U.S. patent application number 12/584684 was filed with the patent office on 2010-03-11 for homologous recombination-based dna cloning methods and compositions.
This patent application is currently assigned to GenScript Corporation. Invention is credited to Wenzhu Chen, Weiqiang Liu, Tao Wang, Zhuying Wang, Ping Yang, Ping Yang, Fang Liang Zhang.
Application Number | 20100062495 12/584684 |
Document ID | / |
Family ID | 41799620 |
Filed Date | 2010-03-11 |
United States Patent
Application |
20100062495 |
Kind Code |
A1 |
Liu; Weiqiang ; et
al. |
March 11, 2010 |
Homologous recombination-based DNA cloning methods and
compositions
Abstract
Methods and compositions for cloning a donor DNA molecule into
an acceptor vector at a predetermined location are described. The
methods are based on homologous recombination mediated by in vitro
treatment of the donor DNA and the acceptor vector with an enzyme
cocktail containing an exonuclease and a single-stranded DNA
binding protein.
Inventors: |
Liu; Weiqiang; (Nanjing,
CN) ; Yang; Ping; (Princeton, NJ) ; Wang;
Tao; (Nanjing, CN) ; Yang; Ping; (Nanjing,
CN) ; Wang; Zhuying; (Monmouth Junction, NJ) ;
Chen; Wenzhu; (Nanjing, CN) ; Zhang; Fang Liang;
(Fanwood, NJ) |
Correspondence
Address: |
PANITCH SCHWARZE BELISARIO & NADEL LLP
ONE COMMERCE SQUARE, 2005 MARKET STREET, SUITE 2200
PHILADELPHIA
PA
19103
US
|
Assignee: |
GenScript Corporation
Piscataway
NJ
|
Family ID: |
41799620 |
Appl. No.: |
12/584684 |
Filed: |
September 10, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61095877 |
Sep 10, 2008 |
|
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Current U.S.
Class: |
435/91.4 ;
435/196 |
Current CPC
Class: |
C12N 15/66 20130101 |
Class at
Publication: |
435/91.4 ;
435/196 |
International
Class: |
C12N 15/64 20060101
C12N015/64; C12N 9/16 20060101 C12N009/16 |
Claims
1. A method of cloning a donor DNA molecule into an acceptor vector
at a predetermined location, the method comprising: a) preparing an
extended donor DNA molecule by adding to the 5'-end and the 3'-end
of the donor DNA molecule a first sequence and a second sequence,
respectively, wherein each of the first and second sequences,
independently, is at least 12 nucleotides in length and is at least
90% identical to a first region and a second region of the acceptor
vector, respectively; b) providing a reaction mixture comprising:
i) the acceptor vector; ii) the extended donor DNA molecule; and
iii) an enzyme cocktail comprising an exonuclease and a
single-stranded DNA binding protein; c) incubating the reaction
mixture to obtain an intermediate product; d) transforming a cell
with the intermediate product to obtain a transformed cell; and e)
culturing the transformed cell under conditions to produce a
recombinant DNA molecule comprising the donor DNA located between
the first region and the second region.
2. The method according to claim 1, wherein the acceptor vector is
a circular vector or a linearized vector.
3. The method according to claim 1, wherein the first region and
the second region are present at the 3'-end and the 5'-end of the
linearized vector, respectively.
4. The method according to claim 1, wherein the first and the
second sequences are 100% identical to the first and the second
regions, respectively.
5. The method according to claim 1, wherein the extended donor DNA
molecule is prepared by a polymerase chain reaction (PCR), using a
first PCR primer comprising the first sequence and a second PCR
primer comprising the complement of the second sequence.
6. The method according to claim 1, wherein the exonuclease is
selected from the group consisting of Escherichia coli exonuclease
I, Escherichia coli exonuclease III, Escherichia coli exonuclease
VII, bacteriophage lambda exonuclease, and bacteriophage
T7-exonuclease Gene 6, or a combination thereof.
