgagcgcaac
gcaattaata cgcgtaccgc 240tagccaggaa gagtttgtag aaacgcaaaa
aggccatccg tcaggatggc cttctgctta 300gtttgatgcc tggcagttta
tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 360acaacgttca
aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
420caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat
ttgatgcctg 480gcagttccct actctcgcgt taacgctagc atggatgttt
tcccagtcac gacgttgtaa 540aacgacggcc agtcttaagc tcgggcccca
aataatgatt ttattttgac tgatagtgac 600ctgttcgttg caacaaattg
atgagcaatg cttttttata atgccaactt tgtacaaaaa 660agcaggctcc
gcggccgcct tgtttaactt taagaaggag cccttcaccn nnnnnaaggg
720tgggcgcgcc gacccagctt tcttgtacaa agttggcatt ataagaaagc
attgcttatc 780aatttgttgc aacgaacagg tcactatcag tcaaaataaa
atcattattt gccatccagc 840tgatatcccc tatagtgagt cgtattacat
ggtcatagct gtttcctggc agctctggcc 900cgtgtctcaa aatctctgat
gttacattgc acaagataaa aatatatcat catgaacaat 960aaaactgtct
gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga
1020aacgtcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt
ataaatgggc 1080tcgcgataat gtcgggcaat caggtgcgac aatctatcgc
ttgtatggga agcccgatgc 1140gccagagttg tttctgaaac atggcaaagg
tagcgttgcc aatgatgtta cagatgagat 1200ggtcagacta aactggctga
cggaatttat gcctcttccg accatcaagc attttatccg 1260tactcctgat
gatgcatggt tactcaccac tgcgatcccc ggaaaaacag cattccaggt
1320attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag
tgttcctgcg 1380ccggttgcat tcgattcctg tttgtaattg tccttttaac
agcgatcgcg tatttcgtct 1440cgctcaggcg caatcacgaa tgaataacgg
tttggttgat gcgagtgatt ttgatgacga 1500gcgtaatggc tggcctgttg
aacaagtctg gaaagaaatg cataaacttt tgccattctc 1560accggattca
gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg
1620gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat
accaggatct 1680tgccatccta tggaactgcc tcggtgagtt ttctccttca
ttacagaaac ggctttttca 1740aaaatatggt attgataatc ctgatatgaa
taaattgcag tttcatttga tgctcgatga 1800gtttttctaa tcagaattgg
ttaattggtt gtaacactgg cagagcatta cgctgacttg 1860acgggacggc
gcaagctcat gaccaaaatc ccttaacgtg agttacgcgt cgttccactg
1920agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
ttctgcgcgt 1980aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
gtggtttgtt tgccggatca 2040agagctacca actctttttc cgaaggtaac
tggcttcagc agagcgcaga taccaaatac 2100tgtccttcta gtgtagccgt
agttaggcca ccacttcaag aactctgtag caccgcctac 2160atacctcgct
ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
2220taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
gctgaacggg 2280gggttcgtgc acacagccca gcttggagcg aacgacctac
accgaactga gatacctaca 2340gcgtgagcat tgagaaagcg ccacgcttcc
cgaagggaga aaggcggaca ggtatccggt 2400aagcggcagg gtcggaacag
gagagcgcac gagggagctt ccagggggaa acgcctggta 2460tctttatagt
cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
2520gtccaggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
ggttcctggc 2580cttttgctgg ccttttgctc acatgtt
2607725543DNAArtificialPlasmid 72gacggatcgg gagatctccc gatcccctat
ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg
cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca
acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag
gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc
tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg
ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac
tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa
tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca
ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca
aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt
acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
gctggctagt 900taagctatca acaagtttgt acaaaaaagc aggctccgcg
gccgcccctt caccatgnnn 960nnnnnnaagg gtgggcgcgc cgacccagct
ttcttgtaca aagtggttga tctagagggc 1020ccgcggttcg aaggtaagcc
tatccctaac cctctcctcg gtctcgattc tacgcgtacc 1080ggttagtaat
gagtttaaac gggggaggct aactgaaaca cggaaggaga caataccgga
1140aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacgggt
gttgggtcgt 1200ttgttcataa acgcggggtt cggtcccagg gctggcactc
tgtcgatacc ccaccgagac 1260cccattgggg ccaatacgcc cgcgtttctt
ccttttcccc accccacccc ccaagttcgg 1320gtgaaggccc agggctcgca
gccaacgtcg gggcggcagg ccctgccata gcagatctgc 1380gcagctgggg
ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
1440gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
cccgctcctt 1500tcgctttctt cccttccttt ctcgccacgt tcgccggctt
tccccgtcaa gctctaaatc 1560ggggcatccc tttagggttc cgatttagtg
ctttacggca cctcgacccc aaaaaacttg 1620attagggtga tggttcacgt
agtgggccat cgccctgata gacggttttt cgccctttga 1680cgttggagtc
cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
1740ctatctcggt ctattctttt gatttataag ggattttggg gatttcggcc
tattggttaa 1800aaaatgagct gatttaacaa aaatttaacg cgaattaatt
ctgtggaatg tgtgtcagtt 1860agggtgtgga aagtccccag gctccccagc
aggcagaagt atgcaaagca tgcatctcaa 1920ttagtcagca accaggtgtg
gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 1980catgcatctc
aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
2040aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
ttatttatgc 2100agaggccgag gccgcctctg cctctgagct attccagaag
tagtgaggag gcttttttgg 2160aggcctaggc ttttgcaaaa agctcccggg
agcttgtata tccattttcg gatctgatca 2220agagacagga tgaggatcgt
ttcgcatgat tgaacaagat ggattgcacg caggttctcc 2280ggccgcttgg
gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
2340tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
tcaagaccga 2400cctgtccggt gccctgaatg aactgcagga cgaggcagcg
cggctatcgt ggctggccac 2460gacgggcgtt ccttgcgcag ctgtgctcga
cgttgtcact gaagcgggaa gggactggct 2520gctattgggc gaagtgccgg
ggcaggatct cctgtcatct caccttgctc ctgccgagaa 2580agtatccatc
atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
2640attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
aagccggtct 2700tgtcgatcag gatgatctgg acgaagagca tcaggggctc
gcgccagccg aactgttcgc 2760caggctcaag gcgcgcatgc ccgacggcga
ggatctcgtc gtgacccatg gcgatgcctg 2820cttgccgaat atcatggtgg
aaaatggccg cttttctgga ttcatcgact gtggccggct 2880gggtgtggcg
gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
2940tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
ccgattcgca 3000gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
gcgggactct ggggttcgcg 3060aaatgaccga ccaagcgacg cccaacctgc
catcacgaga tttcgattcc accgccgcct 3120tctatgaaag gttgggcttc
ggaatcgttt tccgggacgc cggctggatg atcctccagc 3180gcggggatct
catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
3240gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
tcactgcatt 3300ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
tgtctgtata ccgtcgacct 3360ctagctagag cttggcgtaa tcatggtcat
agctgtttcc tgtgtgaaat tgttatccgc 3420tcacaattcc acacaacata
cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 3480gagtgagcta
actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
3540tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
ttgcgtattg 3600ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
ggtcgttcgg ctgcggcgag 3660cggtatcagc tcactcaaag gcggtaatac
ggttatccac agaatcaggg gataacgcag 3720gaaagaacat gtgagcaaaa
ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 3780tggcgttttt
ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
3840agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
ggaagctccc 3900tcgtgcgctc tcctgttccg accctgccgc ttaccggata
cctgtccgcc tttctccctt 3960cgggaagcgt ggcgctttct caatgctcac
gctgtaggta tctcagttcg gtgtaggtcg 4020ttcgctccaa gctgggctgt
gtgcacgaac
cccccgttca gcccgaccgc tgcgccttat 4080ccggtaacta tcgtcttgag
tccaacccgg taagacacga cttatcgcca ctggcagcag 4140ccactggtaa
caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
4200ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
ctgctgaagc 4260cagttacctt cggaaaaaga gttggtagct cttgatccgg
caaacaaacc accgctggta 4320gcggtggttt ttttgtttgc aagcagcaga
ttacgcgcag aaaaaaagga tctcaagaag 4380atcctttgat cttttctacg
gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 4440ttttggtcat
gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
4500gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
caatgcttaa 4560tcagtgaggc acctatctca gcgatctgtc tatttcgttc
atccatagtt gcctgactcc 4620ccgtcgtgta gataactacg atacgggagg
gcttaccatc tggccccagt gctgcaatga 4680taccgcgaga cccacgctca
ccggctccag atttatcagc aataaaccag ccagccggaa 4740gggccgagcg
cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
4800gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
gttgccattg 4860ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
ttcattcagc tccggttccc 4920aacgatcaag gcgagttaca tgatccccca
tgttgtgcaa aaaagcggtt agctccttcg 4980gtcctccgat cgttgtcaga
agtaagttgg ccgcagtgtt atcactcatg gttatggcag 5040cactgcataa
ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
5100actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
tgcccggcgt 5160caatacggga taataccgcg ccacatagca gaactttaaa
agtgctcatc attggaaaac 5220gttcttcggg gcgaaaactc tcaaggatct
taccgctgtt gagatccagt tcgatgtaac 5280ccactcgtgc acccaactga
tcttcagcat cttttacttt caccagcgtt tctgggtgag 5340caaaaacagg
aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
5400tactcatact cttccttttt caatattatt gaagcattta tcagggttat
tgtctcatga 5460gcggatacat atttgaatgt atttagaaaa ataaacaaat
aggggttccg cgcacatttc 5520cccgaaaagt gccacctgac gtc
5543735173DNAArtificialPlasmid 73gacggatcgg gagatctccc gatcccctat
ggtgcactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg
cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca
acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag
gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc
tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg
ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggag
tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa
tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca
ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca
aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt
acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
gctggctagt 900taagctatca acaagtttgt acaaaaaagc aggctccgcg
gccgcccctt caccatgnnn 960nnnnnnaagg gtgggcgcgc cgacccagct
