U.S. patent application number 11/523422 was filed with the patent office on 2007-03-29 for method for ligating nucleic acids and molecular cloning.
This patent application is currently assigned to Stratagene California. Invention is credited to John Bauer, Carsten-Peter Carstens, Richard Gibbs, Alan Greener, Henry Ji, Joseph A. Sorge.
Application Number | 20070072297 11/523422 |
Document ID | / |
Family ID | 24044371 |
Filed Date | 2007-03-29 |
United States Patent
Application |
20070072297 |
Kind Code |
A1 |
Ji; Henry ; et al. |
March 29, 2007 |
Method for ligating nucleic acids and molecular cloning
Abstract
The invention provides methods of covalently joining nucleic
acid molecules and methods of molecular cloning. The methods
provide either sequential or simultaneous ligation of flanking or
vector nucleic acid molecules to nucleic acid insert molecules by
topoisomerase and DNA ligase. The methods provide for directional
and non-directional covalent joining and cloning of nucleic acid
molecules.
Inventors: |
Ji; Henry; (San Diego,
CA) ; Greener; Alan; (San Diego, CA) ; Sorge;
Joseph A.; (Wilson, WY) ; Bauer; John; (San
Diego, CA) ; Gibbs; Richard; (Houston, TX) ;
Carstens; Carsten-Peter; (La Jolla, CA) |
Correspondence
Address: |
PALMER & DODGE, LLP;KATHLEEN M. WILLIAMS / STR
111 HUNTINGTON AVENUE
BOSTON
MA
02199
US
|
Assignee: |
Stratagene California
|
Family ID: |
24044371 |
Appl. No.: |
11/523422 |
Filed: |
September 18, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10057050 |
Jan 25, 2002 |
7109178 |
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11523422 |
Sep 18, 2006 |
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09513710 |
Feb 25, 2000 |
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10057050 |
Jan 25, 2002 |
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Current U.S.
Class: |
435/455 ;
435/91.1 |
Current CPC
Class: |
C12P 19/34 20130101;
C12N 15/66 20130101; C12N 9/00 20130101; C12N 15/64 20130101; C12N
15/10 20130101 |
Class at
Publication: |
435/455 ;
435/091.1 |
International
Class: |
C12N 15/09 20060101
C12N015/09; C12P 19/34 20060101 C12P019/34 |
Claims
1. A method of covalently joining a nucleic acid insert molecule to
first and second nucleic acid flanking molecules to form a ligated
molecule, the method comprising (A) incubating: said insert
molecule, wherein one end of said insert molecule comprises a
5'-hydroxyl group and the other end comprises a 5'-phosphate group,
and a said first flanking molecule, wherein one end only of said
first flanking molecule comprises a covalently bound topoisomerase
polypeptide, under conditions which permit their covalent joining
to form a ligated nucleic acid comprising a said insert molecule
positioned adjacent to a said first flanking molecule, (B)
incubating a said ligated nucleic acid of step (A) with phosphatase
under conditions which permit removal of a 5'-phosphate group from
said ligated nucleic acid; and (C) incubating a product of step (B)
with a said second flanking molecule, wherein one end only of said
second flanking molecule comprises a covalently bound topoisomerase
polypeptide, under conditions which permit covalent joining to form
a ligated molecule comprising a said insert molecule positioned
between a said first and a said second flanking molecule.
2. The method of claim 1 wherein a said first and a said second
nucleic acid flanking molecules comprise a left and a right vector
arm, respectively, such that a said insert molecule is flanked by a
said left vector arm and a said right vector arm.
3. The method of claim 2, wherein said left and right vector arms
each comprise a free end that is not joined to an insert molecule,
said method further comprising the step of: joining the free ends
of said vector arms to each other by a method selected from the
group consisting of nucleic acid ligase mediated ligation,
complementary sequence annealing, topoisomerase mediated ligation,
in vitro site-specific recombination, in vivo site-specific
recombination, and in vivo homologous recombination.
4. A method of covalently joining a nucleic acid insert molecule to
first and second nucleic acid flanking molecules to form a ligated
molecule, the method comprising incubating: said insert molecule
and said flanking molecules, wherein one end of said insert
molecule comprises a 5'-hydroxyl group and the other end comprises
a 5'-phosphate group, wherein one end only of said first flanking
molecule comprises a covalently bound topoisomerase polypeptide and
wherein one end of said second flanking molecule comprises a ligase
substrate site, under conditions which permit their covalent
joining to form a ligated molecule comprising a said insert
molecule positioned between a said first and a said second flanking
molecule.
5. The method of claim 4 wherein a said first and a said second
nucleic acid flanking molecules comprise a left and a right vector
arm, respectively, such that a said insert molecule is flanked by a
said left vector arm and a said right vector arm.
6. The method of claim 5, wherein said left and right vector arms
each comprise a free end that is not joined to an insert molecule,
said method further comprising the step of: joining the free ends
of said vector arms to each other by a method selected from the
group consisting of nucleic acid ligase mediated ligation,
complementary sequence annealing, topoisomerase mediated ligation,
in vitro site-specific recombination, in vivo site-specific
recombination, and in vivo homologous recombination.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/057,050, filed Jan. 25, 2002, which will issue as U.S. Pat.
No. 7,109,178 on Sep. 19, 2006, and which is a Continuation of U.S.
application Ser. No. 09/513,710, filed Feb. 25, 2000, the
entireties of each of which are incorporated herein by
reference.
TECHNICAL FIELD
[0002] The invention relates to methods of covalently joining
nucleic acid molecules and methods of molecular cloning of nucleic
acid molecules.
BACKGROUND OF THE INVENTION
[0003] Construction of recombinant nucleic acid molecules requires
two enzymatic steps. First, site-specific restriction endonuclease
digestion or PCR amplification are used to generate linear nucleic
acid molecules with defined termini. Second, the linear molecules
are covalently joined at their termini in the presence of a ligase
enzyme. Methods of covalently joining and cloning nucleic acid
molecules that require only one step or that eliminate the use of
restriction endonucleases or ligases would be advantageous over the
traditional method of constructing recombinant nucleic acid
molecules.
SUMMARY OF THE INVENTION
[0004] It is an object of the invention to provide methods of
covalently joining nucleic acid molecules. It is a further object
of the invention to provide methods of cloning nucleic acid
molecules. This and other objects of the invention are provided by
one or more of the embodiments described below.
[0005] One embodiment of the invention provides a method of
covalently joining a nucleic acid insert molecule to first and
second nucleic acid flanking molecules to form a ligated molecule.
The method comprises incubating the insert molecule and the
flanking molecules under conditions which permit their covalent
joining to form a ligated molecule wherein an insert molecule is
positioned between the first and the second flanking molecule. Each
end of the insert molecule comprises a 5'-hydroxyl group. One end
only of each of the first and second flanking molecules comprises a
covalently bound topoisomerase polypeptide.
[0006] Another embodiment of the invention provides a method of
covalently joining a nucleic acid insert molecule to first and
second nucleic acid flanking molecules to form a ligated molecule.
The method comprises incubating an insert molecule, wherein one end
of the insert molecule comprises a 5'-hydroxyl group and the other
end comprises a 5'-phosphate group, with the first flanking
molecule, wherein one end only of the first flanking molecule
comprises a covalently bound topoisomerase polypeptide. The
incubation is done under conditions which permit their covalent
joining to form a ligated nucleic acid wherein the insert molecule
is positioned adjacent to the first flanking molecule. This ligated
nucleic acid is incubated with phosphatase under conditions which
permit removal of a 5'-phosphate group from the ligated nucleic
acid. The ligated nucleic acid is incubated with the second
flanking molecule. One end only of the second flanking molecule
comprises a covalently bound topoisomerase polypeptide. The
incubation is done under conditions which permit covalent joining
to form a ligated molecule where the insert molecule is positioned
between the first and the second flanking molecule.
