U.S. patent application number 11/045686 was filed with the patent office on 2005-10-20 for rapid and enzymeless cloning of nucleic acid fragments.
Invention is credited to Chen, Shizhong, Felgner, Philip L., Liang, Xiaowu, Teng, Andy, Xia, Dongyuan.
Application Number | 20050233422 11/045686 |
Document ID | / |
Family ID | 25271962 |
Filed Date | 2005-10-20 |
United States Patent
Application |
20050233422 |
Kind Code |
A1 |
Liang, Xiaowu ; et
al. |
October 20, 2005 |
Rapid and enzymeless cloning of nucleic acid fragments
Abstract
A method for cloning a nucleic acid fragment into a vector by
flanking the fragment with first and second adapter sequences, and
contacting the fragment with the vector having sequences homologous
to the first and second adapter sequences under conditions such
that the nucleic acid fragment is incorporated into the vector by
homologous recombination in vivo in a host cell. Additionally, a
method for selecting for a successful transformation of a vector by
a nucleic acid insert. Also, systems for cloning a nucleic acid
fragment into a vector without at least one of a restriction
enzyme, a ligase, a gyrase, a single stranded DNA binding protein,
or other DNA modifying enzymes. Further, a kit for cloning a
nucleic acid fragment into a vector.
Inventors: |
Liang, Xiaowu; (La Jolla,
CA) ; Teng, Andy; (Fullerton, CA) ; Chen,
Shizhong; (San Diego, CA) ; Xia, Dongyuan;
(Powell, OH) ; Felgner, Philip L.; (Rancho Santa
Fe, CA) |
Correspondence
Address: |
PAUL, HASTINGS, JANOFSKY & WALKER LLP
P.O. BOX 919092
SAN DIEGO
CA
92191-9092
US
|
Family ID: |
25271962 |
Appl. No.: |
11/045686 |
Filed: |
January 28, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11045686 |
Jan 28, 2005 |
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10125789 |
Apr 16, 2002 |
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6936470 |
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10125789 |
Apr 16, 2002 |
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09836436 |
Apr 17, 2001 |
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Current U.S.
Class: |
435/91.2 ;
435/455 |
Current CPC
Class: |
C07K 2319/60 20130101;
C07K 2319/23 20130101; C12N 15/66 20130101; C07K 2319/43 20130101;
C12N 15/10 20130101; C07K 2319/42 20130101; C07K 2319/21
20130101 |
Class at
Publication: |
435/091.2 ;
435/455 |
International
Class: |
C12P 019/34; C12N
015/85 |
Claims
1-43. (canceled)
44. A system for cloning a nucleic acid fragment into a vector
without at least one of a restriction enzyme, a ligase, a gyrase, a
topoisomerase, or a single stranded DNA binding protein, the system
comprising a nucleic acid fragment flanked by first and second
adapter sequences and a vector having sequences homologous to the
first and second adapter sequences wherein the nucleic acid
fragment is adapted to incorporate into the vector by
recombination.
45. The system of claim 44, wherein the nucleic acid fragment
flanked by the first and the second adapter sequences is generated
by PCR and without the use of at least one of a restriction enzyme,
a ligase, a gyrase, a single stranded DNA binding protein, a
topoisomerase, or any other DNA modifying enzyme.
46. The system of claim 44, wherein the nucleic acid fragment
flanked by the first and the second adapter sequences is a
transcriptionally active PCR fragment.
47. The system of claim 44, wherein the recombination comprises
homologous recombination.
48. A system for cloning a nucleic acid fragment into a bacterium
without the use of a restriction enzyme, a ligase, a gyrase, or a
single stranded DNA binding protein, the system comprising a
nucleic acid fragment flanked by first and second adapter sequences
and a bacterium bearing a vector, the vector having sequences
homologous to the first and second adapter sequences, wherein the
nucleic acid fragment is adapted to incorporate into the vector
within the bacterium by recombination.
49. A kit for cloning a nucleic acid fragment into a vector
comprising reagents for amplification of the nucleic acid fragment,
wherein the reagents upon amplification provide for a nucleic acid
fragment flanked by first and second adapter sequences, a vector, a
competent cell, or a competent cell bearing the vector, and the
competent cell is ready to be transformed by electroporation or
chemical transformation.
50-74. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to methods, systems and kits
for fast and enzymeless cloning of nucleic acid fragments into
vectors and for forced cloning selection for successful
transformation.
[0003] 2. Description of the Related Art
[0004] Traditional molecular cloning involves the use of
recombinant DNA technology to propagate DNA fragments inside a
foreign host. Generally, the DNA fragments are isolated from cDNA
libraries or chromosomes and subcloned into a vector utilizing
various enzymes. For example, a small amount (i.e., 0.01-0.03
.mu.g) of isolated DNA fragment is contacted with a small amount
(i.e., 0.01 .mu.g) of linearized vector. Using enzymes, such as
ligases, the fragments are ligated.
[0005] The DNA fragment-containing vector is introduced into a host
cell according to various methods of transformation. For example,
one tenth to one half of the ligation mix can be electroporated
into a cell, such as E. Coli. Generally, a large number of cells,
such as 1.times.10.sup.8, is used to increase the ratio of cells to
DNA fragment-containing vector to enhance the probability of
obtaining a cell with the desired clone. For example, the ration
might be 0.02-0.2 fg/cell.
[0006] A selection marker is usually included in the vector to
increase the probability that the host cell has the DNA
fragment-containing vector. Following introduction into the host
cell and selection of the host cell containing the vector, the DNA
fragment within the vector can then be replicated along with the
host cell DNA. The DNA fragment-containing vector then can be
isolated and purified from the host cell and transfected into
animal cells or tissues for functional analysis of the encoded gene
product.
[0007] Although the traditional enzymatic cloning methods have
advantages such as pinpoint accuracy, they also have significant
drawbacks. As mentioned, the methods require the use various
enzymes that can be very expensive. In addition, the same DNA
fragment has to be enzymatically treated every time it is
introduced into a different vector. All of the vector may not be
effectively cut by the enzymes, which can result in a higher number
of background cells. Also, the methods involve slow and laborious
processes. Selection of host cells containing the DNA
fragment-containing vector entails significant labor and is still
an uncertain process. Traditional cloning methods, even in
conjunction with the use of polymerase chain reaction (PCR), are
still time consuming, costly and difficult to automate.
[0008] The present invention provides simple and rapid methods,
systems and kits for cloning nucleic acid fragments.
SUMMARY OF THE INVENTION
[0009] Embodiments of the present invention relate to methods for
cloning a nucleic acid fragment into a vector by flanking the
fragment with first and second adapter sequences. The fragment can
be contacted with the vector having sequences homologous to the
first and second adapter sequences under conditions such that the
nucleic acid fragment is incorporated into the vector by
recombination in a host cell. The first and second adapter
sequences can be unique. The fragment can be directionally
incorporated into the vector. The recombination can occur in vivo
in the host cell.
[0010] The nucleic acid fragment can be generated by polymerase
chain reaction (PCR). The first and second adapter sequences can be
incorporated to the nucleic acid fragment by PCR. The resulting
nucleic acid fragment can be a transcriptionally active PCR
fragment.
[0011] The first and second adapter sequences further can include a
functional element. The functional element can include, for
example, a promoter, a terminator, a nucleic acid fragment encoding
a selection marker gene, a nucleic acid fragment encoding a known
protein, a fusion tag, a nucleic acid fragment encoding a portion
of a selection marker gene, a nucleic acid fragment encoding a
growth promoting protein, a nucleic acid fragment encoding a
transcription factor, a nucleic acid fragment encoding an
autofluorescent protein (e.g. GFP), and the like.
[0012] The nucleic acid fragment can include an additional element,
such as, for example, an operably linked promoter, a termination
sequence, an operon, a fusion tag, a signal peptide for
intracellular or intercellular trafficking, a peptide, a protein,
an antisense sequence, a ribozyme, a protein binding site, and the
like.
[0013] The promoter can be, for example, a promoter from a plant or
a plant pathogen, such as cauliflower mosaic virus, from a mammal
or a mammalian pathogen, such as CMV, SV40, MMV, HIV, from a
fungus, such as yeast (Gal 4 promoter), from a bacterium or a
bacterial phage, for example, lambda, T3, T7, SP6 and the like. The
terminator sequence can be derived from a plant, a procaryotyic
source or a eukaryotic source, such as SV40, bovine growth hormone,
rabbit beta-globin i, and the like. The operon can be, for example,
lac operon, Tet/on operon, Tet/off operon, trp operon. The fusion
tag can include 6.times. to 10.times. his-tag, GST tag, fluorescent
protein tag, Flag tag, HA tag, and the like. The protein can
include an enzyme, a receptor, a transcription factor, a
lymphokine, a hormone, an antigen, and the like.
[0014] The vector can be, for example a plasmid, a cosmid, a
bacterial artificial chromasome (BAC), and the like. The plasmid
can be CoE1, PR100, R2, pACYC, and the like. The plasmid can be
maintained in the host cell under the selection condition selecting
for the functional selection marker. The vector can also include a
functional selection marker, which for example can be a resistance
gene for kanamycin, amplicillin, blasticidin, carbonicillin,
tetracycline, chloramphenicol, and the like. The vector further can
include a dysfunctional selection marker that lacks a critical
element, and wherein the critical element is supplied by said
nucleic acid fragment upon successful homologous recombination. The
dysfunctional selection marker can be, for example, kenamycin
resistance gene, kanamycin resistance gene, ampicillin resistance
gene, blasticidin resistance gene, carbonicillin resistance gene,
tetracycline resistance gene, chloramphenicol resistance gene, and
the like. Further, the dysfunctional selection marker can be, for
example, a reporter gene, such as the lacZ gene, and the like.