ttcttgtaca aagtggttga tctagagggc 1020ccgcggttcg aaggtaagcc
tatccctaac cctctcctcg gtctcgattc tacgcgtacc 1080ggttagtaat
gagtttaaac gggggaggct aactgaaaca cggaaggaga caataccgga
1140aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacgggt
gttgggtcgt 1200ttgttcataa acgcggggtt cggtcccagg gctggcactc
tgtcgatacc ccaccgagac 1260cccattgggg ccaatacgcc cgcgtttctt
ccttttcccc accccacccc ccaagttcgg 1320gtgaaggccc agggctcgca
gccaacgtcg gggcggcagg ccctgccata gcagatctgc 1380gcagctgggg
ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
1440gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
cccgctcctt 1500tcgctttctt cccttccttt ctcgccacgt tcgcaggctt
tccccgtcaa gctctaaatc 1560ggggcatccc tttagggttc cgatttagtg
ctttacggca cctcgacccc aaaaaacttg 1620attagggtga tggttcacgt
agtgggccat cgccctgata gacggttttt cgccctttga 1680cgttggagtc
cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
1740ctatctcggt ctattctttt gatttataag ggattttggg gatttcggcc
tattggttaa 1800aaaatgagct gatttaacaa aaatttaacg cgaattaatt
ctgtggaatg tgtgtcagtt 1860agggtgtgga aagtccccag gctccccagc
aggcagaagt atgcaaagca tgcatctcaa 1920ttagtcagca accaggtgtg
gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 1980catgcatctc
aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
2040aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
ttatttatgc 2100agaggccgag gccgcctctg cctctgagct attccagaag
tagtgaggag gcttttttgg 2160aggcctaggc ttttgcaaaa agctcccggg
agcttgtata tccattttcg gatctgatca 2220gcacgtgttg acaattaatc
atcggcatag tatatcggca tagtataata cgacaaggtg 2280aggaactaaa
ccatggccaa gcctttgtct caagaagaat ccaccctcat tgaaagagca
2340acggctacaa tcaacagcat ccccatctct gaagactaca gcgtcgccag
cgcagctctc 2400tctagcgacg gccgcatctt cactggtgtc aatgtatatc
attttactgg gggaccttgt 2460gcagaactcg tggtgctggg cactgctgct
gctgcggcag ctggcaacct gacttgtatc 2520gtcgcgatcg gaaatgagaa
caggggcatc ttgagcccct gcggacggtg ccgacaggtg 2580cttctcgatc
tgcatcctgg gatcaaagcc atagtgaagg acagtgatgg acagccgacg
2640gcagttggga ttcgtgaatt gctgccctct ggttatgtgt gggagggcta
agcacttcgt 2700ggccgaggag caggactgac acgtgctacg agatttcgat
tccaccgccg ccttctatga 2760aaggttgggc ttcggaatcg ttttccggga
cgccggctgg atgatcctcc agcgcgggga 2820tctcatgctg gagttcttcg
cccaccccaa cttgtttatt gcagcttata atggttacaa 2880ataaagcaat
agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
2940tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
cctctagcta 3000gagcttggcg taatcatggt catagctgtt tcctgtgtga
aattgttatc cgctcacaat 3060tccacacaac atacgagccg gaagcataaa
gtgtaaagcc tggggtgcct aatgagtgag 3120ctaactcaca ttaattgcgt
tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 3180ccagctgcat
taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
3240ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
gagcggtatc 3300agctcactca aaggcggtaa tacggttatc cacagaatca
ggggataacg caggaaagaa 3360catgtgagca aaaggccagc aaaaggccag
gaaccgtaaa aaggccgcgt tgctggcgtt 3420tttccatagg ctccgccccc
ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 3480gcgaaacccg
acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
3540ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
cttcgggaag 3600cgtggcgctt tctcatagct cacgctgtag gtatctcagt
tcggtgtagg tcgttcgctc 3660caagctgggc tgtgtgcacg aaccccccgt
tcagcccgac cgctgcgcct tatccggtaa 3720ctatcgtctt gagtccaacc
cggtaagaca cgacttatcg ccactggcag cagccactgg 3780taacaggatt
agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
3840taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
agccagttac 3900cttcggaaaa agagttggta gctcttgatc cggcaaacaa
accaccgctg gtagcggttt 3960ttttgtttgc aagcagcaga ttacgcgcag
aaaaaaagga tctcaagaag atcctttgat 4020cttttctacg gggtctgacg
ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 4080gagattatca
aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
4140aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
tcagtgaggc 4200acctatctca gcgatctgtc tatttcgttc atccatagtt
gcctgactcc ccgtcgtgta 4260gataactacg atacgggagg gcttaccatc
tggccccagt gctgcaatga taccgcgaga 4320cccacgctca ccggctccag
atttatcagc aataaaccag ccagccggaa gggccgagcg 4380cagaagtggt
cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
4440tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
ctacaggcat 4500cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
tccggttccc aacgatcaag 4560gcgagttaca tgatccccca tgttgtgcaa
aaaagcggtt agctccttcg gtcctccgat 4620cgttgtcaga agtaagttgg
ccgcagtgtt atcactcatg gttatggcag cactgcataa 4680ttctcttact
gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
4740gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
caatacggga 4800taataccgcg ccacatagca gaactttaaa agtgctcatc
attggaaaac gttcttcggg 4860gcgaaaactc tcaaggatct taccgctgtt
gagatccagt tcgatgtaac ccactcgtgc 4920acccaactga tcttcagcat
cttttacttt caccagcgtt tctgggtgag caaaaacagg 4980aaggcaaaat
gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
5040cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
gcggatacat 5100atttgaatgt atttagaaaa ataaacaaat aggggttccg
cgcacatttc cccgaaaagt 5160gccacctgac gtc
51737469DNAArtificialPrimer 74ttgtacaaaa aagcaggctc cgcggccgcc
ttgtttaact ttaagaagga gcccttcacc 60atgnnnnnn
697552DNAArtificialPrimer 75ggccgccttg tttaacttta agaaggagcc
cttcaccgac tatgtacagt tg 527631DNAArtificialPrimer 76ggccgccccc
ttcaccgact atgtacagtt g 317728DNAArtificialPrimer 77cgcgcccacc
cttgacatag tacagttg 287814PRTArtificialPartial amino acid sequence
of pENTR-dTOPO and pcDNAGW-dTOPO 78Leu Tyr Lys Lys Ala Gly Ser Ala
Ala Ala Pro Phe Thr Met 1 5 10 7913PRTArtificialPartial amino acid
sequence of pENTR/SD-dTOPO, pENTR-dTOPO and pcDNAGW-dTOPO 79Leu Gly
Gly Arg Ala Asp Pro Ala Phe Leu Tyr Lys Val 1 5 10
8015DNAArtificialPrimer 80cccttcacca tgnnn
158115DNAArtificialPrimer 81gcttttttat actaa
158221DNAArtificialPrimer 82caactttttt atacaaagtt g
218325DNAArtificialPrimer 83agcctgcttt tttgtacaaa cttgt
2584233DNAArtificialPrimer 84tacaggtcac taataccatc taagtagttg
attcatagtg actggatatg ttgtgtttta 60cagtattatg tagtctgttt tttatgcaaa
atctaattta atatattgat atttatatca 120ttttacgttt ctcgttcagc
ttttttgtac aaagttggca ttataaaaaa gcattgctca 180tcaatttgtt
gcaacgaaca ggtcactatc agtcaaaata aaatcattat ttg
23385100DNAArtificialPrimer 85caaataatga ttttattttg actgatagtg
acctgttcgt tgcaacaaat tgataagcaa 60tgctttttta taatgccaac tttgtacaaa
aaagcaggct 10086125DNAArtificialPrimer 86acaagtttgt acaaaaaagc
tgaacgagaa acgtaaaatg atataaatat caatatatta 60aattagattt tgcataaaaa
acagactaca taatactgta aaacacaaca tatccagtca 120ctatg
1258727DNAArtificialPrimer 87agcctgcttt tttatactaa cttgagc
278827DNAArtificialPrimer 88gttcagcttt tttatactaa gttggca
278927DNAArtificialPrimer 89agcctgcttt tttatactaa gttggca
279027DNAArtificialPrimer 90gttcagcttt tttatactaa cttgagc