[0007] In still another embodiment of the invention a method of
covalently joining a nucleic acid insert molecule to first and
second nucleic acid flanking molecules to form a ligated molecule
is provided. The method comprises incubating an insert molecule and
flanking molecules under conditions which permit their covalent
joining to form a ligated molecule wherein an insert molecule is
positioned between the first and the second flanking molecule. One
end of the insert molecule comprises a 5'-hydroxyl group and the
other end comprises a 5'-phosphate group. One end only of the first
flanking molecule comprises a covalently bound topoisomerase
polypeptide and one end of the second flanking molecule comprises a
ligase substrate site.
[0008] Any of the first and second nucleic acid flanking molecules
can together comprise a pair of left and right vector arms.
Further, the ends of the vector arms not covalently joined to the
insert can be covalently or non-covalently joined to each other by
a method selected from the group consisting of nucleic acid ligase
mediated ligation, complementary sequence annealing, topoisomerase
mediated ligation, in vitro site-specific recombination, in vivo
site-specific recombination, and in vivo homologous
recombination.
[0009] In still another embodiment of the invention a method of
molecular cloning is provided. The method comprises incubating a
nucleic acid insert molecule comprising a 5'-hydroxyl group at one
end and a 5'-phosphate at the other end, and a linear cloning
vector. The linear cloning vector comprises a covalently bound
topoisomerase polypeptide at one end only and a ligation substrate
site at the other end. The incubation is done under conditions
sufficient for their covalent joining to form a ligated circular
vector. The ligated circular vector is transformed into a host
cell.
[0010] Another embodiment of the invention provides a method for
molecular cloning. The method comprises incubating a nucleic acid
insert molecule where each end of the insert molecule comprises a
5'-hydroxyl group with a first and a second linear arm where one
end only of each of the first and second linear arms comprises a
covalently bound topoisomerase and the other end comprises a
cloning substrate site. The incubation is done under conditions
sufficient for their covalent joining to form a ligated insert
molecule where the insert molecule is positioned between the first
and the second linear arm. The ligated insert molecule is
transformed into a host cell.
[0011] Even another embodiment of the invention provides a method
for molecular cloning. A nucleic acid insert molecule, wherein one
end of the insert molecule comprises a 5'-hydroxyl group and the
other end comprises a 5'-phosphate group, and a first linear arm,
wherein one end only of the first linear arm comprises a covalently
bound topoisomerase polypeptide and the other end comprises a
cloning substrate site are incubated together. The incubation is
done under conditions which permit their covalent joining to form a
ligated nucleic acid wherein the insert molecule is positioned
adjacent to the first linear arm. The ligated nucleic acid is
incubated with phosphatase under conditions which permit removal of
a 5'-phosphate group from the ligated nucleic acid. The ligated
nucleic acid is incubated with a second linear vector arm, wherein
one end only of the second linear vector arm comprises a covalently
bound topoisomerase polypeptide and the other end comprises a
cloning substrate site. The incubation is done under conditions
which permit covalent joining to form a ligated insert molecule
wherein the insert molecule is positioned between the first and the
second linear vector arm. The ligated insert molecule is
transformed into a host cell.
[0012] In yet another embodiment of the invention a method for
molecular cloning is provided comprising incubating a nucleic acid
insert molecule, wherein one end of the insert molecule comprises a
5'-hydroxyl group and the other end comprises a 5'-phosphate group;
a first linear arm, wherein one end only of the first linear arm
comprises a covalently bound topoisomerase polypeptide and the
other end comprises a cloning substrate site; and a second linear
arm, wherein one end of the second linear arm comprises a ligase
substrate site and the other end comprises a cloning substrate
site. The incubation is done under conditions which permit their
covalent joining to form a ligated insert molecule wherein the
insert molecule is positioned between the first and the second
linear arm. The ligated insert molecule is transformed into a host
cell.
[0013] The cloning substrate site can be selected from the group
consisting of a cos site, a LIC site, and a loxP site.
[0014] Where the cloning substrate site is loxP, the method can
further comprise incubating in vitro the ligated insert molecule
with a Cre recombinase and a circular plasmid comprising a loxP
site. The incubation is done under conditions sufficient for
site-specific recombination to form a circular plasmid comprising
the ligated insert molecule. The circular plasmid comprising the
ligated insert molecule is transformed into a host cell.
[0015] Where the cloning substrate site is loxP the method can
further comprise transforming the ligated insert molecule into a
host cell comprising a circular plasmid comprising a loxP site,
wherein the cell expresses Cre recombinase. The transformation is
done under conditions sufficient for site-specific recombination to
form a circular plasmid comprising the ligated insert molecule
within the cell.
[0016] Where the cloning substrate site is a site for homologous
recombination with a circular plasmid vector the transformation
step further comprises transforming the ligated insert molecule
into a host cell comprising a circular plasmid vector. The circular
plasmid vector comprises a site for homologous recombination with
the ligated insert molecule, and the host cell is recA+. The
transformation is done under conditions sufficient for homologous
recombination to form a circular plasmid comprising the ligated
insert molecule within the host cell.
[0017] The first linear arm can comprise a left lambda arm
comprising at one end only a covalently bound topoisomerase. The
second linear arm can comprise a right lambda arm comprising at one
end only a covalently bound topoisomerase.
[0018] As used herein, the term "join" or "joining" refers to both
covalent and noncovalent attachment of one nucleic acid to another,
or one end of a nucleic acid to another end of a nucleic acid.
"Covalent" joining refers to the attachment of one end of a nucleic
acid strand to another end of a nucleic acid strand via a phosphate
bond or to attachment of one end of a double-stranded nucleic acid
to another double-stranded end via phosphate bonding on one or both
strands. "Non-covalent" joining refers to attachment of one end of
a nucleic acid to another end via annealing of a single-stranded
regions to each other; that is, no phosphate bond is generated in
this embodiment.
[0019] "Ligate" or "ligated" refers to the covalent joining of two
ends of one or more nucleic acid molecules.
[0020] "Complementary annealing" refers to annealing, or the
pairing of bases, of complementary regions of one or more nucleic
acids, and thus to the formation of hydrogen bonds and other
non-covalent interactions between pairs of bases.
[0021] A "topoisomerase" is a polypeptide that is capable of
covalently joining to at least one strand of a nucleic acid
molecule and ligating that strand to another strand, as described
hereinbelow. Topoisomerase according to the invention comprises
type I topoisomerases.
[0022] "Bound to" refers to a covalent bonding of a topoisomerase
polypeptide to a nucleic acid molecule.
[0023] "Nucleic acid molecule" refers to a double-stranded nucleic
acid, unless otherwise specified.
[0024] "One end only" refers to the presence of a topoisomerase
polypeptide at one end of a nucleic acid molecule, where the
nucleic acid molecule contains two ends.
[0025] The term "site" is meant to designate a contiguous stretch
of nucleotides, e.g., 1-100 bases in length, usually 5-25 bases in
length, e.g., 8-16 bases, that is susceptible to (i.e., a substrate
for) modification by an enzyme that modifies nucleic acids, e.g., a
ligase or a restriction enzyme.
[0026] A "cloning substrate site", as used herein, is a site
occurring on a nucleic acid molecule for the covalent or
non-covalent joining of nucleic acid sequences or for
recombination. Examples of cloning substrate sites include cos
sites, LIC sites, sites for site-specific recombination, such as
lambda attachment elements or loxP sites, sites for homologous
recombination, and ligation substrate sites.
[0027] A "ligation substrate site", as used herein, is a site
occurring on a nucleic acid molecule of the invention that is
capable of becoming covalently joined to another nucleic acid
molecule in the presence of a ligase enzyme, such as DNA
ligase.