[0015] The vector can include a negative selection element
detrimental to host cell growth. The negative selection element can
be disabled by the nucleic acid fragment upon successful
recombination. The negative selection element can be inducible. The
negative selection element can be, for example, a mouse GATA-1
gene. The vector can include a dysfunctional selection marker and a
negative selection element.
[0016] The host cell can be a bacterium. The bacterium can be
capable of in vivo recombination. Examples of a bacterium include
JC8679, TB1, DH5.alpha., DH5, HB101, JM101, JM109, LE392, and the
like.
[0017] The first and second adapter sequences can each be at least
11 bp, 15 bp, 20 bp, 25 bp, or 30 bp and the like. Further, the
first and second adapter sequences can each be at least 35 bp, 40
bp, or 45 bp, and the like. Still further, the first and second
adapter sequences can each be at least 50 bp, 60 bp, or greater
than 60 bp, and the like.
[0018] The contacting can include transforming a host cell with the
vector and the nucleic acid fragment. The transformation can
include, for example, electroporation, more preferably chemical
transformation, and the like.
[0019] In other embodiments, the host cell can be a bacterium
bearing the vector. The bacterium can be capable of in vivo
recombination. The bacterium can be, for example, JC8679, TB1,
DH5.alpha., DH5, HB101, JM101, JM109, LE392, and the like. The
contacting of the vector and the nucleic acid fragment can include
transforming the host cell bearing the vector with the nucleic acid
fragment.
[0020] The vector that is borne in the host cell can be a plasmid.
The plasmid can include a functional selection marker, such as, for
example, a resistance gene for kanamycin, ampicillin, blasticidin,
carbonicillin, tetracycline, chloramphenicol, and the like. The
plasmid can include a dysfunctional selection marker that lacks a
critical element, and wherein the critical element is supplied by
said nucleic acid fragment upon successful recombination. The
dysfunctional selection marker can be, for example a resistance
gene for kanamycin, kenamycin, ampicilin, blasticidin,
carbonicillin, tetracycline, chloramphenicol, and the like.
Further, the dysfunctional selection marker can be, for example, a
reporter gene, such as the lacZ gene, and the like.
[0021] Further, the vector borne in the host cell can include a
negative selection element detrimental to host cell growth, and the
negative selection element can be disabled by the nucleic acid
fragment upon successful homologous recombination. The negative
selection element can be inducible, for example. The negative
selection element can be, for example GATA-1 gene. The vector can
include a dysfunctional selection marker and a negative selection
element.
[0022] The recombination can include for example, homologous
recombination or any other like process. In some embodiments at
least 65%, 70%, 75%, or 85% of the cells have undergone successful
recombination. More preferably, 90% or 95% of the cells have
undergone successful recombination. Still more preferably 96%, 97%,
98%, 99% or 100% of the cells have undergone successful
recombination.
[0023] The vector can be a linearized vector, which can be prepared
by the digestion of a vector and purification of digested vector.
The purification can include chromatography and/or PCR. Also, the
vector can be prepared by successive rounds of digestion.
[0024] The cell, the nucleic acid fragment(s) and the vector can be
present at an amount of about 2.times.10.sup.7, 0.4-2.0 .mu.g, and
0.05-0.1 .mu.g respectively, for example.
[0025] Other embodiments of the present invention relate to methods
for selecting for successful transformation of a vector by a
nucleic acid insert. The methods can provide a nucleic acid insert
flanked by first and second adapter sequences that is adapted for
recombining with homologous sequences in a vector. The vector can
have a dysfunctional selection marker lacking a critical element
and the nucleic acid insert contains the critical element. The
nucleic acid insert can be contacted with the vector to effect
recombination at homologous sites such that the critical element is
supplied to the vector by the nucleic acid insert and the
dysfunctional selection marker is restored to a functional one. The
successfully restored selection marker can be selected for based
upon growth of a host containing the successfully recombined vector
that allows the host to grow or be identified in a selective
environment. The recombining can be by recombination, such as for
example, homologous recombination, and the like.
[0026] Further embodiments of the present invention relate to
methods for selecting for successful transformation of a vector by
a nucleic acid insert. The methods can include providing a nucleic
acid insert flanked by first and second adapter sequences that is
adapted for recombining with homologous sequences in a vector. The
vector can include a negative selection element detrimental to cell
growth. The nucleic acid insert can be contacted with the nucleic
acid insert to effect recombination at homologous sites such that
the negative selection element is disabled. Successful
transformation can be selected for based on the absence of a
functional negative selection element. The negative selection
element can be inducible, for example. The selection step can
include inducing the negative selection element. Methods utilizing
the negative selection element further can include the methods for
selecting for successful transformation of a vector by a nucleic
acid insert, wherein the vector includes a dysfunctional selection
marker lacking a critical element and the nucleic acid insert
contains the critical element, as discussed above. The negative
selection element can be disabled by insertion of a sequence
encoding a selection marker.
[0027] Other embodiments of the present invention relate to systems
for cloning a nucleic acid fragment into a vector without at least
one of a restriction enzyme, a ligase, a gyrase, a topoisomerase, a
single stranded DNA binding protein, or other DNA modifying
enzymes. The system can include a nucleic acid fragment flanked by
first and second adapter sequences and a vector having sequences
homologous to the first and second adapter sequences wherein the
nucleic acid fragment is adapted to incorporate into the vector by
recombination. The recombination can include homologous
recombination or any other like process. The nucleic acid fragment
flanked by the first and the second adapter sequences can be
generated by PCR without the use of at least one of a restriction
enzyme, a ligase, a gyrase, a topoisomerase, a single stranded DNA
binding protein, or any other DNA modifying enzyme. The nucleic
acid fragment flanked by the first and the second adapter sequences
can be a transcriptionally active PCR fragment.
[0028] Still further embodiments relate to systems for cloning a
nucleic acid fragment into a bacterium without the use of at least
one of a restriction enzyme, a ligase, a gyrase, a topoisomerase, a
single stranded DNA binding protein, or any other DNA modifying
enzyme. The system can include a nucleic acid fragment flanked by
first and second adapter sequences and a bacterium bearing a
vector, the vector having sequences homologous to the first and
second adapter sequences, wherein the nucleic acid fragment is
adapted to incorporate into the vector within the bacterium by
recombination, such as for example, homologous recombination.
[0029] Embodiments also relate to kits for cloning a nucleic acid
fragment into a vector. The kits can include reagents for
amplification of the nucleic acid fragment or fragments, wherein
the reagents upon amplification can provide for a nucleic acid
fragment or fragments flanked by first and second adapter
sequences; and can further include a vector, a competent cell, or a
competent cell bearing the vector, and the like. The competent cell
can be ready to be transformed by electroporation, chemical
transformation, and the like. The competent cell or the competent
cell bearing the vector can be a bacterium. The bacterium can be
capable of in vivo recombination.
[0030] Further embodiments relate to methods of generating a
substantially background-free linearized vector preparation. The
methods can include providing a circular vector that includes a
restriction enzyme cleavage site, wherein the site is flanked by
homologous sequences; linearizing the vector with a restriction
enzyme; and purifying the linearized vector to a purity. The purity
can be substantially 98%, 99%, or 100%, for example, full length
vector.
[0031] The purification can include chromatography, which can
include for example, affinity chromatography. The affinity
chromatography can include capturing an undigested vector, said
undigested vector comprising a binding molecule in a cloning site
such that the binding molecule is not present on the linearized
vector due to cleavage by at least one restriction enzyme. The
binding molecule can include a PNA binding sequence, for example.
The affinity chromatography can include capturing only the
linearized vector, where the linearized vector includes a binding
site. For example, the binding site can include an end of the
vector that is exposed by the restriction enzyme cleavage, wherein
the end is captured by a complementary probe on the affinity
column.
[0032] The purification also can include PCR amplification of the
linearized vector. The purification can include PCR amplification
of the linearized vector and chromatography purification. Further,
multiple rounds of digestions can be included. The purification can
result in substantially 98%, 99%, or 100% linearized vector
composition, for example.
[0033] The linearizing step can include cleaving the vector at one
site, two sites or more on the vector.
[0034] Still other embodiments relate to methods of introducing
more than one nucleic acid fragment into a vector within a cell.
The methods can include providing a first nucleic acid fragment
that includes a first coding sequence flanked by a first and a
second homologous sequence, wherein the first and second homologous
sequences are added to the first coding sequence by PCR. The
methods further can include providing a second nucleic acid
fragment that can include a second coding sequence flanked by a
third and a fourth homologous sequence, wherein the third and
fourth homologous sequences are added to the second coding sequence
by PCR. Further, the methods can include providing a linearized
vector comprising a first end and a second end, wherein the first
and second ends are respectively homologous to the first homologous
sequence on the first nucleic acid fragment and to the third
homologous sequence on the second nucleic acid fragment. Also, the
methods can include introducing the nucleic acid fragments and the
linearized vector into the cell under conditions such that the
nucleic acid fragments are incorporated into the vector by
recombination in the cell. The recombination can include homologous
recombination or the like.
[0035] The methods can include culturing the recombinant cell. The
methods can further include selecting a cell that has undergone
successful recombination. The selecting can include growing the
cell under selective conditions.