7. The method according to claim 1, wherein the single-stranded DNA
binding protein is selected from the group consisting of extreme
thermostable single-strand DNA binding protein (ET SSB), RecA, T4
Gene 32 Protein, Thermus thermophilus RecA (Tth RecA) and
Escherichia coli single-strand DNA binding protein (SSB), or a
combination thereof.
8. The method according to claim 7, wherein the enzyme cocktail
comprises an enzyme combination selected from the group consisting
of Escherichia coli exonuclease I and RecA; Escherichia coli
exonuclease III and RecA; Escherichia coli exonuclease VII and
RecA; bacteriophage lambda exonuclease and RecA; bacteriophage
T7-exonuclease Gene 6 III and RecA; Escherichia coli exonuclease I
and Tth RecA; Escherichia coli exonuclease III and Tth RecA;
Escherichia coli exonuclease VII and Tth RecA; bacteriophage lambda
exonuclease and Tth RecA; bacteriophage T7-exonuclease Gene 6 III
and Tth RecA; Escherichia coli exonuclease I and ET SSB;
Escherichia coli exonuclease I and T4 Gene 32 Protein; Escherichia
coli exonuclease I and Escherichia coli SSB; Escherichia coli
exonuclease III and ET SSB; Escherichia coli exonuclease III and T4
Gene 32 Protein; Escherichia coli exonuclease III and Escherichia
coli SSB; Escherichia coli exonuclease VII and ET SSB; Escherichia
coli exonuclease VII and T4 Gene 32 Protein; and Escherichia coli
exonuclease VII and Escherichia coli SSB.
9. The method according to claim 1, wherein the reaction mixture
comprises about 1 to about 100 mg/l of the exonuclease and about 1
to about 100 mg/l of the single-stranded DNA binding protein.
10. The method according to claim 1, wherein the reaction mixture
further comprises about 1 to about 10 g/l of
tris(hydroxymethyl)aminomethane (Tris), about 0.1 to about 10 g/l
of NaCl, about 0.1 to about 10 g/l of ethylenediaminetetraacetic
acid (EDTA), about 0.1 to about 10 g/l of MgCl.sub.2, about 10 to
about 200 g/l of glycerol, about 10 to about 50 g/l of bovine serum
albumin (BSA), about 0.1 to about 10 g/l of
adenosine-5'-triphosphate (ATP), about 0.1 to about 10 g/l of
dithiothreitol (DTT), and wherein the pH of the reaction mixture is
about 5.0 to about 9.0.
11. The method according to claim 1, wherein in step (c), the
incubation is conducted at a temperature of about 10.degree. C. to
about 38.degree. C. for about 15 minutes to about 60 minutes.
12. The method according to claim 1, wherein the cell is an
Escherichia coli cell and the acceptor vector is a plasmid
comprising an origin of replication that directs replication of the
plasmid in the Escherichia coli cell.
13. The method according to claim 1, wherein the transformed cell
expresses a marker gene from the recombinant DNA molecule that
allows the selection or screening of the transformed cell.
14. A composition for use in cloning a donor DNA molecule into an
acceptor vector at a predetermined location, comprising: a) an
enzyme cocktail comprising an exonuclease and a single-stranded DNA
binding protein; and b) a reaction buffer.
15. The composition of claim 14, wherein the exonuclease is
selected from the group consisting of Escherichia coli exonuclease
I, Escherichia coli exonuclease III, Escherichia coli exonuclease
VII, bacteriophage lambda exonuclease, and bacteriophage
T7-exonuclease Gene 6.
16. The composition of claim 14, wherein the single-stranded DNA
binding protein is selected from the group consisting of extreme
thermostable single-strand DNA binding protein, RecA, T4 Gene 32
Protein, Thermus thermophilus RecA (Tth RecA), and Escherichia coli
single-strand DNA binding protein.