[0028] A "vector arm" or a "linear arm", as used herein, is a
linear nucleic acid molecule, and is preferably a portion or
fragment of a bacteriophage or plasmid genome.
[0029] "Directional" cloning refers to a cloning method in which,
by selecting steps in the method, one can obtain a desired
orientation of a given nucleic acid molecule upon cloning into
another nucleic acid molecule or between two other nucleic acid
molecules; as used herein, "orientation" may refer to 5' to 3' with
reference to a given open reading frame or a given control region
or a known sequence. Thus, for example, an insert molecule may
contain an open reading frame having a 5'-3' orientation with
respect to transcription and the insert molecule may be
directionally cloned between a left and right vector arms such that
the ligated (cloned) molecule comprises, from 5' to 3': left vector
arm, 5' insert 3', right vector arm. "Non-directional" cloning
refers to cloning methods which produce a ligated molecule in which
the insert, for example, appears between the two arms in either
orientation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 shows the non-directional covalent joining of an
insert molecule with 5'-OH groups on each end to a right vector arm
and a left vector arm each comprising a topoisomerase polypeptide
on one end only.
[0031] FIG. 2 shows the directional covalent joining of an insert
molecule with a 5'-OH group on one end and a 5'-phosphate group on
the other end to a right vector arm and a left vector arm each
comprising a topoisomerase polypeptide on one end only.
[0032] FIG. 3 shows the directional covalent joining of an insert
molecule with a 5'-OH group on one end and a 5'-phosphate group on
the other end to a left vector arm comprising a topoisomerase
polypeptide on one end only and a right vector arm comprising a
ligation substrate site on one end.
[0033] FIG. 4 shows the directional cloning of an insert molecule
with a 5'-OH group on one end and a 5'-phosphate group on the other
end to a linear vector molecule. The linear vector molecule
comprises a topoisomerase molecule on one end only and a ligation
substrate site on the other end.
[0034] FIG. 5 shows the non-directional cloning of an insert
molecule with 5'-OH groups on each end to a right vector arm and a
left vector arm each comprising a topoisomerase polypeptide on one
end only and a cloning substrate site, cos, on the other end.
[0035] FIG. 6 shows the directional cloning of an insert molecule
with a 5'-OH group on one end and a 5'-phosphate group on the other
end to vector molecules comprising a topoisomerase polypeptide on
one end only and a cloning substrate site, cos, on the other
end.
[0036] FIG. 7 shows the non-directional cloning of an insert
molecule with 5'-OH groups on each end to a right vector arm and a
left vector arm each comprising a topoisomerase polypeptide on one
end only and a cloning substrate site, LIC, on the other end.
[0037] FIG. 8 shows the directional cloning of an insert molecule
with a 5'-OH group on one end and a 5'-phosphate group on the other
end to vector molecules comprising a topoisomerase polypeptide on
one end only and a cloning substrate site, LIC, on the other
end.
[0038] FIG. 9 shows the directional cloning of an insert molecule
with a 5'-OH group on one end and a 5'-phosphate group on the other
end to vector molecules comprising a topoisomerase polypeptide on
one end only of a lambda vector arm.
[0039] FIG. 10 shows the non-directional cloning of an insert
molecule with 5'-OH groups on each end to a right plasmid arm and a
left plasmid arm each comprising a topoisomerase polypeptide on one
end only.
[0040] FIG. 11 shows the directional cloning of an insert molecule
with a 5'-OH group on one end and a 5'-phosphate group on the other
end to vector molecules comprising a topoisomerase polypeptide on
one end only.
[0041] FIG. 12 shows the non-directional cloning of an insert
molecule with 5'-OH groups on each end to a right vector arm and a
left vector arm each comprising a topoisomerase polypeptide on one
end only and a cloning substrate site, a loxP site, on the other
end.
[0042] FIG. 13 shows the directional cloning of an insert molecule
with a 5'-OH group on one end and a 5'-phosphate group on the other
end to vector molecules comprising a topoisomerase polypeptide on
one end only and a cloning substrate site, a loxP site, on the
other end.
[0043] FIG. 14 shows the non-directional cloning of an insert
molecule with 5'-OH groups on each end to a right vector arm and a
left vector arm each comprising a topoisomerase polypeptide on one
end only and a cloning substrate site, a site for homologous
recombination, on the other end.
[0044] FIG. 15 shows the directional cloning of an insert molecule
with a 5'-OH group on one end and a 5'-phosphate group on the other
end to vector molecules comprising a topoisomerase polypeptide on
one end only and a cloning substrate site, a site for homologous
recombination, on the other end.
DETAILED DESCRIPTION OF THE INVENTION
Insert Polynucleotide Molecules
[0045] Insert polynucleotide molecules comprise isolated and
purified double-stranded DNA, double-stranded RNA, or
double-stranded DNA/RNA hybrid nucleic acids. An insert molecule
can be a full-length molecule or a fragment of a full-length
molecule. Further, an insert molecule can be naturally-occurring,
i.e., found in nature or recombinant.
[0046] Preferably, insert polynucleotides are isolated free of
other components, such as proteins and lipids. Insert
polynucleotides can be made by a cell and isolated or can be
synthesized in the laboratory, for example, using an automatic
synthesizer or an amplification method such as PCR. Where an insert
polynucleotide is prepared by PCR, the insert is generated using a
pair of primers comprising a 3'-primer and a 5'-primer. Both the
3'-primer and the 5'-primer can comprise a 5'-hydroxyl group to
produce an insert with 5'-hydroxyl groups (5'-OH) on both ends.
Alternatively, one of the primers can comprise a 5'-hydroxyl group
and one can comprise a 5'-phosphate group to produce an insert with
a 5'-OH group on one end and a 5'-phosphate (5'-P) group on the
other end. Optionally, both the 3'-primer and the 5'-primer can
comprise a 5'-phosphate group to produce an insert with 5'-P groups
on both ends.
Molecules Flanking an Insert Molecule
[0047] An insert polynucleotide molecule can be covalently joined
to several types of molecules, such as a double-stranded DNA, a
double-stranded RNA, and a double-stranded DNA/RNA hybrid molecule.
Preferably, an insert polynucleotide molecule is covalently joined
to a vector molecule or to vector molecules such as a linear arm of
a plasmid or bacteriophage. Vectors suitable for ligation of an
insert molecule include bacteriophage, such as bacteriophage
lambda, including, but not limited to lambda insertion vectors such
as Lambda ZAP.RTM.II vector, ZAP Express.RTM. vector, Lambda
ZAP.RTM.-CMV vector (Stratagene), lambda gt10, and lambda gt11.
Lambda replacement vectors, for example Lambda FIX.RTM.II vector,
Lambda DASH.RTM.II vector, and Lambda EMBL3 and Lambda EMBL4
(Stratagene) can also be used as vectors.
[0048] Both prokaryotic and eukaryotic linear plasmids can be used
as vectors. See e.g., Meinhardt et al. (1997) Appl. Microbiol.
Biotechnol. 47:329-36; Fukuhara, (1995) FEMS Microbiol. Lett.
131:1-9; Hinnebusch & Tilly, (1993) Mol Microbiol. 10:917-22.
For example, the plasmid prophage N15 of E. coli is a suitable
linear plasmid vector. See Rybchin & Svarchevsky (1999) Mol.
Microbiol. 33:895-903.
[0049] Vector nucleic acid polynucleotides, such as bacteriophage
and plasmids can be isolated and purified from cells carrying these
elements according to methods well known in the art. See e.g.