[0036] In some embodiments at least 50%, 60%, 65% or 70% of the
cells have undergone successful recombination. Preferably, at least
75%, 80%, 85%, 90% of the cells have undergone successful
recombination. More preferably, at least 95%, 99% or 100% of the
cells have undergone successful recombination.
[0037] In some embodiments more than two nucleic acid fragments can
be incorporated into a vector. For example 3, 4, 5, or more nucleic
acid fragments can be designed. Each fragment will have appropriate
homologous sequences to ensure directional incorporation into the
vector.
[0038] The linearized vector can be prepared by the digestion of a
vector and purification of the digested vector. The digestion can
include cutting of the vector with a restriction enzyme. A vector
can be prepared by PCR amplification so that digestion is not
required. The purification can include chromatography. The
purification can include PCR amplification of the linearized
vector. The purification can result in a substantially 98%, 99%,
99.5%, or 100% linearized vector composition. The linearized vector
of any of the embodiments of the invention can be prepared
according to any of the methods described herein.
[0039] The first or said second homologous sequence can each
include at least about 11, 15, 20, 21, 22, 23, 24, or 25 bases.
Further each can include 30, 35, 40, 45, 50, or more bases, for
example.
[0040] The introducing step can include chemical insertion of the
nucleic acid fragments and the linearized vector into the cell. The
chemical insertion can include co-introduction of the vector and
the nucleic acid fragments. Any other introduction method can be
used, such as electroporation, for example.
[0041] The cell, the nucleic acid fragments and the linearized
vector can be present at an amount of about 2.times.10.sup.7,
0.4-2.0 .mu.g, and 0.05-0.1 .mu.g respectively, for example.
[0042] Further embodiments relate to systems for cloning more than
one nucleic acid fragment into a vector without at least one of a
restriction enzyme, a ligase, a gyrase, a topoisomerase, a single
stranded DNA binding protein or the like; the system can include
more than one nucleic acid fragment each flanked by first and
second adapter sequences, and a vector having sequences homologous
to the adapter sequences on the 5' terminal nucleic acid fragment
and the 3' terminal nucleic acid fragment, respectively, wherein
the one or more nucleic acid fragment is adapted to incorporate
into the vector by recombination.
[0043] Embodiments relate to kits for cloning at least one nucleic
acid fragment into a vector comprising reagents for amplification
of the nucleic acid fragment, wherein the reagents upon
amplification provide for at least one nucleic acid fragment
flanked by first and second adapter sequences, a vector, a
competent cell, or a competent cell bearing the vector, and the
competent cell is ready to be transformed by electroporation or
chemical transformation.
[0044] Still other embodiments relate to methods of high throughput
cloning that do not require colony selection. The embodiments
described herein can be used in high through-put cloning because in
some embodiments, little or no colony selection is required due to
the high efficiency of cloning. The methods can include introducing
a vector and one or more nucleic acid fragments designed to
directionally recombine within a host cell, as described herein.
The amount of cells, vector and fragment(s) can be those amounts
discussed herein, for example. The cells are grown and no selection
is necessary due to the high efficiency of recombination and
cloning. For example, substantially 99%-100% of the cells have the
correct vector and insert(s). Thus, cloning can be done rapidly in
a high throughput manner. The cloned DNA can then be further
utilized and/or manipulated as necessary. Thus, certain embodiments
relate to methods for generating a plurality of recombinant
constructs. The methods can include the steps of introducing into a
host organism a linearized polynucleotide vector and a linearized
polynucleotide vector insert, wherein the insert and the vector
have respective regions of homology at ends thereof, under
conditions favoring assembly of the vector and the insert into a
circular recombinant construct in the host organism, such that such
assembly occurs in at least about 95% of the host organisms;
repeating the introducing step with the same or different vector
and a different vector insert a plurality of times to produce a
plurality of host organisms containing different recombinant
constructs; and creating a collection of such host organisms by
replicating the host organisms without a selection step. In some of
the embodiments, assembly occurs in at least about 96, 97, 98, 99,
or 100% of the host organisms.
[0045] The various specific features discussed above also can be
used in the other embodiments discussed below and combined with
each other in various combinations.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] FIG. 1 depicts an embodiment of the present invention
related to fast and enzymeless cloning into a vector.
[0047] FIG. 2 illustrates one embodiment related to cloning a
nucleic acid fragment into a vector.
[0048] FIG. 3 illustrates an example of generating protein
fusions.
[0049] FIG. 4 illustrates one embodiment related to generating and
cloning a nucleic acid fragment into a vector.
[0050] FIG. 5 depicts an exemplary receptor plasmid vector.
[0051] FIG. 6 illustrates one example of generating purified
linearized vector.
[0052] FIG. 7 illustrates one embodiment relating to selection of a
successfully transformed host.
[0053] FIG. 8 illustrates an exemplary vector, phCMV3/Xi.
[0054] FIG. 9 illustrates an exemplary vector, phCMV2/Xi.
[0055] FIG. 10 illustrates an exemplary vector, phCMV1/Xi.
[0056] FIG. 11 illustrates an exemplary vector, pIX1/Xi.
[0057] FIG. 12 illustrates an exemplary vector, pIX2/Xi.
[0058] FIG. 13 illustrates an exemplary vector, pIX3/Xi.
DETAILED DESCRIPTION OF THE INVENTION
[0059] The present invention, in at least certain embodiments,
overcomes many of the above-described drawbacks of traditional
cloning. The present invention includes methods, systems and kits
for fast and simple molecular cloning of a nucleic acid fragment or
more than one nucleic acid fragment directly into a vector without
the use of enzymes, such as restriction endonuclease to cut the
fragment(s), DNA ligase or any other DNA modifying enzyme. The
invention includes methods for generating substantially
background-free linearized vector. The present invention also
includes simple and fast methods for selecting host cells
containing the desired DNA fragment and vector.
[0060] The present invention generally provides methods, systems
and kits for cloning a nucleic acid fragment into a vector by
homologous recombination within a host cell. However, it should be
noted that the present invention is not limited to one particular
theory or mode of operation. It is not entirely clear, for example,
that a homologous recombination mechanism will always be
responsible for ligating the inserted fragment(s). Thus, the
nucleic acid fragment may be cloned into a vector within a host
cell by another intracellular mechanism besides or in addition to
homologous recombination. For example, some other repair process in
a cell may permit the fragment to be cloned into the vector as
described herein. When the common sequences on both the 5' and 3'
ends of the nucleic acid fragment are complimentary with terminal
sequences in a linearized empty vector, and the fragment and
linearized vector are introduced, by electroporation or more
preferably by chemical transformation, for example, together into a
host cell, they recombine resulting in a new expression vector with
the fragment directionally inserted. In alternative embodiments the
host cell can include the linearized empty vector so that only the
nucleic acid fragment is introduced into the host cell. It should
be noted that in alternative embodiments of the present invention
the vector can be circularized, and as used herein a vector can be
either linearized or circular. The host cell is converted into an
expression vector through some mechanism within the host cell, such
as for example, homologous recombination or some other repair or
ligation process. In principle this approach can be applied
generally as an alternative to conventional cloning methods.
[0061] One embodiment of the present invention includes a method
for cloning a nucleic acid fragment flanked by first and second
adapter sequences into a vector having homologous first and second
adapter sequences. The nucleic acid fragment incorporates into the
vector by recombination within a host cell. As used herein the term
"recombination" is meant to broadly include any interaction that
facilitates the incorporation of a nucleic acid fragment with a
vector. The interaction can be in vivo or in vitro. Examples of
recombination include homologous recombination, DNA repair
mechanisms, and the like. Thus, in some embodiments the fragment or
fragments may incorporate by homologous recombination or by some
other intracellular mechanism.
[0062] More specifically, referring now to the embodiment of the
present invention depicted in FIG. 1, a nucleic acid fragment 10 is
flanked by a first adapter sequence 12 and a second adapter
sequence 14. The nucleic acid fragment 10 also includes a coding
region 26, which will be discussed more fully below. A vector 16
also has a first vector adapter sequence 18 and a second vector
adapter sequence 20, which sequences are respectively homologous to
the first and second adapter sequences 12, 14 of the nucleic acid
fragment 10.
[0063] FIG. 2 illustrates in further detail some of the embodiments
of the invention. A nucleic acid fragment 102 includes a first
adapter sequence 106 and a second adapter sequence 108, which flank
a coding region 104. The nucleic acid fragment 102 also includes a
coding region 104, which will be discussed more fully below. A
vector 110 also includes a first vector adapter sequence 112 and a
second vector adapter sequence 114, which sequences are
respectively homologous to the first and second adapter sequences
106, 108 of the nucleic acid fragment 102.
[0064] The nucleic acid fragment 102 and the vector 110 are
introduced 116 into a host cell 118, such as E. coli. Suitable
cells are discussed more fully below. The introduction step 116 can
include any method that permits a sufficient quantity of fragment
102 and vector 110 to be introduced into a host cell 118. Such
methods are discussed more fully below. In preferred embodiments
the introduction step includes chemical transformation, as
discussed more fully below. Once introduced into the cell, the
nucleic acid fragment 102 incorporates 120 into or with the vector
118, for example by homologous recombination or any other process
In some embodiments the ratio of nucleic acid fragment 102 and
vector 110 to host cell 118 can be increased. This can permit a
higher number of nucleic acid fragments 102 and vectors 110 to be
introduced into an individual host cell 118. In some cases this may
increase the frequency of inter-molecular reactions, such as in
vivo homologous recombination or any other intracellular process or
repair mechanism. Conversely, in some cases an increased number of
host cells 118 in comparison to nucleic acid fragment 102 and/or
vector 110 can result in a decrease in the frequency of
incorporation of the nucleic acid fragment 102 with the vector 110.