17. The composition according to claim 16, wherein the enzyme
cocktail comprises an enzyme combination selected from the group
consisting of Escherichia coli exonuclease I and RecA; Escherichia
coli exonuclease III and RecA; Escherichia coli exonuclease VII and
RecA; bacteriophage lambda exonuclease and RecA; bacteriophage
T7-exonuclease Gene 6 III and RecA; Escherichia coli exonuclease I
and Tth RecA; Escherichia coli exonuclease III and Tth RecA;
Escherichia coli exonuclease VII and Tth RecA; bacteriophage lambda
exonuclease and Tth RecA; bacteriophage T7-exonuclease Gene 6 III
and Tth RecA; Escherichia coli exonuclease I and ET SSB;
Escherichia coli exonuclease I and T4 Gene 32 Protein; Escherichia
coli exonuclease I and Escherichia coli SSB; Escherichia coli
exonuclease III and ET SSB; Escherichia coli exonuclease III and T4
Gene 32 Protein; Escherichia coli exonuclease III and Escherichia
coli SSB; Escherichia coli exonuclease VII and ET SSB; Escherichia
coli exonuclease VII and T4 Gene 32 Protein; and Escherichia coli
exonuclease VII and Escherichia coli SSB.
18. A kit for use in cloning a donor DNA molecule into an acceptor
vector at a predetermined location, the kit comprising: a) the
composition of claim 14; and b) instructions on using the
composition in the cloning.
19. The kit according to claim 18, further comprising a competent
cell for use in the cloning.
20. A system for use in cloning a donor DNA molecule into an
acceptor vector at a predetermined location, the system comprising:
a) the acceptor vector; b) an extended donor DNA molecule
comprising a first sequence and a second sequence at the 5'-end and
the 3'-end of the donor DNA molecule, respectively, wherein each of
the first and second sequences, independently, is at least 12
nucleotides in length and is at least 90% identical to a first
region and a second region of the acceptor vector, respectively; c)
an enzyme cocktail comprising an exonuclease and a single-stranded
DNA binding protein; and d) a cell transformable with an
intermediate product formed after incubating a reaction mixture
comprising (a), (b) and (c), whereby the transformed cell produces
a recombinant DNA molecule that comprises the donor DNA located
between the first and the second regions.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is entitled to and claims the benefit of
the priority pursuant to 35 U.S.C. .sctn.119(e) of U.S. Provisional
Patent Application No. 61/095,877, filed Sep. 10, 2008, the
disclosure of which is hereby incorporated by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] Embodiments of the present invention relate to methods and
compositions for molecular cloning, particularly, cloning of a
donor DNA into an acceptor vector at a predetermined location.
[0004] 2. Background of the Invention
[0005] In molecular biology research and biotechnology industry,
there is a constant need of cloning a desired DNA molecule into a
vector, preferably at a predetermined location. A conventional
method for cloning a donor DNA into an acceptor vector at a
predetermined location, such as a plasmid, usually involves six
major steps: (i) digesting the acceptor vector DNA with one or two
restriction endonucleases and purifying the linearized vector; (ii)
treating the linearized vector with Calf Intestinal Phosphatase
(CIP) to minimize self-recircularization of the linearized vector
without the donor DNA during ligation; (iii) amplifying the donor
DNA by a polymerase chain reaction (PCR) using PCR primers that
will add to the 5'- and 3'-ends of the amplified donor DNA
restriction enzyme recognition site(s) for the one or two
restriction endonucleases used to linearize the vector DNA; (iv)
digesting the amplified donor DNA with the same restriction
endonucleases used to linearize the vector DNA and purifying the
digested donor DNA; (v) ligating the purified donor DNA and the
purified linearized vector using DNA ligase; and (vi) transforming
the ligation products into recipient cells, such as competent
Escherichia coli cells, and selecting transformants containing the
desired cloning product where the donor DNA is inserted in the
vector at the desired cloning site. The conventional cloning method
is cumbersome and time-consuming. It has relatively low cloning
efficiency. It is also limited by the availability of suitable
restriction enzyme recognition sites on the vector and the donor
DNA.
[0006] Recombination-based methods have been used to expedite
cloning. For example, a recombineering-based method for generating
conditional knockout mutations was described by Pentao Liu, et al.