MOLECULAR CLONING: A LABORATORY MANUAL (Sambrook et al., eds., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, 1989) and
Ausubel (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel et al.,
eds., John Wiley & Sons, New York, 1987)). Additionally, many
bacteriophage and plasmid vectors are commercially available. The
bacteriophage or plasmid nucleic acid can be prepared, if necessary
by cleavage with an appropriate restriction enzyme such that the
digested bacteriophage or plasmid nucleic acid is compatible with
an insert molecule.
[0050] Preferably, an insert molecule is covalently joined to right
and left lambda linear vector arms such that the insert molecule is
positioned between right and left lambda linear vector arms. In
lambda insertion vectors, a left vector arm can comprise lambda
nucleic acids occurring to the left of the insertion site and a
right vector arm can comprises lambda nucleic acids occurring to
the right of the insertion. In lambda replacement vectors, a left
lambda arm comprises lambda nucleic acid occurring to the left of
the nucleic acids to be replaced by the insert nucleic acids and a
right lambda arm comprises lambda nucleic acids occurring to the
right of the nucleic acids to replaced by the insert nucleic acids.
Lambda vectors can vary in nucleic acid sequence; however, the left
arm can typically comprise the head and tail genes A-J, while the
right arm can typically comprise from p.sub.R through a cosR site
of a lambda genome.
[0051] Preferably, the vector or flanking molecule to which the
insert is to be covalently joined is a linear molecule comprising a
topoisomerase covalently linked to only one end of the linear
molecule. A double-stranded DNA, double-stranded RNA, or
double-stranded DNA/RNA molecule with one topoisomerase molecule
bound to one end of the DNA or RNA molecule is a univalent
molecule. DNA topoisomerases catalyze a conversion in the linking
number of a double-stranded DNA molecule. The linking number is the
number of times one DNA strand crosses over the second DNA strand
in space. Type 1 topoisomerases act by making a transient break in
one strand of a nucleic acid. A type 1 topoisomerase first binds to
a nucleic acid and nicks one strand of the nucleic acid. A stable
complex is formed where the free 3'-phosphate end of the nicked
strand is covalently bound to a tyrosine residue of the enzyme. The
second strand is pulled through the gap in the first strand and the
gap is then sealed by the enzyme. The gap can be sealed at the same
bond originally nicked or the complex can combine with a
heterologous nucleic acid, such as an insert molecule, that has a
5'-hydroxy end. Where the complex is combined with a heterologous
nucleic acid, a recombinant molecule is formed.
[0052] Type 1 topoisomerases include, but are not limited to E.
coli topoisomerase I (Keck et al., (1999) Nat. Strut. Biol. 6:900),
E. coli topoisomerase III (Mondragon et al., (1999) Structure Fold.
Des. 7:1373), S. cerevisiae topoisomerase III (Kim et al., (1992)
J. Biol. Chem. 267:17178), human topoisomerase III (Hanai et al.,
(1996) Proc. Natl. Acad. Sci. 93:3653), the type I topoisomerase
from chloroplasts (Mukherjee et al. (1994) 269:3793; Fukata et al.
(1991) J. Biochem (Tokyo) 109:127), thermophilic reverse gyrases
(Nadal et al., (1994) J. Biol. Chem. 269:5255; Slesarev et al.,
(1991) J. Biol. Chem. 266:12321; Bouthier de la Tour et al., (1991)
J. Bact. 173:3921), thermophilic D. amylolyticus topoisomerase III
(Slesarev et al., (1991) J. Biol. Chem. 266:12321), and vaccinia
DNA topoisomerase I (Shuman et al., (1987) Proc. Natl. Acad. Sci.
84:7478). Site-specific type I DNA topoisomerases are particularly
useful in the invention. Site-specific type I DNA topoisomerases
include vaccinia topoisomerase and pox virus topoisomerases.
[0053] A topoisomerase enzyme can be covalently linked to a vector
or flanking molecule by, for example, the method of Heyman et al.
(Genome Res. (1999) 9:383). Briefly, Vaccinia DNA topoisomerase
cleaves the phosphodiester backbone of one strand of a nucleic acid
at a consensus pentopyrimidine element: 5'-C/TCCTT-3' (SEQ ID
NO:1). This element can be added onto the end of a vector or
flanking molecule. Vaccinia topoisomerase can then be incubated
with the vector or flanking molecule such that the topoisomerase
becomes covalently bound to the underlined T in the C/TCCTT
sequence. Optionally, nuclease treatment, such as exonuclease III
treatment can be used to remove single strand ends from the element
such that a blunt-ended insert fragment with topoisomerase bound to
the molecule is formed.
[0054] Optionally, the molecule to which the insert is to be
covalently joined is a linear molecule comprising a ligation
substrate site at a first end of the linear molecule. A ligation
substrate site comprises a site for nucleic acid ligation that is
mediated by a ligase enzyme. A ligation substrate site can comprise
any double-stranded nucleic acid that has blunt ends or protruding
termini that can be covalently joined to another nucleic acid
molecule in the presence of a ligase enzyme. Preferably, the
ligation substrate site comprises a 5'-phosphate group and is
complementary to one end of an insert molecule. A ligation
substrate site can be produced by, for example cleaving a
double-stranded nucleic acid molecule with a restriction enzyme
that produces blunt-ended termini, 5'-protruding ends, or
3'-protruding ends and purifying the nucleic acid molecule. A
ligation between a linear molecule comprising a ligation substrate
site and an insert molecule takes place in the presence of a ligase
enzyme such as bacteriophage T4 DNA ligase or Pfu DNA ligase
(Stratagene). Preferably, the vector or flanking molecule to which
the insert is to be covalently joined is a linear molecule
comprising a topoisomerase covalently linked to only one end of the
molecule or a ligation substrate site at one end of the linear
molecule. The second end of the linear molecule preferably
comprises a cloning substrate site such as, a cos site, a LIC site,
a site-specific recombination site (such as a loxP site or lambda
attachment element), a homologous recombination site or a ligation
substrate site.
[0055] A bacteriophage lambda genome has cos sites at the ends of
the genome. See, LAMBDA II (Roger W. Hendrix, ed., Cold Spring
Harbor Laboratory Press) 1983; Higgins et al., (1995) J. Mol. Biol.
252:31; Higgins et al., (1994) EMBO J. 13:6152; Cue et al., (1993)
J. Mol. Biol. 234:594; Cue et al., (1993) Proc. Natl. Acad. Sci.
USA 90:9290. Cleavage occurs at a left cos site (as defined on a
standard lambda map) to generate a free end that is inserted into a
capsid. The insertion of nucleic acid continues until a right cos
site is encountered. Cleavage occurs at the right cos site to
generate the second end. Any nucleic acid molecule that is
contained between two cos sites can be packaged. A nucleic acid
molecule comprising a cos site, a fragment of a cos site, a mutant
of a cos site, or a variant of a cos site can be isolated from a
preparation of bacteriophage lambda DNA or may synthesized in the
laboratory. A nucleic molecule comprising a cos site can be ligated
to the end of the molecule to which the insert is to be covalently
joined. Alternatively, a cos site can be added to the end of a
molecule to which the insert is to be covalently joined using
standard molecular biology cloning techniques such as PCR. In the
methods of the invention distal ends (i.e., the ends of vector arms
not covalently joined to an insert molecule) of vector arms
containing terminal cos sites can be readily annealed to one
another in E. coli host cells by virtue of their explicit sequence.
cos sites do not appreciably anneal in vitro at room
temperature.
[0056] A ligation-independent cloning (LIC) site can be any size,
but is preferably 12 to 13 nucleotides or longer. Sites longer than
12-13 nucleotides may work more efficiently, e.g., up to 24 bases,
or up to 48 bases or longer. See Aslanidis and de Jong, (1990)
Nucleic Acids Res. 18:6069. The 12-13 nucleotide terminus can
comprise any nucleic acid sequence; however, preferably one or none
of the nucleotides of a 3' strand of the 12-13 nucleotide terminus
is an adenosine. A nucleic molecule comprising a LIC site can be
ligated to the ends of the vector or flanking molecule to which the
insert is to be covalently joined. Alternatively, a LIC site can be
added to the end of a vector or flanking molecule to which the
insert is to be covalently joined using standard molecular biology
cloning techniques, such as by PCR.