In preferred embodiments, the quantity of nucleic acid fragment 102
can be 0.4-2.0 .mu.g and the quantity of vector 110 can be 0.05-0.1
.mu.g. Further, in preferred embodiments that quantity of host cell
118 can be about 2.times.10.sup.7 cells. In more preferred
embodiments the ratio of total DNA molecules to host cell 118 can
be about 20 fg to 100 fg per host cell.
[0065] Still further embodiments of the invention relate to
methods, systems and kits for generating a protein fusion. More
than one nucleic acid fragment can be cloned into a vector within a
cell. For example, referring to FIG. 3, primers 130, 132 are
designed, each with a sequence that is specific for a first coding
region 134 and a sequence that adds an overlapping region 136, 138.
PCR is performed and a first nucleic acid fragment 148 is
generated, which includes the first coding region flanked by the
overlapping regions 136, 138. The overlapping region 136 can be
designed to be homologous to a first vector adapter sequence 154 on
a vector 152. The overlapping region 138 can be designed to be
homologous to an overlapping region 145 on a second nucleic acid
fragment 150.
[0066] The second nucleic acid fragment 150 can be generated in a
manner similar to the first nucleic acid fragment 148. Primers 140,
142 are designed, each with a sequence that is specific for a
second coding region 144 and a sequence that adds an overlapping
region 145, 146. PCR is performed and a second nucleic acid
fragment 150 is generated, which includes the second coding region
144 flanked by the overlapping regions 145, 146. Overlapping region
146 can be designed to be homologous to a second vector adapter
sequence 156 on vector 152. Overlapping region 145 can be designed
to be homologous to overlapping region 138 on the first nucleic
acid fragment 148.
[0067] The first nucleic acid fragment 148, the second nucleic acid
fragment 150, and the vector 152 are introduced into a host cell
(not shown). As discussed more fully below, the introduction can be
accomplished by any appropriate method, such as for example,
chemical transformation, electroporation, and the like. Also, as
discussed more fully herein, any suitable host cell can be
used.
[0068] Once introduced into the host cell, the first nucleic acid
fragment 148 and the second nucleic acid fragment 150 incorporate
with the vector 152 by a process. For example, the process can be
homologous recombination, another cellular repair process, and the
like. FIG. 3 illustrates an embodiment where two nucleic acid
fragments are cloned into a vector.
[0069] Other embodiments of the invention relate to cloning more
than two nucleic acid fragments into a vector. As described above,
each successive nucleic acid fragment can be designed with an
overlapping region that is homologous to an overlapping region on
the next nucleic acid fragment. Without being limited to any
particular theory, presumably, once inside a host cell the
homologous regions facilitate a recombination or repair process
that causes the nucleic acid fragments to be joined and also to be
incorporated into a vector.
[0070] In embodiments of the present invention the homologous first
and second adapter sequences, the overlapping regions, or the
homologous sequences of any of the embodiments can be at least 11
bp. In other embodiments the homologous first and second adapter
sequences can be at least 15 or 20 bp. Further in embodiments the
homologous first and second adapter sequences can be at least 25,
30 or 35 bp. The homologous first and second adapter sequences can
be at least 40 bp. Also, the homologous first and second adapter
sequences can be at least 50 bp. In preferred embodiments the
homologous first and second adapter sequences are at least 60 bp.
In more preferred embodiments the homologous first and second
adapter sequences are at greater than 60 bp.
[0071] Certain embodiments of the invention relate to high
efficiency cloning. For example, in some embodiments, regardless of
the length of the adapter sequence or overlap region, a high
percentage of host cells result that have the vector with nucleic
acid fragment(s) incorporated into the vector. For the
above-mentioned lengths, there can be an efficiency or percentage
of cells with vector and correct nucleic acid fragment(s) insert of
about 50%, more preferably, about 60% or about 70%, still more
preferably, about 75% or about 85%. In more preferred embodiments
the percentage of cells with the correct cloning vector and nucleic
acid insert(s) can be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or 100%, for example. As another example, in some
embodiments, a homologous adapter sequence or overlap region of at
least 20, 25, 30 or about 30 bp can result in 85% of the resulting
cells having the vector with the nucleic acid fragment incorporated
therein.
[0072] As mentioned above, the nucleic acid fragment of any of the
embodiments of the invention can also include a coding region for a
sequence or gene of interest. For example, a coding region is
depicted in FIG. 1 as 26, in FIG. 2 as 104, and in FIG. 3 as 134
and 144. As used herein coding region refers generically to a
region of a nucleic acid fragment that can encode, for example, an
operably linked promoter, a termination sequence, an operon, a
fusion tag, a signal peptide for intracellular or intercellular
trafficking, a peptide, a protein, an antisense sequence, a
ribozyme, a protein binding site, and the like.
[0073] In further embodiments, the coding region can encode any
polypeptide or protein of interest. These can include enzymes,
receptors, transcription factors, lymphokines, hormones, antigens,
antibodies, fragments of any of the aforementioned, and the like.
In some embodiments, the coding region can encode a polypeptide or
protein of unknown function or portions of the same. In one
embodiment the coding region can include a gene encoding a product
which is absent or present at reduced levels in an organism.
Nonlimiting examples of these gene products are the cystic fibrosis
transmembrane regulator (CFTR), insulin, dystrophin, interleukin-2,
interleukin-12, erythropoietin, gamma interferon, and granulocyte
macrophage colony stimulating factor (GM-CSF). In some embodiments,
the coding region can encode a functional motif or domain of a
polypeptide or protein. These can include DNA binding domains,
transcription activation domains, catalytic domains of kinases,
phosphatases, and other enzymes or receptors, ligand binding domain
of receptors, transmembrane domains of membrane-bound proteins or
polypeptides, variable and constant domains of antibodies,
protein-protein interacting domains, and the alike. As noted above,
one of skill in the art need only know the terminal sequences of
the coding region gene of interest in order to generate a nucleic
acid fragment from a natural source or library comprising the gene
with the first and second adapter sequences.
[0074] The nucleic acid fragment(s) with adapter or homologous
regions can be generated by methods well known to those of skill in
the art. Referring to the embodiment of the invention depicted in
FIG. 4, a gene of interest 26 with known 5' and 3' sequences
undergoes PCR along with overlapping 5' and 3' priming
oligonucleotides 30, 32. The priming oligonucleotides can be
obtained by methods known in the art, including manufacture by
commercial suppliers. A primary fragment 34 with adapter sequences
is generated. The adapter sequences flanking the gene of interest
can be homologous to sequences on a vector, another primary
fragment with adapter sequences, or to sequences from other 5' or
3' fragments to be used in a subsequent PCR, as will be discussed
more fully below. The method depicted in FIG. 4 is more fully
described in U.S. patent application Ser. No. 09/535,262, "Methods
for Generating Transcriptionally Active DNA Fragments," which is
hereby incorporated by reference in its entirety.
[0075] The nucleic acid fragment(s) from any other embodiment can
also include a functional element. In some embodiments, an adapter
sequence or overlapping region can include the functional element.
In one embodiment the first and second adapter sequences, such as
for example, 12, 14 of FIG. 1, can include the functional element.
FIG. 4 illustrates one method for generating a nucleic acid
fragment with functional elements. The primary fragment generated,
as discussed above, has flanking sequences homologous to sequences
on a 5' fragment 36 and a 3' fragment 40, respectively. The 5' and
3' fragments 36, 40 include functional elements 46 and 50, as well
as a first and a second adapter sequence homologous to sequences on
the primary fragment 34. A 5' primer 42 and 3' primer 44 for PCR
can also be included. All undergo PCR. The resulting fragment 52
has a new 5' element 54 and a new 3' element 56 that include a
functional element and terminal flanking sequences homologous to
sequences on a vector. As noted above, the method is more fully
described in U.S. patent application Ser. No. 09/535,262, "Methods
for Generating Transcriptionally Active DNA Fragments," Liang, et
al, which is hereby incorporated by reference in its entirety. For
purposes of the present invention "transcriptionally active PCR
fragment" or "transcriptionally active DNA fragment" refers to a
nucleic acid fragment having a promoter and terminator sequence
included therewith such that the fragment can be transcribed within
a host cell. Depending upon the adapter sequences, the resulting
vectors are useful for a variety of different applications.
[0076] One of skill in the art can readily configure orientations
and generate nucleic acid fragments with such functional elements
by methods well known in the art. In some embodiments, for example,
the functional element can be a promoter, a terminator, a nucleic
acid fragment encoding a selection marker gene, a nucleic acid
fragment encoding a known protein, such as a fusion tag, a nucleic
acid fragment encoding a portion of a selection marker gene, a
nucleic acid fragment encoding a growth promoting protein, a
nucleic acid fragment encoding a transcription factor, a nucleic
acid fragment encoding an autofluorescent protein (e.g. GFP), and
the like.