(Genome Res (2003) 13: 476-484). The method uses a phage-based E.
coli homologous recombination system without the need for
restriction enzymes or DNA ligases. In particular, the method uses
homologous recombination mediated by the .lamda. phage Red
proteins, to subclone DNA from bacterial artificial chromosomes
(BACs) into high-copy plasmids by gap repair, and together with Cre
or Flpe recombinases, to introduce loxP or FRT sites into the
subcloned DNA. Longer than 45-55-bp regions of homology are used in
the method. Like several other recombination-based methods, the
method depends on specific sequences within the acceptor vector and
the expression of specific phage proteins in the host cell, thus
restricts the user to particular vectors and host cells.
[0007] Another example of recombination-based cloning is developed
by Clontech Laboratories, Inc. (Mountain View, Calif. 94043), as
In-Fusion.TM. PCR Cloning Kits. The Kits purport to allow cloning
of any PCR fragment into any linearized vector in a single step
without restriction digestion of the PCR fragment, ligation or
blunt-end polishing. The In-Fusion.TM. system allows to fuse the
ends of the PCR fragment to the homologous ends of a linearized
vector. The 3' and 5' regions of homology are generated by adding
15 by extensions to both PCR primers that precisely match the ends
of the linearized vector. The method consists of 30 minutes
incubation of the linearized vector with the PCR fragment and
In-Fusion.TM. enzyme, followed by transformation of E. coli. The
In-Fusion.TM. enzyme is a proprietary protein that converts the
double-stranded extensions into single-stranded DNA and fuses these
regions to the corresponding ends of the linearized vector. While
the In-Fusion.TM. system does allow rapid directional cloning of
PCR product without the restrictions of vectors and host cells, it
depends on the proprietary In-Fusion.TM. enzyme, thus restricts the
user to the In-Fusion.TM. PCR Cloning Kits or similar systems sold
by Clontech or its affiliates.
[0008] Therefore, there is a need for a novel fast and simple
method for cloning a donor DNA in a predetermined location of a
vector. Such method is described in the present application. The
method according to embodiments of the present invention has all
the privileges of the In-Fusion.TM.PCR Cloning Kits with even
higher cloning efficiency using an enzyme cocktail instead of the
In-Fusion.TM. enzyme.
BRIEF SUMMARY OF THE INVENTION
[0009] In one general aspect, embodiments of the present invention
relate to a method of cloning a donor DNA molecule into an acceptor
vector at a predetermined location. The method comprises:
[0010] a) preparing an extended donor DNA molecule by adding to the
5'-end and the 3'-end of the donor DNA molecule a first sequence
and a second sequence, respectively, wherein each of the first and
second sequences, independently, is at least 12 nucleotides in
length and is at least 90% identical to a first region and a second
region of the acceptor vector, respectively;
[0011] b) providing a reaction mixture comprising [0012] i) the
acceptor vector; [0013] ii) the extended donor DNA molecule; and
[0014] iii) an enzyme cocktail comprising an exonuclease and a
single-stranded DNA binding protein;
[0015] c) incubating the reaction mixture to obtain an intermediate
product;
[0016] d) transforming a cell with the intermediate product to
obtain a transformed cell; and
[0017] e) culturing the transformed cell under conditions to
produce a recombinant DNA molecule comprising the donor DNA located
between the first region and the second region.
[0018] In an embodiment according to the present invention, the
extended donor DNA is prepared by polymerase chain reaction
(PCR).
[0019] In another general aspect, embodiments of the present
invention relate to a composition for use in cloning a donor DNA
molecule into an acceptor vector at a predetermined location. The
composition comprises:
[0020] a) an enzyme cocktail comprising an exonuclease and a
single-stranded DNA binding protein; and
[0021] b) a reaction buffer.
[0022] An embodiment of the present invention also relates to a kit
for use in cloning a donor DNA molecule into an acceptor vector at
a predetermined location. The kit comprises the composition
according to embodiments of the present invention and instructions
on using the kit in the cloning.