[0057] Where the second end of a linear molecule comprises a LIC
site, a ligated insert/vector molecule will be formed that
comprises LIC ends at each end of the ligated insert/vector
molecule. The insert can then be joined to a LIC ready vector.
Aslanidis et al., (1994) PCR Methods Appl. 4:172; Aslanidis and de
Jong (1990) Nucleic Acids Res. 18:6069. Briefly, the ligated
insert/vector molecule is subjected to treatment with, for example,
Pfu DNA polymerase in the presence of dATP. In the absence of dTTP,
dGTP, and dCTP, the 3'- to 5'-exonuclease activity of Pfu DNA
polymerase removes 12 to 13 nucleic acids from the 3'-ends of the
ligated insert/vector molecule. This activity continues until the
first adenine is encountered. This produces a ligated insert/vector
molecule with 5'-extended single-stranded tails that are
complementary to the single-stranded tails of a LIC ready vector.
The ligated insert/vector molecule will anneal to the LIC ready
vector without further enzymatic treatment.
[0058] The second end of the linear molecule can further comprise a
site for homologous recombination or a site for site-specific
recombination. Homologous recombination is a recombination event
occurring between homologous sequences of nucleic acids. The
enzymes responsible for homologous recombination can use any pair
of homologous sequences as substrates, although some types of
nucleic acid sequences can be favored over others. Sites for
homologous recombination comprise nucleic acid sequences that are
homologous to the nucleic acid sequences of a cloning vector, such
as a circular plasmid. The sites can insert (or integrate) into a
cloning vector by homologous recombination, thereby inserting or
displacing a nucleic acid sequence, or deleting a nucleic acid
sequence altogether.
[0059] To create a homologous recombinant plasmid cloning vector, a
plasmid cloning vector is prepared which contains homologous
recombination nucleic acid sites that are substantially homologous
to those sites occurring on the ligated insert/vector of interest.
Substantially homologous nucleic acid sequences are those nucleic
acid sequences that share sufficient nucleic acid sequence homology
to provide for sufficient homologous recombination between a
ligated insert/vector sequence and a plasmid cloning vector.
Sufficient nucleic acid sequence homology is the amount which
provides for homologous recombination at a frequency which allows
for detection of plasmid cloning vectors in which homologous
recombination and integration of the ligated vector/insert has
occurred. Substantially homologous nucleic acid sequences
preferably share regions with about 60% to 100% nucleic acid
sequence homology, and more preferably about 75% to 100% homology
in the nucleic acid sequence. A site for homologous recombination
can be present in the plasmid cloning vector in two or more copies.
The homologous recombination sites in the plasmid cloning vector
are of sufficient length for successful homologous recombination
with a ligated insert/vector molecule. Typically, each homologous
recombination site is at least 30, 75, 100, 150, 250, 500, or 1000
base pairs. The ligated insert/vector sequence comprises these
substantially homologous recombination sites at both the 5'- and
3-' ends. The ligated insert/vector sequence is transformed into a
host cell, such as an E. coli cell that contains the plasmid
cloning vector. Preferably, the host cell is RecA+. Rec A is the
product of the recA locus of E. coli and is a protein that is
involved in recombination.
[0060] In addition to homologous recombination as described above,
enzyme-assisted site-specific integration systems are known in the
art and can be applied to integrate a ligated nucleic acid
insert/vector molecule at a predetermined location in a cloning
vector molecule. Site-specific recombination is a recombination
event between specific pairs or sequences. The recombination event
involves specific sequences of nucleic acids comprising a short
stretch of homology necessary for the recombination event. The
enzymes involved in the recombination event will act only on this
particular pair of target sequences. Examples of such
enzyme-assisted integration systems include the Cre
recombinase/loxP target system (e.g., as described in Baubonis and
Sauer (1993) Nucl. Acids Res. 21:2025; and Fukushige and Sauer,
(1992) Proc. Natl. Acad. Sci. USA 89:7905). A loxP site (locus of
crossing over) comprises two 13 base pair inverted repeats
separated by an 8 base pair asymmetric spacer region:
TABLE-US-00001 ATAACTTCGTATA ATGTATGC TATACGAAGTTAT (SEQ ID
NO:2)
[0061] A loxP site of the invention comprises variants and mutants
of this sequence that function to produce site-specific
recombination. Cre is a 38 kDa recombinase protein from
bacteriophage P1 which mediates intramolecular and intermolecular
site-specific recombination between loxP sites. Sauer, (1993)
Methods Enzymol. 225:890. A loxP site is an asymmetrical nucleotide
sequence and two lox sites on the same DNA molecule can have the
same or opposite orientation with respect to one another. See U.S.
Pat. No. 4,959,317. Where two loxP sites occur in the same
orientation on a nucleic acid molecule, recombination between the
loxP sites results in the deletion of the nucleic acid segment
located between the two loxP sites and a connection between the
resulting ends of the original nucleic acid molecule. The deleted
nucleic acid molecule will form a circular molecule of nucleic
acid. The original nucleic acid molecule and the circular nucleic
acid molecule will each contain a single loxP site. Where two loxP
sites occur in opposite orientations on the same nucleic acid
molecule recombination will result in an inversion of the
nucleotide sequence of the nucleic acid segment located between the
two loxP sites. Further, where two loxP sites occur on each of two
nucleic acid segments, reciprocal exchange of nucleic acid segments
proximate to the loxP sites can occur.
Methods of Covalently Joining
[0062] Insert polynucleotide molecules comprising a 5'-OH group on
each end or a 5'-OH on one end and a 5'-phosphate group on the
other end can be covalently joined to flanking polynucleotide
molecules such that non-directional or directional covalent joining
is achieved. Where an insert polynucleotide molecule has a 5'-OH
group on each end non-directional covalent joining of the insert to
flanking polynucleotide molecules results. For example, where an
insert polynucleotide (I) with a 5'-OH group at each end is
covalently joined to flanking molecules, for example, a left vector
arm (LVA) and a right vector arm (RVA) each with a topoisomerase
polypeptide covalently joined at only one end, the result will be
non-directional covalent joining of the molecules. A LVA, RVA, and
insert molecule are incubated together under conditions sufficient
to permit their topoisomerase-mediated covalent joining to form a
covalently joined nucleic acid molecule where the insert molecule
is positioned between the LVA and RVA. Four different covalently
joined products (ligated insert/vector molecules) will result:
LVA-I-LVA, RVA-I-RVA, LVA-I-RVA, and RVA-I-LVA. Only the LVA-I-RVA
and RVA-I-LVA products are viable replication competent entities.
Where an insert polynucleotide has a 5'-OH group on one end and a
5'-phosphate group on the other end directional covalent joining of
the insert to flanking polynucleotide molecules can result. For
example, where an insert polynucleotide is covalently joined to a
flanking molecules such as a LVA and a RVA, each comprising a
topoisomerase covalently bound to only one end, directional
covalent joining of the molecules can result. A first vector arm,
for example, a LVA is covalently joined to an insert molecule at
the 5'-OH end by incubating a LVA and an insert molecule together
under conditions sufficient to permit topoisomerase-mediated
covalent joining to form a ligated nucleic acid molecule where the
insert molecule is positioned adjacent to a LVA to create
LVA-I-phosphate. The 5'-phosphate end of the insert is unable to be
ligated to the LVA or RVA because either the LVA or RVA has a
3'-phosphate, which is the site to which a topoisomerase
polypeptide is joined to the LVA and RVA. The LVA-I-5'-phosphate is
treated with phosphatase, under conditions which permit removal of
a 5'-phosphate group from the ligated nucleic acid resulting in a
LVA-I-5'-OH molecule. The LVA-I-5'-OH molecule is then covalently
joined to the RVA to form LVA-I-RVA by incubating a LVA-I-5'-OH
molecule with a RVA under conditions which permit topoisomerase
covalent joining to form a ligated molecule where the insert
molecule is positioned between a RVA and a LVA (a ligated
insert/vector molecule).