[0077] Nucleic acid fragments flanked by adapter sequences suitable
for the purposes of the present invention can be generated using
the TAP Express.TM. system (Gene Therapy Systems, San Diego,
Calif.). The TAP Express.TM. uses nested PCR to append adapter
sequences, which can include additional sequences such as a
promoter and a terminator sequence, onto PCR fragments so that they
become transcriptionally active and can be used directly in vitro
and in vivo transfection experiments. The TAP Express.TM. system
can be used to generate a large numbers of genes that can be
conveniently amplified and introduced into functional assays in a
single day, a task that is impractical or impossible using
conventional cloning methodology.
[0078] As used herein, the term "promoter" is a DNA sequence which
extends or is located upstream from the transcription initiation
site and is involved in binding of RNA polymerase, or a DNA
sequence which locates downstream from the transcription start site
and is involved in binding of RNA polymerase III, and the like. The
promoter may contain several short (<10 base pair) sequence
elements that bind transcription factors, generally dispersed over
>200 base pairs. A promoter that contains only elements
recognized by general and upstream factors is usually transcribed
in any cell type. Such promoters may be responsible for expression
of cellular genes that are constitutively expressed (sometimes
called housekeeping genes). There are also tissue-specific
promoters limited to particular cell types, such as the human
metallothionein (MT) promoter that is upregulated by heavy metal
ions and glucocorticoids. The promoter can be selected based upon
consideration of the desired use for the nucleic acid fragment. One
skilled in the art easily can select an appropriate promoter
according the uses of the nucleic acid fragment. For example, if
the nucleic acid sequence encodes a gene with potential utility in
human cells, then a promoter capable of promoting transcription in
mammalian cells can be selected. Other examples of a promoter
includes a promoter from a plant or a plant pathogen, such as
cauliflower mosaic virus, and the like. The promoter can be from a
mammal or a mammalian pathogen such as CMV, SV40, MMV, HIV, and the
like. In other examples the promoter can be from a fungus such as a
yeast (Gal 4 promoter), and the like, while in other examples it
can be from bacteria or bacterial phage, for example lambda, T3,
T7, SP6, and the like.
[0079] As used herein, the term "terminator" is a DNA sequence
represented at the end of the transcript that causes RNA polymerase
to terminate transcription. This occurs at a discrete site
downstream of the mature 3' end, which is generated by cleavage and
polyadenylation. For example, the terminator sequence can be
derived from a plant, a procaryotyic or a eukaryotic source, such
as SV40, bovine growth hormone, rabbit beta-globin i, and the
like.
[0080] As used herein, the term "operon" is a controllable unit of
transcription consisting of a number of structural genes
transcribed together. An operon can contain at least two distinct
regions, the operator and the promoter. Examples of operons include
the lac operon, Tet/on operon, Tet/off operon, trp operon, and the
like.
[0081] Term "fusion tag" is used herein to refer generally to a
nucleic acid sequence encoding a molecule used to quantify,
capture, purify, visualize, etc., the expressed protein to which
the fusion tag is fused or attached. Examples of fusion tags
include 6.times. or 8.times. his-tag, GST tag, fluorescent protein
tag, Flag tag, HA tag, and the like.
[0082] It should be noted that in some embodiments, the vector can
include a promoter, an operon, a terminator, a fusion tag, and the
like. In such cases, the nucleic acid fragment may or may not
include any of the same in addition to the vector.
[0083] In one embodiment the nucleic acid fragment(s) and the
vector are introduced together into a host cell. Within the host
cell the nucleic acid fragment incorporates into the vector by in
vivo homologous recombination. The homologous sequence between the
nucleic acid fragment and the vector can be recognized by the DNA
recombination and repair mechanism (e.g., in E. coli) and joined
together. In other embodiments, the incorporation can occur by any
other reaction or process, such as another intracellular repair
mechanism.
[0084] In another embodiment, the vector first can be mixed with
the competent host cell. The host cell can be, for example, frozen
away immediately. The competent host cell/vector mixture can be
aliquotted and kept frozen. The transformation can be performed
thawing the aliquot or using non frozen host cell, and adding only
the desired nucleic acid fragment or PCR product to the host cell
bearing the vector.
[0085] In another embodiment the nucleic acid fragment, can be
introduced into the host cell bearing the vector. For example, the
vector may be replicated with the host cell. Once the nucleic acid
fragment is introduced into the host cell bearing the vector, it
incorporates into the vector by in vivo by homologous
recombination.
[0086] As used herein, the vector, including, for example, any
vector described in FIGS. 1-3 can be a plasmid, a cosmid, a
bacterial artificial chromasome (BAC), or the like. Examples of a
plasmid include CoE1, PR100, R2, pACYC, and the like. FIG. 5
depicts one example of a plasmid that can be used in the present
invention. The plasmid can include a functional or intact selection
marker for growth. For example, FIG. 5 illustrates a plasmid vector
60 that includes an intact selection marker 62 for growth. Examples
of a functional selection marker include a resistance gene for
kanamycin, kenamycin, ampicillin, blasticidin, carbonicillin,
tetracycline, chloramphenicol, and the like. The vector can be
maintained in the host cell under the selection condition selecting
for the functional selection marker. Other detailed examples of
vectors are illustrated in FIGS. 8-13, including vectors suitable
for mammalian expression.
[0087] In certain embodiments, the vector can be a linear vector.
The vector can be linearized by any suitable method familiar to one
of skill in the art. For example, a circular vector can be treated
with at least one restriction endonuclease, or it may have a
restriction site recognized by an endogenous endonuclease in the
target organism/cell. In that case, the endonuclease cleaves the
vector at a desired location thereby resulting in a linearized
vector. The vector can be maintained in a linear state by any
suitable method, including by maintaining the conditions of the
medium to favor linearization. The linear vector can also be
prepared by PCR using primers for that vector. The vector can be
amplified using PCR.
[0088] Other embodiments of the invention relate to methods of
generating substantially background free linearized vector. As used
herein substantially-background free can mean a vector preparation
that is at least about 80% linearized vector with the remaining
portion being uncut or partially cut circular vector, for example.
In other embodiments substantially-background free can mean at
least about 85% linearized vector, more preferably, about 90% or
93% linearized vector. In still more preferred embodiments it can
mean about 94%, 95%, 96%, 97%, 98%, or 99% linearized vector. In
even more preferred embodiments, it can mean substantially 100%
linearized vector. The term substantially 100% full length vector
can mean that the purity of linearized vector is at least about
99.0 to 99.9% linearized vector. Linearized vector can be generated
by providing a circular vector. The circular vector can have at
least one restriction site. In preferred embodiments, the circular
vector can have two or more restriction sites. The site or sites
can be flanked by an adapter or homologous sequence that is
homologous to sequence on the nucleic acid fragment that is to be
inserted. The circular vector is cut at the at least one
restriction site thereby causing it to be linearized. The
linearized vector can be treated with phosphatase, such as calf
intestine alkaline phosphatase, or DNA polymerase, such as T4 DNA
polymerase, to prevent self ligation of the compatible sticky ends.
The linearized vector can then be purified to a substantially
background free purity, for example, or more preferably to a purity
of substantially 100% full length vector.
[0089] The purification can be done by any suitable method by the
skilled artisan. For example, the purification can include
chromatography. The chromatography can be positive or negative
chromatography, gel or other matrix chromatography, and the like.
The digested vector can be captured. Alternatively, the undigested
vector can be captured. One example of this is depicted in FIG. 5,
which is discussed more fully below. The linearized vector can be
captured. It can include a sticky end that will bind to a probe on
a column, for example. The linearized vector can include a binding
site that can be bound, for example. One of skill in the art will
appreciate that any kind of chromatography can be designed and
used.
[0090] Referring now to FIG. 6, uncut vector 160 includes a peptide
nucleic acid (PNA) binding site 162 and restriction sites for
enzyme 1 164 and enzyme 2 166. PNA binding sites and PNAs are well
known in the art. For example, PNA binding and binding sites are
disclosed in U.S. Pat. No. 6,280,977, issued on Aug. 28, 2001, and
entitled METHOD FOR GENERATING TRANSCRIPTIONALLY ACTIVE DNA
FRAGMENTS; and in U.S. Pat. No. 6,165,720, issued on Dec. 26, 2000,
and entitled CHEMICAL MODIFICATION OF DNA USING PEPTIDE NUCLEIC
ACID CONJUGATES; both of which are hereby incorporated by reference
in their entirety. The vector is treated with enzyme 1 and enzyme 2
resulting in composition that includes linearized vector 168, PNA
binding fragment 170 and uncut vector 160. The composition is run
through a PNA affinity column. Uncut vector and PNA binding
fragment 170 are captured by the column. Linearized vector 168
passes through and can be collected. Any steps of the process can
be repeated.
[0091] The purification step can also include PCR amplification of
the linearized vector. Each successive round of PCR can increase
the amount of linearized vector. The PCR purification can be
coupled with any of the other purification techniques, including
the chromatography techniques.
[0092] Further, embodiments of the present invention include
methods for selecting for successful transformation of a vector by
a nucleic acid fragment. In one embodiment of the present invention
the vector can include a dysfunctional selection marker that lacks
a critical element. Upon successful in vivo homologous
recombination, the lacked critical element is supplied to the
vector by the nucleic acid fragment. As the homologous
recombination and repair mechanism is not yet well characterized,
it is of a relatively low frequency, making the identification of
the intended recombined vector difficult. The inclusion of a
critical element necessary for the viability of the host cell will
facilitate selection of the intended vector, because only the
correctly recombined vector can survive with the host, while a host
only carrying either the insert or the vector alone cannot survive.