[0023] In yet another general aspect, embodiments of the present
invention relate to a system for use in cloning a donor DNA
molecule into an acceptor vector at a predetermined location. The
system comprises:
[0024] a) the acceptor vector;
[0025] b) an extended donor DNA molecule comprising a first
sequence and a second sequence at the 5'-end and the 3'-end of the
donor DNA molecule, respectively, wherein each of the first and
second sequences, independently, is at least 12 nucleotides in
length and is at least 90% identical to a first region and a second
region of the acceptor vector, respectively;
[0026] c) an enzyme cocktail comprising an exonuclease and a
single-stranded DNA binding protein; and
[0027] d) a cell transformable with an intermediate product formed
after incubating a reaction mixture comprising (a), (b) and (c),
whereby the transformed cell produces a recombinant DNA molecule
that comprises the donor DNA located between the first region and
the second region.
[0028] Other aspects, features and advantages of the invention will
be apparent from the following disclosure, including the detailed
description of the invention and its preferred embodiments and the
appended claims.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0029] The foregoing summary, as well as the following detailed
description of the invention, will be better understood when read
in conjunction with the appended drawings. For the purpose of
illustrating the invention, there are shown in the drawings
embodiments which are presently preferred. It should be understood,
however, that the invention is not limited to the precise
arrangements and instrumentalities shown.
[0030] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0031] In the drawing:
[0032] FIG. 1 is a schematic representation of a method according
to an embodiment of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0033] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention pertains.
Otherwise, certain terms used herein have the meanings as set in
the specification. All patents, published patent applications and
publications cited herein are incorporated by reference as if set
forth fully herein. It must be noted that as used herein and in the
appended claims, the singular forms "a," "an," and "the" include
plural reference unless the context clearly dictates otherwise.
[0034] As used herein, "sequence" means the linear order in which
monomers occur in a polymer, for example, the order of amino acids
in a polypeptide or the order of nucleotides in a
polynucleotide.
[0035] As used herein, the term "nucleotide sequence", "nucleic
acid" or "polynucleotide" refers to the arrangement of either
deoxyribonucleotide or ribonucleotide residues in a polymer in
either single- or double-stranded form. Nucleic acid sequences can
be composed of natural nucleotides of the following bases: T, A, C,
G, and U, and/or synthetic analogs of the natural nucleotides. In
the context of the present invention, adenosine is abbreviated as
"A", cytidine is abbreviated as "C", guanosine is abbreviated as
"G", thymidine is abbreviated as "T", and uridine is abbreviated as
"U". A polynucleotide can be a single-stranded or a double-stranded
nucleic acid. Unless otherwise indicated, a polynucleotide is not
defined by length and thus includes very large nucleic acids, as
well as short ones, such as an oligonucleotide.
[0036] Conventional notation is used herein to describe
polynucleotide sequences. The left-hand end of a single-stranded
polynucleotide sequence is the 5'-end. The left-hand end of a
double-stranded polynucleotide sequence is the 5'-end of the plus
strand, which is depicted as the top strand of the double strands,
and the right-hand end of the double-stranded polynucleotide
sequence is the 5'-end of the minus strand, which is depicted as
the bottom strand of the double strands. The direction of 5' to 3'
addition of nucleotides to nascent RNA transcripts is referred to
as the transcription direction. A DNA strand having the same
sequence as an mRNA is referred to as the "coding strand." Sequence
on a DNA strand which is located 5' to a reference point on the DNA
is referred to as "upstream sequence"; sequence on a DNA strand
which is 3' to a reference point on the DNA is referred to as
"downstream sequence."
[0037] As used herein, a "complement of a nucleotide sequence" is a
nucleic acid sequence that is 100% complementary to the nucleotide
sequence.
[0038] "Sequence identity", as known in the art, is the
relationship between two or more polypeptide sequences or two or
more polynucleotide sequences, as determined by comparing the
sequences. As used herein, "identity", in the context of the
relationship between two or more nucleic acid sequences or two or
more polypeptide sequences, refers to the percentage of nucleotide
or amino acid residues, respectively, that are the same when the
sequences are optimally aligned and analyzed. The identity is
calculated along with the percentage of identical matches between
the two sequences over the reported aligned region, including any
gaps in the length. Analysis can be carried out manually or using
sequence comparison algorithms. For sequence comparison, typically
one sequence acts as a reference sequence, to which a queried
sequence is compared. When using a sequence comparison algorithm,
test and reference sequences are input into a computer,
sub-sequence coordinates are designated, if necessary, and sequence
algorithm program parameters are designated.