[0063] Alternatively, an insert polynucleotide comprising a 5'-OH
group on one end and a 5'-phosphate group on the other end can be
covalently joined in a directional manner to a flanking nucleic
acid molecule comprising a topoisomerase polynucleotide on only one
end and to a second flanking molecule comprising a ligation
substrate site on one end. For example, an insert molecule can be
covalently joined to a flanking nucleic acid molecule, such as a
LVA, comprising a topoisomerase polypeptide on only one end and to,
for example, a RVA comprising a ligation substrate end on one end.
The insert LVA, and RVA are covalently joined by
topoisomerase-mediated joining and ligase-mediated joining under
conditions sufficient to form a ligated nucleic acid where the
insert molecule is positioned between a LVA and a RVA to form
LVA-I-RVA (a ligated insert/vector molecule). This reaction can
take place in one step.
[0064] After the ligated insert/vector molecule described above has
been constructed, the two vector arms can be non-covalently or
covalently joined to one another, at the ends distal to the
covalently attached topoisomerase polypeptide or ligation substrate
site (i.e., at their free ends), by a number of methods such that a
circular molecule is formed. For example, the ends of the ligated
insert/vector molecule can comprise ligase substrate sites or
complementary nucleic acid sequences such that the ends can be
joined by ligase enzyme mediated ligation or complementary sequence
annealing. Further, where the ends of the ligated insert/vector
molecule comprise 5'-OH groups the ends can be joined by
topoisomerase mediated ligation using a polynucleotide comprising a
topoisomerase polypeptide at both ends of the polynucleotides. See
e.g. U.S. Pat. No. 5,766,891. Further, where the ends of the
ligated insert/vector molecule comprise in vitro or in vivo
site-specific recombination sites or in vivo homologous
recombination sites the ligated insert/vector molecule can be
recombined into a circular plasmid containing the same
recombination sites.
[0065] The methods of directional and non-directional covalently
joining of nucleic acid molecules are useful in, for example,
end-labeling, ligand tagging, and molecular cloning.
Methods of Molecular Cloning
[0066] Insert polynucleotide molecules comprising a 5'-OH group on
each end or a 5'-OH on one end and a 5'-phosphate group on the
other end can be cloned into vector molecules such that
non-directional or directional cloning is achieved.
[0067] Non-directional cloning can be accomplished by cloning a
polynucleotide insert molecule comprising 5'-OH groups at both ends
of the molecule into a nucleic acid vector. For example, an insert
polynucleotide (I) with a 5'OH group at each end can be cloned into
a vector, such as a left vector arm (LVA) and a right vector arm
(RVA) where each vector arm has a topoisomerase polypeptide
covalently joined at only one end of the vector arm. The result
will be non-directional covalent joining of the molecules.
Preferably, the LVA and RVA molecules have a cloning substrate
site, such as a cos site, a LIC site, a loxP site, a site for
homologous recombination, a site for site-specific recombination,
or a ligase substrate site at the other end of the molecule. A LVA,
RVA, and insert molecule are incubated together under conditions
sufficient for topoisomerase-mediated covalent joining of the
molecules to form a ligated nucleic acid wherein the insert
molecule is positioned between the LVA and RVA. Four different
covalently joined products will result: LVA-I-LVA, RVA-I-RVA,
LVA-I-RVA, and RVA-I-LVA (ligated insert/vector molecules). Only
the LVA-I-RVA and RVA-I-LVA products are viable replication
competent entities.
[0068] Directional cloning can be accomplished by cloning a
polynucleotide insert molecule comprising a 5'-OH group at one end
of the molecule and a 5'-phosphate group at the other end into a
nucleic acid vector. For example, an insert polynucleotide (I) with
a 5'-OH group at one end and a 5'-phosphate at the other end can be
cloned into a linear cloning vector, where the linear cloning
vector has a topoisomerase polypeptide covalently joined at one end
and a ligation substrate site at the other end. The insert
polynucleotide, the linear cloning vector, and a ligase are
incubated together under conditions sufficient for their covalent
joining to form a ligated circular vector (a ligated insert/vector
molecule). The circular vector can then be transformed into a host
cell.
[0069] Directional cloning can also be accomplished by cloning an
insert polynucleotide having a 5'-OH group on one end and a
5'-phosphate group on the other end into a vector where the vector
comprises, for example, two vector arm molecules comprising a
topoisomerase polynucleotide at only one end and a cloning
substrate site at the other end. For example, a first vector arm,
LVA, is covalently joined to an insert molecule at the 5'-OH end by
incubating a LVA and an insert molecule together under conditions
sufficient to permit topoisomerase-mediated covalent joining to
form a ligated nucleic acid molecule where the insert molecule is
positioned adjacent to a LVA to create LVA-I-phosphate. The
5'-phosphate end of the insert is unable to be ligated to the LVA
or RVA because a topoisomerase polypeptide is joined to the LVA and
RVA at the 5'-phosphate. The LVA-I-5'-phosphate is treated with
phosphatase, under conditions which permit removal of a
5'-phosphate group from the ligated nucleic acid resulting in a
LVA-I-5'-OH molecule. The LVA-I-5'-OH molecule is then covalently
joined to the RVA to form LVA-I-RVA by incubating a LVA-I-5'-OH
molecule with a RVA under conditions which permit topoisomerase
covalent joining to form a ligated molecule where the insert
molecule is positioned between a RVA and a LVA (a ligated
insert/vector molecule). Preferably, the cloning substrate site is
a cos site, a LIC site, a loxP site, a site for homologous
recombination, a site for site-specific recombination, or a
ligation substrate site.
[0070] Alternatively, directional cloning can be accomplished with
an insert polynucleotide comprising a 5'-OH group on one end and a
5'-phosphate group on the other end and two vector molecules. One
vector molecule comprises a topoisomerase polynucleotide on only
one end and a cloning substrate site on the other end. The other
vector molecule comprises a ligation substrate site on one end and
a cloning substrate site on the other end. An insert, a first
vector molecule comprising a topoisomerase polypeptide at one end
and a cloning substrate site at the other end, such as a LVA, and a
second vector molecule such as a RVA comprising a ligation
substrate site at one end and a cloning substrate at the other end
are covalently joined by topoisomerase-mediated joining and
ligase-mediated joining under conditions sufficient to form a
ligated nucleic acid where the insert molecule is positioned
between the LVA and the RVA vector molecules (a ligated
insert/vector molecule). Preferably, the cloning substrate site is
a cos site, a LIC site, a loxP site, a site for homologous
recombination, a site for site-specific recombination, or a
ligation substrate site.
[0071] Where the ligated insert/vector molecule comprises cos sites
at each end, the linear molecule can be transformed directly into a
host cell. Where the ligated insert/vector molecule comprises LIC
ends at each end, the LIC ends can be annealed to a circular
plasmid vector with LIC compatible ends. The circular molecule can
be transformed into a host cell. Where the ligated insert/vector
molecule comprises loxP sites on both ends, the ligated
insert/vector molecule can be recombined into a circular plasmid in
vitro in the presence of Cre recombinase. The recombinant circular
plasmid can then be transformed into a host cell. Alternatively, a
ligated insert/vector molecule with loxP sites at both ends of the
molecule can be directly transformed into a host cell, such as E.
coli harboring a plasmid suitable for site-specific recombination.