This embodiment of the present invention can be referred to as
"forced cloning." As an example, FIG. 5 illustrates a plasmid
vector 60 having a dysfunctional or crippled marker 64 for
recombination selection. Examples of a dysfunctional selection
marker include an incomplete sequence of a resistance gene, for
example kanamycin, kenamycin, ampicillin, blasticidin,
carbonicillin, tetracycline, chloramphenicol, and the like.
Additional examples include reporter genes, such as the lacZ gene,
and the like. As used herein reporter gene refers to a gene that is
used to locate or identify another gene. Other dysfunctional
selection markers can include genes encoding products necessary for
a metabolic or cellular pathway, and the like. One of skill in the
art can easily select other useful "dysfunctional" selection
markers based upon knowledge and skill common in the art.
[0093] The incomplete sequence, lacking a critical element, is
completed by insertion of the lacked sequence or critical element
upon a successful homologous recombination. In some embodiments the
incomplete sequence can be missing at least a portion of a protein
coding region, or, e.g., all or part of a regulatory element such
as a promoter or termination sequence. The missing portion can be a
major portion of the critical element or selection marker, or only
a minor portion (e.g., one or more critical nucleotide
residues).
[0094] A successfully transformed host can also be selecting for by
a negative selection method. The vector can include a negative
selection element. A negative selection element can be a sequence
that encodes a molecule that is detrimental to growth of the host
cell, such as, for example, the mouse GATA-1 gene product which is
toxic to some cells. The toxic gene can be interrupted or replaced
by a nucleic acid sequence that is correctly incorporated into the
vector by homologous recombination. Only cells with a successfully
incorporated "interrupting" sequence survive, because only those
cells lack the toxic gene product. The negative selection element
can also encode for molecules that block cell metabolism or prevent
efficient transcription, and the like. One of skill in the art can
easily select other elements that will work with the present
invention.
[0095] In embodiments of the present invention the host cell can be
a bacterium. In preferred embodiments the bacterium is capable of
in vivo recombination. Examples of a bacterium include JC8679, TB1,
DH5.alpha., DH5, HB101, JM101, JM109, LE392, and the like. In some
embodiments the bacterium may lack certain recombinase enzymes,
such as RecT or RecE. For example, proper cloning has been observed
in cells that typically are considered to be lacking in RecT, RecE
or any other recombinase enzyme.
[0096] This may be due, at least in part, to the favorable methods
of the embodiments of the present invention. It may also be due, at
least in part to, the presence of other repair or ligation
mechanisms or other recombinases within the particular host cell.
In one embodiment the host cell can bear the vector. In preferred
embodiments the host cell can be a bacterium. In more preferred
embodiments the bacterium is capable of in vivo recombination.
Examples of a bacterium that can bear the vector, as described
above, can include JC8679, TB1, DH5.alpha., DH5, HB101, JM101,
JM109, LE392, and the like. In embodiments where the host cell
bears the vector, only the nucleic acid fragment is introduced into
the host cell, for example, by electroporation or chemical
transformation.
[0097] As mentioned above, the nucleic acid fragment(s) and the
vector can be introduced together into the host cell.
Alternatively, the vector first can be introduced into the cell
followed by a later introduction of the nucleic acid fragment(s) or
simply the nucleic acid fragment(s) can be introduced into the host
cell in order to transform the cell. In some embodiments relating
to protein fusion, the vector and all of the nucleic acid fragments
can be introduced together. Alternatively, the vector and fragments
can be introduced individually by successive procedures, or
combinations of vector and fragment(s) can be introduced followed
by introduction of fragment(s). Further, the host cell can include
a vector that replicates with the cell, thus obviating the need to
introduce a vector into the host cell. In preferred embodiments the
nucleic acid fragment(s) and/or vector can be introduced by
electroporation, chemical transformation, and the like. In one
preferred embodiment the nucleic acid fragment and the vector are
introduced into an E. Coli cell by high efficiency electroporation.
For example, in "high efficiency electroporation," as used herein,
each microgram of a supercoiled plasmid, when delivered into a cell
(such as E. Coli, for example) by electroporation, would be able to
produce 10.sup.10 or more colonies. In more preferred embodiments,
the vector and nucleic acid fragment(s) can be introduced by
chemical methods. Such methods are well known in the art, and
Example 8 below provides an exemplary method. For example, as
mentioned above, the amount of insert can be about 0.4-2.0 .mu.g.
The amount of vector can be about 0.05-0.1 .mu.g. The amount of E.
coli cells can be about 2.times.10.sup.7. Further, the ratio of
total DNA to E. coli can be about 20-100 fg/cell.
[0098] In another embodiment the present invention also includes
high efficiency electroporation-competent cells. Other preferred
embodiments relate to chemically competent cells, such as E. coli,
for example. These cells are capable of withstanding the conditions
of chemical transformation, including the quantity of nucleic acid
introduced into the cells. These cells significantly facilitate the
introduction of insert and vector into the host cells, thus
improving the efficiency of recombination, which is a bi-molecule
reaction, that is exponentially dependent on the amount of
substrate (the fragment and vector). Examples of these cells
include JC8679, TB1, HB101, DH5.alpha., DH5, JM101, JM109, and
LE392 and the like.
[0099] As discussed briefly above, the present invention also
includes methods and systems for forced cloning. Traditionally, a
cloning vector will include a selection marker, such as a
resistance gene, so that only a host cell having a properly
incorporated DNA insert and vector will grow in a selective medium.
However, the host cell may incorporate a vector having the
resistance gene without the desired insert or with only a portion
of the insert. Thus, host cell colonies will have to be screened,
potentially at a significant time, material and labor cost, in
order to identify a colony having the proper vector and insert.
[0100] Embodiments of the present invention relate to methods for
selecting for the successful transformation of a vector by a
nucleic acid insert. As an exemplary embodiment, referring to FIG.
1, the vector 16 is prepared with a dysfunctional selection marker
that lacks a critical element. The nucleic acid fragment 10 can
include the critical element. As used herein, the term "critical
element" can refer to any sequence on the nucleic acid fragment 10
that, upon incorporation with the vector 16, restores functionality
to a selection marker. For example, the critical element can be a
promoter, a terminator, a nucleic acid fragment encoding a
selection marker gene, a nucleic acid fragment encoding a known
protein such as fusion tag, a nucleic acid fragment encoding a
portion of a selection marker gene, a nucleic acid fragment
encoding a growth promoting protein, a nucleic acid fragment
encoding a transcription factor, a nucleic acid fragment encoding
an autofluorescent protein (e.g. GFP), and the like. The resulting
vector within the transformed host cell allows the host cell to
grow in a selective medium. Thus, only host cells that are properly
transformed with vector and nucleic acid fragment will grow. These
embodiments minimize the need for subsequent, labor intensive and
time consuming identification and selection of transformed
cells.
[0101] In one embodiment, the vector can have a dysfunctional
antibiotic resistance gene. For example, the vector can be prepared
having an interrupted antibiotic resistance gene. The nucleic acid
fragment is engineered to restore the functional antibiotic
resistance gene upon incorporation into the vector by homologous
recombination. The host cell having the "restored" vector can then
be plated in a selective growth media. Any host cell lacking the
"restored" vector will be unable to grow in the selective
media.
[0102] In addition to the embodiments described above related to
positive selection, embodiments of the present invention include
methods, systems and kits relating to negative selection for a
successful transformant. In one such embodiment, the vector can
have a negative selection element that is detrimental to cell
growth. For example, the negative selection element can be sequence
that encodes a molecule that is toxic to the cell, a molecule that
stops or prevents transcription, a molecule that is otherwise
detrimental to growth of the host cell, and the like. When a
nucleic acid fragment incorporates with the vector by homologous
recombination within the host cell, the negative selection element
is disabled. Disabling the negative selection element allows the
host cell to grow, thus only cells with proper insertion of the
nucleic acid fragment into the vector will survive and be
selected.
[0103] The negative selection element can be inducible. For
example, the vector can have a functional suicide gene or other
negative selection element. The suicide gene can be replaced or
disabled upon incorporation of the nucleic acid fragment into the
vector by homologous recombination.
[0104] For example, referring to FIG. 7, a vector 76 can be
prepared having a negative selection element 80, in this embodiment
a mouse GATA-1 transcription factor gene. The negative selection
element can be inserted between the first adapter sequence 82 and
the second adapter sequence 84. The first and second adapter
sequences 82, 84 have regions homologous to the ends of a nucleic
acid fragment 68 that is to be cloned. The nucleic acid fragment 68
can be generated by PCR or any other suitable method, as discussed
herein. The nucleic acid fragment 68 can encode some gene of
interest 70 as discussed above. The fragment 68 includes a first
adapter sequence 72 and a second adapter sequence 74, which are
homologous to the first and second adapter sequences 82, 84 on the
vector 76. The first and second adapter sequences 72, 74, as
discussed above can also include additional elements, such as
sequences encoding a promoter, a terminator, an operon, a fusion
tag, and the like.
[0105] The negative selection element 80, in this case the GATA-1
gene, is under the control of TAC promoter inducible by IPTG, and
its product is able to bind to the bacterial origin of replication,
therefore resulting in a rapid arrest of cell growth. The nucleic
acid fragment 68 upon incorporation into the vector 76 by
homologous recombination will replace the negative selection
element 80, thus enabling the host cell to grow in a selective
media. Any host cells lacking the recombined vector will be unable
to grow in the selective media.