[0039] Optimal alignment of sequences for comparison can be
conducted by methods known in the art, such as the homology
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
Smith & Waterman, Adv. Appl. Math. 2:482 (1981) or Pearson
& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by
computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by
visual inspection. One example of algorithm that is suitable for
determining percent sequence identity and sequence similarity is
the BLAST algorithm, which is described in Altschul et al, J. Mol.
Biol. 215:403-410 (1990). Software for performing BLAST analyses is
publicly available online through the National Center for
Biotechnology Information (NCBI) web site.
[0040] As used herein, "recombinant" refers to a polynucleotide, a
polypeptide encoded by a polynucleotide, a cell, a viral particle
or an organism that has been modified using molecular biology
techniques to something other than its natural state.
[0041] As used herein, a "recombinant DNA molecule" refers to a DNA
molecule that does not exist as a natural product, but is produced
using molecular biology techniques.
[0042] As used herein, a "transformed cell", "transfected cell",
"transformant" and "recombinant cell" all refer to a cell that has
had introduced into it a recombinant polynucleotide sequence. For
example, transformed cells can contain at least one nucleotide
sequence that is not found within the native (non-transformed) form
of the cell or can express native genes that are otherwise
abnormally expressed, under-expressed, or not expressed at all.
Transformed cells can also contain genes found in the native form
of the cell wherein the genes are modified and re-introduced into
the cell by artificial means. The term encompasses cells that
contain the recombinant polynucleotide sequence either on a vector,
or integrated into a cell chromosome.
[0043] Recombinant DNA sequence can be introduced into host cells
using any suitable method including, for example, electroporation,
calcium phosphate precipitation, microinjection, transformation,
biolistics and viral infection. Recombinant DNA may or may not be
integrated (covalently linked) into chromosomal DNA making up the
genome of the cell. For example, the recombinant DNA can be
maintained on an episomal element, such as a plasmid.
Alternatively, with respect to a stably transformed or transfected
cell, the recombinant DNA has become integrated into the chromosome
so that it is inherited by daughter cells through chromosome
replication. This stability is demonstrated by the ability of the
stably transformed or transfected cell to establish cell lines or
clones comprised of a population of daughter cells containing the
exogenous DNA. It is further understood that the term "transformed
cell" refers not only to the particular subject cell, but also to
the progeny or potential progeny of such a cell. Because certain
modifications can occur in succeeding generations due to either
mutation or environmental influences, and in such circumstances,
such progeny may not, in fact, be identical to the parent cell, but
are still included within the scope of the term as used herein.
[0044] As used herein, the term "vector" refers to a nucleic acid
molecule capable of transporting another nucleic acid to which it
has been linked. One type of vector is a plasmid, which refers to a
circular double stranded DNA loop into which additional DNA
segments can be inserted. Another type of vector is a viral vector,
wherein additional DNA segments can be inserted. Other types of
vectors include, but are not limited to, bacterial artificial
chromosomes (BACs), yeast artificial chromosomes (YACs), and
phagemid. Certain vectors are capable of autonomous replication in
a host cell into which they are introduced (e.g., bacterial vectors
having a bacterial origin of replication and episomal mammalian
vectors). Other vectors (e.g., non-episomal mammalian vectors) are
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors, expression vectors, are capable
of directing the expression of genes to which they are operably
linked. In general, vectors of utility in recombinant DNA
techniques are often in the form of plasmids. These plasmids can be
single, low, medium or high copy plasmids. Examples of such vectors
are described, for example, by Sambrook et al. (Molecular Cloning,
Laboratory Manual, 2nd Edition (1989), Cold Spring Harbor
Laboratory Press) and Ioannou et al. (Nature Genet. 6 (1994),
84-89) or references cited therein. However, the invention is
intended to also include other forms of vectors, such as viral
vectors (e.g., replication defective retroviruses, adenoviruses and
adeno-associated viruses), BACs, YACs, and phagemid, which serve
equivalent functions.