279127DNAArtificialPrimer 91gttcagcttt tttgtacaaa gttggca
279227DNAArtificialPrimer 92agcctgcttt tttgtacaaa gttggca
279325DNAArtificialPrimer 93gttcagcttt tttgtacaaa cttgt
259425DNAArtificialPrimer 94acccagcttt cttgtacaaa gtggt
259527DNAArtificialPrimer 95gttcagcttt cttgtacaaa gttggca
279627DNAArtificialPrimer 96acccagcttt cttgtacaaa gttggca
279725DNAArtificialPrimer 97gttcagcttt cttgtacaaa gtggt
259822DNAArtificialPrimer 98caactttatt atacaaagtt gt
229927DNAArtificialPrimer 99gttcaacttt attatacaaa gttggca
2710024DNAArtificialPrimer 100caactttatt atacaaagtt ggca
2410125DNAArtificialPrimer 101gttcaacttt attatacaaa gttgt
2510222DNAArtificialPrimer 102caacttttct atacaaagtt gt
2210327DNAArtificialPrimer 103gttcaacttt tctatacaaa gttggca
2710424DNAArtificialPrimer 104caacttttct atacaaagtt ggca
2410525DNAArtificialPrimer 105gttcaacttt tctatacaaa gttgt
2510622DNAArtificialPrimer 106caacttttgt atacaaagtt gt
2210727DNAArtificialPrimer 107gttcaacttt tgtatacaaa gttggca
2710824DNAArtificialPrimer 108caacttttgt atacaaagtt ggca
2410925DNAArtificialPrimer 109gttcaacttt tgtatacaaa gttgt
2511022DNAArtificialPrimer 110caactttttc gtacaaagtt gt
2211127DNAArtificialPrimer 111gttcaacttt ttcgtacaaa gttggca
2711224DNAArtificialPrimer 112caactttttc gtacaaagtt ggca
2411325DNAArtificialPrimer 113gttcaacttt ttcgtacaaa gttgt
2511422DNAArtificialPrimer 114caactttttg gtacaaagtt gt
2211527DNAArtificialPrimer 115gttcaacttt ttggtacaaa gttggca
2711624DNAArtificialPrimer 116caactttttg gtacaaagtt ggca
2411725DNAArtificialPrimer 117gttcaacttt ttggtacaaa gttgt
2511822DNAArtificialPrimer 118caacttttta atacaaagtt gt
2211927DNAArtificialPrimer 119gttcaacttt ttaatacaaa gttggca
2712024DNAArtificialPrimer 120caacttttta atacaaagtt ggca
2412125DNAArtificialPrimer 121gttcaacttt ttaatacaaa gttgt
2512215DNAArtificialPrimer 122atgggatctg ataaa
1512319DNAArtificialPrimer 123caccatggga tctgataaa
1912443DNAArtificialPrimer 124gactcgtaat acgactcact atagggccct
tattccgata gtg 4312542DNAArtificialPrimer 125agggccctat agtgagtcgt
attacgagtc aaaaaaaaaa aa 4212616DNAArtificialPrimer 126caacactatc
ggaata 1612724DNAArtificialPrimer 127gctcaccatg gatgatgata tcgc
2412824DNAArtificialPrimer 128ggaggagcaa tgatcttgat cttc
2412933DNAArtificialPrimer 129cacggatccg ctcaccatgg atgatgatat cgc
3313033DNAArtificialPrimer 130cacaagcttg gaggagcaat gatcttgatc ttc
3313125DNAArtificialPrimer 131atggctagca aaggagaaga acttt
2513225DNAArtificialPrimer 132ttatttgtag agctcatcca tgcca
2513329DNAArtificialPrimer 133gatgactcgt aatacgactc actataggg
2913424DNAArtificialPrimer 134gatgactcgt aatacgactc acta
2413511DNAArtificialPrimer 135ggccataagg g
1113611DNAArtificialPrimer 136gttccgaagg g
1113711DNAArtificialPrimer 137ggcctaaagg g
1113833DNAArtificialPrimer 138cggaacaaat tgaaattctt cctcgggaag tgg
3313912DNAArtificialPrimer 139ctgatacatg tc
1214048DNAArtificialPrimer 140ttgtacaaaa aagacggctc cgcggccgcc
cccttcacca tgnnnnnn 4814112DNAArtificialPrimer 141cgggggaagt gg
1214245DNAArtificialPrimer 142nnnnnnaagg gtgggcgcgc cgacccagct
ttcttgtaca aagtg 4514314DNAArtificialPrimer 143tcgaaagggc cctt
1414414DNAArtificialPrimer 144ggccaagggc cctt
1414511DNAArtificialPrimer 145gttccgaagg g
1114616DNAArtificialPrimer 146cggaacaagg gccctt
1614716DNAArtificialPrimer 147taggccaagg gccctt
1614811DNAArtificialPrimer 148ggcctaaagg g 11
* * * * *