[0106] The negative selection methods and systems can be combined
with the other systems, methods, and kits, including for example,
forced cloning. The nucleic acid insert can encode a critical
element, as described above, that restores function to a disabled
selection marker, while at the same time disabling a negative
selection element, such as a suicide gene. Alternatively, the
forced cloning methods, systems and kits can be used independently,
in conjunction with the negative selection methods, systems and
kits. In one embodiment, two nucleic acid fragments may be
introduced into the host cell.
[0107] Another embodiment of the present invention relates to a
system for cloning a nucleic acid fragment or fragments into a
vector lacking at least one of the following: a restriction enzyme,
a ligase, a gyrase, a single stranded DNA binding protein, or any
other DNA modifying enzyme. The system can include a nucleic acid
fragment flanked by first and second adapter sequences, and a
vector having sequences homologous to the first and second adapter
sequences. The nucleic acid fragment can be adapted to incorporate
into the vector by homologous recombination or any other suitable
process.
[0108] The nucleic acid fragment flanked by the first and the
second adapter sequences can be generated by PCR without the use of
a restriction enzyme, a ligase, a gyrase, a single stranded DNA
binding protein, or any other DNA modifying enzyme as discussed
above or according to any other method known in the art. The
nucleic acid fragment flanked by the first and the second adapter
sequences can be a transcriptionally active PCR fragment.
[0109] One embodiment of the present invention relates to a system
for cloning a nucleic acid fragment or fragments, into a bacterium
bearing a vector, without the use of a restriction enzyme, a
ligase, a gyrase, a single stranded DNA binding protein, or any
other DNA modifying enzyme. The system can include a nucleic acid
fragment flanked by first and second adapter sequences and a
bacterium bearing a vector having sequences homologous to the first
and second adapter sequences. The nucleic acid fragment is adapted
to incorporate into the vector within the bacterium by homologous
recombination.
[0110] A further embodiment relates to a kit for cloning a nucleic
acid fragment or fragments into a vector. The kit can include
reagents for amplification of the nucleic acid fragment(s).
Suitable reagents may include, for example, TAQ polymerase and/or
PCR reagents such as adapter sequences capable of acting as primers
for nested PCR and including regions of homology to a nucleic
fragment of interest and regions added onto the ends of the nucleic
acid fragment of interest upon successful amplification steps, as
explained in more detail in U.S. application Ser. No. 09/535,262,
discussed above. The reagents upon amplification can provide for a
nucleic acid fragment flanked by first and second adapter
sequences, a vector, a competent cell, or a competent cell bearing
the vector. The competent cell can form a part of the kit and can
be ready to be transformed by electroporation, chemical
transformation, or any like method known in the art. In preferred
embodiments, the competent cell or the competent cell bearing the
vector is bacteria. In other preferred embodiments the bacteria can
be capable of in vivo recombination.
EXAMPLES
Example 1
Generation of Transcriptionally Active PCR Fragment Encoding
Chloramphenicol Acetyltransferase (CAT)
[0111] The following components were combined in a 50 .mu.l
polymerase chain reaction (PCR): primers;
5'CTGCAGGCACCGTCGTCGACTTAACAATGGAGAAAAAAAT- CACTGG3' (SEQ ID NO.
1); and 5'CATCAATGTATCTTATCATGTCTGATTACGCCCCGCCCTGCCA- CTC3,'(SEQ
ID NO. 2) 1 ng of DNA template containing CAT coding region, 200
.mu.M dNTP and I unit Taq DNA polymerase.
[0112] PCR was performed as follows: denaturation at 94.degree. C.
for 30 seconds, annealing for 45 seconds at 55.degree. C. and
extension for 2 minutes at 72.degree. C. for 25 cycles. The PCR
product was analyzed by electrophoresis in 1% agarose gel and
purified using a commercial PCR cleaning kit. A second PCR reaction
was carried out using the product from the first PCR as template.
The reaction mix also contained 5 ng of DNA fragment (800 bp)
comprising a modified promoter sequence from human cytomegalovirus
(Gene Therapy Systems, San Diego, Calif.), 5 ng of DNA fragment
(200 bp) SV40 transcription terminator region, and 400 ng of
primers CMV154 and SV40-2. The PCR was performed under similar
conditions as above except the annealing temperature was raised to
60.degree. C. and the extension time was extended to 3 minutes. The
resulting PCR product was transcriptionally active and was used
directly for transfection of cells in vitro or tissues in vivo.
Example 2
Cloning of Transcriptionally Active PCR Fragment Encoding
Chloramphenicol Acetyltransferase (CAT)
[0113] The PCR fragment of Example 1 was cloned by mixing 0.5 .mu.g
of the final PCR product with 0.1 .mu.g of plasmid pCMVm-SV40-T
that was linearized and had sequences identical to the sequences
flanking the CAT gene in the PCR fragment. The mixed PCR product
and linear vector were transformed into E. coli JC8679 through
electroporation followed by incubation in SOC medium at 37.degree.
C. for 1 hour and plating on a LB/agar plate containing 100
.mu.g/ml Kanamycin for selection over night at 37.degree. C.
Colonies were selected and miniprep DNA was isolated for further
analysis and insertion of the PCR product into the vector.
Example 3
Cloning of PCR Fragment Encoding Chloramphenicol Acetyltransferase
(CAT) Using Chemically Competent Cells
[0114] The 1.sup.st PCR fragment of Example 1 was cloned by mixing
0.5 .mu.g of the final PCR product with 0.1 .mu.g of plasmid
pCMVm-SV40-T that was linearized and had sequences identical to the
sequences flanking the CAT gene in the PCR fragment. The mixed PCR
product and linear vector were transformed into 10.sup.7 E. coli
DH5.alpha. chemically competent cells on ice for 15 minutes
followed by incubation in SOC medium at 37.degree. C. for 1 hour
and plating on a LB/agar plate containing 100 .mu.g/ml Kanamycin
for selection over night at 37.degree. C. Colonies were selected
and miniprep DNA was isolated for further analysis and insertion of
the PCR product into the vector.
Example 4
Vector Linearization
[0115] The pXic-His vector was linearized by Bam HI restriction
enzyme digestion at 37.degree. C. over night (8 units/1 ug DNA).
After digestion the sticky-ends generated by the restriction enzyme
were filled in with Taq DNA polymerase, Super-Mix (Invitrogen, CA),
at 72.degree. C. for 15 minutes. The linearized vector was also
dephosphorylated with alkaline phosphatase (calf intestinal
alkaline phosphatase, CIP; Invitrogen, Calif.) to eliminate
re-circularization of the vector by self-ligation. The linearized
vectors were cleaned with the PCR cleaning kit (Qiagen) and stored
at 0.05 ug/ul in TE buffer. Although there was no obvious
indication of uncut vector based on the agarose gel, after the
above treatment uncut vector was still apparent based on the
presence of a high frequency of colonies lacking the insert (40% of
the colonies lacked insert). The uncut vector was reduced by doing
a second round of restriction digestion as described above. The
background of the "double cut" vector was only 25%.
Example 5
Generation of Linear Vector by PCR
[0116] The background transformation of the uncut vector also can
be limited by using a "PCR vector". The PCR vector was generated
with two primers that matched to the ends of the linearized vector.
The PCR was done with the manufacturer's PCR protocol and
proof-reading enzyme, pfx, from Invitrogen. After PCR production
and cleaning, the PCR vector was stored in TE buffer at 0.05 ug/ul.
This vector was used in place of the linearized vector as describe
in Example 4. A no background recombination cloning was obtained
with this "PCR vector." This 100% efficiency cloning technique can
be used for high throughput cloning.
Example 6
Forced Cloning Using a Suicide Gene
[0117] A plasmid is constructed in such that the toxic gene GATA-1
is under the control of tac (IPTG-inducible) promoter. The
GATA-1-expressing unit is then flanked by TAP promoter (modified
CMV IE promoter/intron, 800 bp) and TAP terminator (SV40
transcription terminator, 200 bp) sequences. A Transcriptionally
active PCR fragment encoding CAT gene is generated using the same
promoter and terminator elements. 2 .mu.g of such TAP fragments is
transformed into competent bacteria cells that contains the
GATA-1/TAC plasmid and prepared in the absence of IPTG. After
transformation, bacteria are plated on a LB plate containing 10
ng/ml IPTG. Only the cells bearing the plasmid in which the
TAC/ATA-1 is replaced by the TAP fragment encoding gene of interest
are able to grow.
Example 7
Linearized Vector Purification
[0118] Uncut vector creates background colonies that contain uncut
vector without insert. Affinity chromatography can be utilized to
remove uncut vector. A PNA binding sequence is inserted into the
poly linker site of the vector and this sequence is excised when
the vector is linearized. In other words, only the uncut vectors
have the PNA binding site and are able to bind the PNA affinity
column. The cut out sites containing the PNA binding site may also
be captured by the column. The linearized vector lacking the PNA
binding site will not bind to the column. After restriction enzyme
digestion, the product is loaded to the PNA affinity column and the
linearized vector collected from the follow through, thus
eliminating the uncut vector from the cut vector (see FIG. 5 as an
example). This procedure can generate a vector leading to up to
100% efficiency, i.e. zero background, for example.
Example 8
[0119] Six different circular vectors were linearized using the
materials as specified in Table 1, below. A portion of the
linearized vectors was treated a second time to further linearize
the vectors using the materials as specified in Table 2, below.