The host cell may be rec A+ or recA-, and is preferably recA-.
Where the covalently joined insert/vector molecule comprises sites
for homologous recombination at each end, the covalently joined
insert/vector molecule can be directly transformed into a suitable
host cell harboring a plasmid suitable for homologous
recombination.
[0072] The covalently joined insert/vector can be transformed into
a prokaryotic or eukaryotic cell. Preferably, the covalently joined
insert/vector is transformed into a prokaryotic host cell, such as
a bacteria cell such as E. coli. Transformation of a ligated
insert/vector molecule into a host cell can be done by any method
known in the art. Methods for transformation of host cells can be
found in Sambrook et al. and Ausubel and include, but are not
limited to transfection, chemical transformation, electroporation,
and lipofection. Where a bacteriophage lambda vector has been used
according to the invention, the ligated insert/lambda vector can be
packaged in vitro and then transfected into host cells, such as
XL1-Blue E. coli. See e.g. Sambrook et al.
[0073] The following are provided for exemplification purposes only
and are not intended to limit the scope of the invention described
in broad terms above. All references cited in this disclosure are
incorporated herein by reference.
EXAMPLES
Example 1
[0074] Inter-Molecular Ligation and Molecular Cloning Using
Univalent Topoisomerase-Bound DNA
[0075] An insert nucleic acid molecule, for example, a PCR product,
can be generated by PCR using a primer set consisting of a
5'-primer and 3'-primer. Two vector nucleic acid molecules, for
example, a left vector arm and a right vector arm are prepared such
that a topoisomerase enzyme (TOPO) is covalently bound only to one
end of a nucleic acid molecule to form a univalent topoisomerase
vector molecule. PCR primers for generating an insert molecule can
be synthesized to possess either a hydroxyl group or phosphate
group at each of the 5'-ends. A hydroxyl group permits ligation to
topoisomerase-bound DNA while a phosphate group prohibits such
ligation.
[0076] For non-directional ligation of a PCR insert molecule to,
for example two vector arms, both PCR primers will possess
5'-hydroxyl groups. The PCR insert can ligate with the vector arms
to form four different types of ligation products: 1) left vector
arm (LVA)-insert molecule (I)-left vector arm (LVA); 2) right
vector arm (RVA)-insert (I)-right vector arm (RVA); 3) LVA-I-RVA;
and 4) RVA-I-LVA. Only the LVA-I-RVA and RVA-I-LVA create viable
replication competent entities (FIG. 1).
[0077] For directional ligation of a PCR insert molecule to, for
example, left and right vector arms, one PCR primer possesses a
5'-hydroxyl group and the other PCR primer possesses a 5'-phosphate
group. The PCR generated insert molecule is generated and is first
ligated to one vector arm, for example, a LVA to create a
LVA-I-5'-phosphate molecule. The 5'-phosphate end of this molecule
is unable to ligate to the LVA or RVA because the vector arm sites
to which the TOPO is bound contain a 3'-phosphate. This molecule is
then dephosphorylated to create to LVA-I-5'-OH. The LVA-I-5'-OH
molecule is then ligated to the other vector arm (RVA) to form
LVA-I-RVA (FIG. 2). Once the ligated insert/vector molecule
described above has been constructed, the two vector arms can be
non-covalently or covalently joined to one another, at the ends
distal to the covalently attached topoisomerase polypeptide (i.e.,
their free ends), by a number of methods such that a circular
molecule is formed. Such methods include, for example, ligase
enzyme mediated ligation, complementary sequence annealing,
topoisomerase mediated ligation, in vitro or in vivo site-specific
recombination, or in vivo homologous recombination.
Example 2
Directional Molecular Cloning Using Topoisomerase and a Ligase
Enzyme
[0078] A nucleic acid insert is generated using, for example, a
pair of PCR primers wherein one primer (P1) has a hydroxyl group at
its 5'-end (OH-P1) and the other primer (P2) has a phosphate group
at its 5'-end (P2-P) (see FIG. 3). The insert molecule is generated
by PCR. A linear vector nucleic acid is prepared such that it has
TOPO bound at one end (univalent TOPO-bound nucleic acid molecule);
the other end of the linear vector nucleic acid comprises a
substrate for ligation (a 3'-OH) to be mediated by a ligase enzyme.
In a single incubation, the PCR insert can be ligated to the
TOPO-end of the linear vector nucleic acid via TOPO-mediated
ligation and to the other end of the linear vector nucleic acid via
a ligase enzyme-mediated reaction. The product of the ligation is
transformed into an appropriate host cell. A cloning event mediated
by both topoisomerase and DNA ligase is unidirectional. The
hydroxyl or phosphate group at the 5'-end of the PCR primers
determines the directionality of the insert.
[0079] A second approach involving a topoisomerase- and
ligase-mediated ligation comprises generation of an insert by for
example, PCR. Where PCR is used to generate an insert, a pair of
PCR primers where one has a hydroxyl group at its 5'-end (HO-P1)
and the other has a phosphate group at its 5'-end (P2-P) (see FIG.
4). A vector, such as two vector nucleic acid arms, can be prepared
such that one vector arm has a TOPO bound at one end (univalent
TOPO-bound DNA molecule) and the other vector arm has a substrate
for ligation at one end. In a single incubation, the PCR insert is
ligated to the one vector arm with a TOPO end via TOPO-mediated
ligation and to the other vector arm with the ligation-ready end
via ligase enzyme-mediated reaction. The product of the ligation is
transformed into an appropriate host cell. The cloning event
mediated by both topoisomerase and DNA ligase is unidirectional.
The hydroxyl or phosphate group at the 5'-end of the PCR primers
determines the directionality. The other ends of the two vector
arms are then joined by any of the methods described above. Using
this cloning method the ligation products comprised of RVA-I-RVA or
LVA-I-LVA should not be formed, but in the event that some do
occur, such ligation products are incapable of subsequent
replication and propagation.
Example 3
Molecular Cloning Using Topoisomerase and cos Ends
[0080] A method of molecular cloning using topoisomerase and cos
ends can comprise a vector, where such a vector may consist of two
vector arms, with each arm consisting of one TOPO-end and one cos
end. cos refers to the cohesive ends present at the termini of
bacteriophage lambda. An insert, such as a PCR insert, can be
generated using primers comprising 5'-OH termini. The PCR insert
can be ligated to a TOPO-end of the two vector arms by DNA
topoisomerase (see FIG. 5). Ligation events that result in
LVA-I-LVA or RVA-I-RVA cannot subsequently be propagated. The
product of the ligation can be transformed into a suitable host.
The distal ends of the vector arms contain terminal cos sites that
are readily annealed to one another in E. coli host cells by virtue
of their explicit sequence. cos sites do not anneal in vitro at
room temperature.
[0081] This method of cloning can be directional or
non-directional. In the case of non-directional cloning, an insert
comprises a 5'-hydroxyl ends and can be ligated to, for example,
two vector arms in a single reaction. For directional cloning, an
insert can be generated by, for example, PCR wherein one PCR primer
has a 5'-hydroxyl group and the other PCR primer has a 5'-phosphate
group. Thus, the resulting PCR insert will contain one 5'-hydroxyl
end and one 5'-phosphate end. The insert is to be ligated
sequentially, first to a left vector arm containing a TOPO bound
end followed by dephosphorylation of the 5'-phosphate of the insert
and then ligation to the right vector arm containing a TOPO bound
end (FIG. 6).
[0082] The ligation product of the insert to the vector is a linear
molecule in vitro with two cos sequences at its end. It is
transformed into a host, such as E. coli more efficiently than a
circular molecule.