[0045] Specifically designed vectors allow the cloning of DNA in
different hosts or the shuttling of DNA between hosts such as
bacteria-yeast or bacteria-animal cells or bacteria-fungal cells or
bacteria-invertebrate cells. Numerous vectors are known to those of
skill in the art and the selection of an appropriate vector is a
matter of choice. For other suitable expression systems for both
prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook
et al. (supra).
[0046] Embodiments of the present invention relate to a quick DNA
cloning method, which is a significant improvement over the
conventional DNA cloning techniques as well as other
recombination-based cloning methods. According to embodiments of
the present invention, virtually any donor DNA molecule can be
cloned into any vector at any predetermined location using a method
that no longer requires several steps in the conventional DNA
cloning method, such as restriction enzyme digestion of the PCR
amplified donor DNA, dephosphorylation of the restriction enzyme
digested acceptor vector with alkaline phosphatase, ligation of the
donor DNA with the acceptor DNA using DNA ligase, etc. Methods
according to embodiments of the invention greatly reduce the time
and costs for cloning, while give the user greater flexibility of
the vectors and host cells to be used in the cloning. The cloning
method according to embodiments of the present invention employs
homologous recombination between a donor DNA molecule and an
acceptor vector.
[0047] In one general aspect of the present invention, a donor DNA
molecule can be cloned into an acceptor vector at predetermined
location using a method that comprises the following steps:
[0048] a) preparing an extended donor DNA molecule by adding to the
5'-end and the 3'-end of the donor DNA molecule a first sequence
and a second sequence, respectively, wherein each of the first and
second sequences, independently, is at least 12 nucleotides in
length and is at least 90% identical to a first region and a second
region of the acceptor vector, respectively;
[0049] b) providing a reaction mixture comprising [0050] i) the
acceptor vector; [0051] ii) the extended donor DNA molecule; and
[0052] iii) an enzyme cocktail comprising an exonuclease and a
single-stranded DNA binding protein;
[0053] c) incubating the reaction mixture to obtain an intermediate
product;
[0054] d) transforming a cell with the intermediate product to
obtain a transformed cell; and
[0055] e) culturing the transformed cell under conditions to
produce a recombinant DNA molecule comprising the donor DNA located
between the first region and the second region.
Acceptor Vector
[0056] The choice and use of acceptor vectors in the present
invention will be readily apparent to those of skill in the art in
view of the present disclosure. The acceptor vector can be any
vector known in the art, such as a plasmid, a BAC, a YAC, a virus,
or a phagemid.
[0057] In an embodiment of the present invention, an acceptor
vector has an origin of replication sequence that allows autonomous
replication of the constructed recombinant DNA inside a transformed
prokaryotic or eukaryotic host cell. For example, the acceptor
vector can have the ColE1 origin of replication for autonomous
replication of the constructed recombinant DNA inside E. coli host
cells, 2.mu. origin of replication for yeast host cells, or a virus
origin of replication, e.g., SV40 origin of replication, for host
cells containing the appropriate replication factors for virus
replicons.
[0058] In another embodiment of the present invention, an acceptor
vector has sequences that facilitate integration or incorporation
of the constructed recombinant DNA onto the genome of the
transformed cell.
[0059] In an embodiment of the present invention, the acceptor
vector contains a sequence that allows the constructed recombinant
DNA to express a marker gene product that is not expressed by the
acceptor vector, thereby allowing selection or screening of
transformed cells containing the recombinant DNA by the selection
or screening of the presence of the marker gene product. This can
be achieved by the design of the recombinant DNA, for example, by
providing missing element of the marker gene through insertion of
the donor DNA.