1TABLE 1 Linearize Vectors for Cloning 50 ug in 1 ml after fill-in
and clean (by PCR) 37 C. O/N 37 C. Final Final Plasmid size plasmid
# WFI Buffer BSA plasmid Cut AM Cut PM Conc(ug/ul) Vol (ul) ID
phCMV Clone pST 4236 bp p0031 820 ul 100 ul 10 ul 30.1 ul EcoRI 20
ul EcoRI 20 ul 0.1 500 #1 EcoRI 1.66 ug/ul Buffer pST-nHA 4261 bp
p0032 779 ul 100 ul 10 ul 31.3 ul Nhel 20 ul Nhel 20 ul 0.1 500 #2
EcoRI 1.6 ug/ul EcoRI EcoRI Buffer 20 ul 20 ul pST-cHA 4248 bp
p0033 781 ul 100 ul 10 ul 29.2 ul Xhol 20 ul Xhol 20 ul 0.1 500 #3
BamHI 1.71 ug/ul BamHI BamHI buffer 20 ul 20 ul for pIX Clone pXIC
3236 bp p0041 768 ul 100 ul 10 ul 42.4 ul Ncol 20 ul Ncol 20 ul 0.1
500 #4 BamHI 1.18 ug/ul BamHI BamHI buffer 20 ul 20 ul pXIC-nHis
3180 bp p0042 780 ul 100 ul 10 ul 70 ul BamHI BamHI 0.1 500 #5
BamHI 0.71 ug/ul 20 ul 20 ul buffer pXIC-cHA 3271 bp p0043 643 ul
100 ul 10 ul 167 ul NcoI 20 ul NcoI 20 ul 0.1 500 #6 BamHI 0.3
ug/ul BamHI BamHI buffer 20 ul 20 ul Note: pST 1.66 ug/ul (mega)
EcoRI 20 units/ul EcoRI buffer pST- 1.6 ug/ul (mega) NheI 10
units/ul Buffer 2 nHA pST-cHA 1.71 ug/ul(mega) BamHI 20 units/ul
BamHI buffer pXIC 1.18 ug/ul (mega) NcoI 10 units/ul Buffer 4 pXIC-
0.71 ug/ul (maxi) XhoI 20 units/ul Buffer 2 nHis pXIC- 0.3 ug/ul
(maxi) cHA Verify the cut completion by gel (load 5 ul, ie. 0.25
ug) Store the cut plasmid at -20 C. or proceed to next step. (2)
Fill-in Add 100 ul Supermix to the cut plasmid 1 ml Incubate at 72
C. for 15 min to fill-in. (3) Clean Clean the Cut plasmid using
Qiagen PCR cleaning kit (10 ug/kit, 5 kits per plasmid) Elute with
100 ul WFI per kit. Combine all S kits (.about.500 ul, .about.0.1
mg/ml). Run gel (load 2.5 ul, .about.0.25 ug) to verify DNA
conc.
[0120]
2TABLE 2 Repeat Linearize Vectors for Cloning 50 ug in 1 ml after
Qiagen PCR clean kit 37 C. O/N 37 C. Final Final Plasmid size
plasmid# WFI Buffer BSA plasmid* cut PM cut AM Conc(ug/ul) Vol (ul)
ID phCMV Clone pST 4236 bp p0031 350 ul 100 ul 10 ul 500 ul EcoRI
20 ul EcoRI 20 ul 0.1 500 #1 EcoRI 0.1 ug/ul Buffer pST-nHA 4261 bp
p0032 310 ul 100 ul 10 ul 500 ul NheI 20 ul NheI 20 ul 0.1 500 #2
EcoRI 0.1 ug/ul EcoRI EcoRI Buffer 20 ul 20 ul pST-cHA 4248 bp
p0033 310 ul 100 ul 10 ul 500 ul XhoI 20 ul XhoI 20 ul 0.1 500 #3
BamHI 0.1 ug/ul BamHI BamHI buffer 20 ul 20 ul for pIX Clone pXIC
3236 bp p0041 310 ul 100 ul 10 ul 500 ul NcoI 20 ul NcoI 20 ul 0.1
500 #4 BamHI 0.1 ug/ul BamHI BamHI buffer 20 ul 20 ul pXIC-nHis
3180 bp p0042 350 ul 100 ul 10 ul 500 ul BamHI BamHI 0.1 500 #5
BamHI 0.1 ug/ul 20 ul 20 ul buffer pXIC-cHA 3271 bp p0043 310 ul
100 ul 10 ul 500 ul NcoI 20 ul NcoI 20 ul 0.1 500 #6 BamHI 0.1
ug/ul BamHI BamHI buffer 20 ul 20 ul Note: pST 1.66 ug/ul (mega)
EcoRI 20 units/ul EcoRI buffer pST- 1.6 ug/ul (mega) NheI 10
units/ul Buffer 2 nHA pST- 1.71 ug/ul (mega) BamHI 20 units/ul
BamHI buffer cHA pXIC 1.18 ug/ul (mega) NcoI 10 units/ul Buffer 4
pXIC- 0.71 ug/ul (maxi) XhoI 20 units/ul Buffer 2 nHis pXIC- 0.3
ug/ul (maxi) cHA *all plasmids had been cut once, filled-in and
cleaned before Verify the cut completion by gel (load 5 ul, ie.
0.25 ug) Store the cut plasmid at -20 C. or proceed to next step.
(2) Clean Clean the cut plasmid using Qiagen PCR cleaning kit (10
ug/kit, 5 kits per plasmid) Elute with 100 ul WFI per kit. Combine
all 5 kits (.about.500 ul, .about.0.1 mg/ml). Run gel (load 2.5 ul,
.about.0.25 ug) to verify DNA conc.
[0121] A PCR fragment encoding CAT was prepared specific for each
linearized vector with 5' and 3' ends complementary to the
corresponding vector. The PCR fragments were cleaned using Qiagen
PCR cleaning kit and eluted with 30 .mu.l 1/2TE for each product.
Ten .mu.l was used for each clone reaction. The following steps
were performed to introduce the linearized vector and corresponding
PCR fragment into a host cell:
[0122] Prechill Eppendorf tubes
[0123] Add 10 .mu.l CAT PCR fragment (Qiagen cleaned)
[0124] Add 1 .mu.l appropriate linear vector (0.05 ug)
[0125] Add 10 .mu.l chemically competent cells. Tap gently
[0126] On ice for 45 min
[0127] Heat shock at 42C for 60 sec
[0128] Add 100 .mu.l SC to recover
[0129] Shake at 225 RPM 37C for 1 hour
[0130] Plate entire volume for (pST Clone) or half volume (for pXic
Clone)
[0131] Incubate Overnight at 37C
[0132] Transformation efficiency was checked by phenol gel. The
following was performed:
[0133] Place 30 .mu.l WFI in each Eppendorf tube
[0134] Add 30 .mu.l Phenol/Chloroform/Isoamyl alcohol to the
tube
[0135] Pick and disperse the colony to each tube. Save the tip.
[0136] Load 20 .mu.l top layer to agarose gel (opalescent and
cloudy, do not need loading buffer). Also include the blank
vector.
[0137] Run at .about.90 volts for 25 min.
[0138] The transformation efficiency after one treatment and two
treatments with restriction enzyme is shown below in Tables 3 and 4
for each clone. The data represent the percentage of cells with the
vector and proper insert.
3TABLE 3 phCMV % transformation after one % transformation after
two Cloning Vector Treatment Treatments pST 88% 89% pST-nHA 63%
100% pST-cHA 89% 100%
[0139]
4TABLE 4 % linear vector after one % linear vector after two pIX
Cloning Vector Treatment Treatments pXIC 38% 67% PXIC-nHis 60% 75%
PST-cHA 50% 78%
Example 9
Clone for In Vitro Transcription & Translation--Kit
Components
[0140] The resulting vectors contain a T7 promoter and they can be
used as templated in cell free in vitro transcription &
translation systems that utilize T7 RNA polymerase to generate
message. The kit can include:
5 ITEM DESCRIPTION QUANTITY PCR Cloning vector One of the linear
Cloning- 24 .mu.l adapted vectors: pIX/Xi, pIX2/Xi, or pIX3/Xi. For
vector maps, see page 14-16. SmartCells .TM. Chemically competent
E. coli 5 .times. 50 .mu.l Chemically provided at 1 .times.
10.sup.9 cfu/.mu.g Competent E. coli transformation efficiency.
SmartCells .TM. Competent E. coli are optimized to clone your PCR
fragments efficiently into the cloning vector. F.sup.- recA1 endA1
hsdR17 supE44 thi-1 gyrA96 relA1 .phi.80lacZ.DELTA.M15
.DELTA.(lacZYA- argF)U169SmartCells .TM. Competent E. coli Genotype
Control template A 700 base pair 10 .mu.l chloramphenicol acetyl (1
ng/.mu.l) transferase gene fragment provided as a control template
for enzymeless, directional PCR cloning Instruction Manual
[0141] While particular embodiments of the invention have been
described in detail, it will be apparent to those skilled in the
art that these embodiments are exemplary rather than limiting, and
the true scope of the invention is that defined in the following
claims.
Sequence CWU 1
1
2 1 46 DNA Artificial Sequence Primer for polymerase chain reaction
1 ctgcaggcac cgtcgtcgac ttaacaatgg agaaaaaaat cactgg 46 2 46 DNA
Artificial Sequence Primer for polymerase chain reaction 2
catcaatgta tcttatcatg tctgattacg ccccgccctg ccactc 46
* * * * *