Example 4
Molecular Cloning Using Topoisomerase and LIC Ends
[0083] A method of molecular cloning using topoisomerase and LIC
ends can comprise a vector, such as two vector arms, each
consisting of one TOPO-end and one LIC end. An insert, such as a
PCR insert, can be generated using primers comprising two 5'-OH
termini. The PCR insert can be ligated to a TOPO-end of the two
vector arms by DNA topoisomerase (see FIG. 7). Ligation events that
result in LVA-I-LVA or RVA-I-RVA cannot subsequently be propagated.
The distal ends of the vector arms contain terminal LIC sites that
are readily annealed to a plasmid comprising LIC compatible
ends.
[0084] This method of cloning can be directional or
non-directional. In the case of non-directional cloning, an insert
comprising 5'-hydroxyl ends and can be ligated to, for example, two
vector arms in a single reaction. For directional cloning, an
insert can be generated by, for example, PCR wherein one PCR primer
has a 5'-hydroxyl group and the other PCR primer has a 5'-phosphate
group. Thus, the resulting PCR insert will contain one 5'-hydroxyl
end and one 5'-phosphate end. The insert is to be ligated
sequentially, first to the left vector arm containing a TOPO bound
end and followed by dephosphorylation of the 5'-phosphate of the
insert and then ligation to the right vector arm containing a TOPO
bound end (FIG. 8).
Example 5
Molecular Cloning into Lambda Vector
[0085] The vector can comprise lambda DNA vector arms (termed left
lambda arm (LLA)) and right lambda arm (RLA)). An insert, such as a
PCR generated insert, can be ligated to the lambda vector arms in a
directional manner or non-directional manner. In the case of
non-directional cloning, a PCR insert can be generated using
5'-hydroxyl PCR primers. The insert can be ligated to two lambda
vector arms in a single reaction. Ligation events resulting in
LLA-I-LLA or RLA-I-RLA cannot subsequently be propagated. For
directional cloning, one PCR primer has a 5'-hydroxyl end and the
other PCR primer has a 5'-phosphate end. Thus, the PCR insert is
comprised of one 5'-hydroxyl end and one 5'-phosphate end. The
insert can be ligated sequentially to the two lambda vector arms
with a dephosphorylation step in between as depicted in FIG. 9. The
ligated lambda construct can be packaged in vitro and transfected
into host cells such as XLI-Blue E. coli. A circular plasmid DNA
containing the insert of interest can be rescued from the lambda
vector using, for example, ZAP technology (Stratagene).
Example 6
Molecular Cloning Into a Linear Plasmid DNA Molecule
[0086] A vector can comprise vector arms of a linear plasmid such
as N15. An insert, such as a PCR generated insert, can be ligated
to the plasmid vector arms in a directional manner or
non-directional manner. In the case of non-directional cloning, a
PCR insert can be generated using 5'-hydroxyl PCR primers. The
insert can be ligated to two plasmid vector arms in a single
reaction (FIG. 10). Ligation events resulting in LVA-I-LVA or
RVA-I-RVA cannot subsequently be propagated. For directional
cloning, one PCR primer has a 5'-hydroxyl end and the other PCR
primer has a 5'-phosphate end. Thus the PCR insert is comprised of
one 5'-hydroxyl end and one 5'-phosphate end. The insert can be
ligated sequentially to the two plasmid vector arms with a
dephosphorylation step in between as depicted in the FIG. 11. The
linear DNA can be transformed directly into E. Coli. Alternatively,
the ligated plasmid construct can be packaged in vitro and
transfected into host cells such as XLI-Blue E. coli. A DNA
containing the insert of interest can be rescued from the vector
using, for example, ZAP technology (Stratagene).
[0087] A vector can also comprise a linear plasmid vector
consisting of a covalently bound topoisomerase polypeptide at one
end and a ligation substrate site at the other end (see FIG. 4).
Incubation of the vector with an insert molecule comprising 5'-OH
group on one end and a 5'-phosphate group on the other end, under
conditions sufficient for topoisomerase-mediated ligation and
ligase enzyme-mediated ligation results in a ligated circular
plasmid comprising the insert molecule. The plasmid can be
transformed into a host cell.
Example 7
Molecular Cloning Using Topoisomerase and Site-Specific
Recombination
[0088] A vector can comprise vector arms that comprise one TOPO-end
and one loxP end. The loxP site can be recombined with a second
loxP site in the presence of a Cre site-specific recombination
protein. An insert, such as a PCR generated insert, can be ligated
to the TOPO-end of the two vector arms. Such cloning can be
directional or non-directional. In the case of non-directional
cloning, an insert, such as a PCR insert can be generated from PCR
primers each comprising 5'-hydroxyl ends. An insert comprising two
5'-OH ends can be ligated to two vector arms in a single reaction
(FIG. 12). For directional cloning, an insert can be generated by,
for example, PCR wherein one PCR primer comprises a 5'-hydroxyl end
and the other PCR primer comprises 5'-phosphate end resulting in an
insert that comprises one 5'-hydroxyl end and one 5'-phosphate end.
The insert can be ligated sequentially to two vector arms with a
dephosphorylation step in between as depicted in FIG. 13. The
ligation product comprises a loxP site at each end of a linear
molecule. The linear molecule can be recombined into a circular
recombinant plasmid in vitro, for example using purified Cre
recombinase or in vivo by, for example transformation into an E.
coli host expressing Cre recombinase and a plasmid that has loxP
sites.
Example 8
Molecular Cloning Using Topoisomerase and Homologous Recombination
in Vivo
[0089] In vivo homologous recombination can be exploited to
transfer a ligated insert/vector of interest into a circular
plasmid vector. Homologous sequences flank a ligated insert/vector
of interest and are substantially identical to sequences of a
plasmid cloning vector. A ligated insert/vector of interest is
recombined into a plasmid cloning vector of choice via homologous
recombination between the homologous sequences flanking the ligated
insert/vector and in the plasmid cloning vector. An insert can be
generated with homologous sequences attached to each end by, for
example, synthesizing PCR primers with homologous vector sequences,
of for example, 30, 75, 100, 150, 200, 250, 500, or 1000 base pairs
and using the PCR primers to generate a ligated insert/vector with
homologous vector sequences flanking the ligated insert/vector of
interest. A ligated insert/vector molecule with homologous
sequences at the ends can also be generated by preparing
topoisomerase-bound homologous sequence elements and employing a
TOPO cloning scheme as outlined in FIGS. 14 and 15 for generating
an insert with homologous sequence elements on each end. A PCR
amplified insert containing TOPO ligated arms can be transformed
into host cells containing a cloning vector wherein homologous
recombination can occur. For efficient in vivo homologous
recombination, a recA+ host strain can be used. To protect a linear
insert from degradation by endogenous exonuclease activities, the
ends of the insert can be modified to either inhibit or prohibit
exonuclease digestion events.
[0090] To achieve site-specific in vivo recombination, lambda
attachment sites can be employed in place of the homologous
sequences described above. In this scenario, lambda attachment
sites flank a ligated insert/vector of interest, which is generated
according to the PCR and TOPO cloning schemes described above. The
ligated insert/vector with the flanking lambda attachment sites is
transformed into host cells containing a cloning vector with lambda
attachment sites. Inside the host cell, the ligated insert/vector
then can be site-specifically recombined into a plasmid cloning
vector between the lambda attachment sites flanking the ligated
insert/vector and those sites in the plasmid cloning vector.
OTHER EMBODIMENTS
[0091] Other embodiments are within the following claims.
Sequence CWU 1
1
2 1 6 DNA Vaccinia virus 1 ctcctt 6 2 34 DNA Artificial sequence
loxP site consensus sequence 2 ataacttcgt ataatgtatg ctatacgaag
ttat 34
* * * * *