[0060] In another embodiment of the present invention, the acceptor
vector contains a sequence that allows the constructed recombinant
DNA to not express a marker gene product that is expressed by the
acceptor vector, thereby allowing selection or screening of
transformed cells containing the recombinant DNA by the selection
or screening of the absence of the marker gene product. This can be
achieved by the design of the recombinant DNA, for example, by
nullifying the marker gene through insertion of the donor DNA.
[0061] The marker gene can be a selectable marker gene, the
expression of which allows the selection of the transformed cells
on a selective medium. Examples of selectable marker genes include,
but are not limited to, those encode proteins that confer
resistance to drugs, such as antibiotics, G418, hygromycin and
methotrexate; and those that encode proteins that confer the
ability to grow in medium lacking what would be an essential
nutrient.
[0062] The marker gene can also be a reporter gene, the expression
of which allows easy screening of the transformed cells using
conventional lab techniques. Examples of reporter gene include, but
are not limited to, those encode green fluorescent protein (GFP),
.beta.-galactosidase, luciferase, chloramphenicol
acetyltransferase, .beta.-glucuronidase, neomycin
phosphotransferase, guanine xanthine phosphoribosyl-transferase,
etc.
[0063] A marker gene differently expressed in the acceptor vector
and the constructed recombinant DNA would allow easy selection or
screening of transformed cells containing the recombinant DNA from
those containing the acceptor vector only. However, such a feature
may be present, but is not required, for a method according to the
present invention, because of the high cloning efficiency provided
by the present method. Transformants containing the recombinant DNA
can be easily identified by PCR screening using methods known in
the art in view of the present disclosure.
[0064] The donor DNA can be cloned into an acceptor vector at any
predetermined location. The cloning location can be chosen
according to the experimental need. The donor DNA can be inserted
into the acceptor vector with or without replacing or deleting a
portion of the vector. If a portion of an autonomously replicating
acceptor vector is deleted as a result of the homologous
recombination, care is taken to ensure that the origin of
replication of the vector remains in the constructed recombinant
DNA.
[0065] Once a location for insertion of the donor DNA molecule is
chosen, sequences of the two regions flanking the chosen location
on the acceptor vector are readily discernable, e.g., by
pre-existing sequence of the vector or by DNA sequencing of the
regions.
[0066] As used herein, the "first region" refers to the sequence
located upstream of the 5'-end of the predetermined insertion site.
When precise insertion of the donor DNA at the 5'-end of the
insertion site is required, the first region is contiguous with the
first or 5' most nucleotide of the predetermined insertion
location.
[0067] As used herein, the "second region" refers to the sequence
located downstream of the 3'-end of the predetermined insertion
site. When precise insertion of the donor DNA at the 3'-end of the
insertion site is required, the second region is contiguous with
the last or 3' most nucleotide of the predetermined insertion
location.
[0068] In an embodiment of the present invention, the acceptor
vector is a plasmid, and the predetermined location for the
insertion of the donor DNA is at a restriction endonuclease
cleavage site, or is between two restriction endonuclease cleavage
sites. The plasmid is digested with the one or two restriction
endonucleases. After the restriction enzyme digestion, the one or
two restriction enzymes are heat-inactivated, and the linearized
plasmid is purified by gel or column purification. The first region
and the second region are the sequences at the two ends of the
linearized plasmid, respectively.
[0069] In another embodiment of the present invention, the acceptor
vector is circular, undigested or uncut with any restriction
enzyme. An uncut plasmid can be used directly in a reaction mixture
for homologous recombination according to an embodiment of the
present invention.
[0070] Preparation of the Extended Donor DNA Molecule
[0071] Any donor DNA can be cloned into a vector using the present
invention. Examples of donor DNA molecules include, without
limitation, cDNA or genomic DNA fragments. The donor DNA molecule
can be a gene encoding a protein of interest. It can also be a
sequence carrying a genetic mutation or genetic lesion of interest,
for performing quick DNA mutagenesis. The genetic mutations or
genetic lesions include, but are not limited to: 1) a deletion of
one or more nucleotides from a target DNA; 2) an addition of one or
more nucleotides to the target DNA; 3) a substitution of one or
more nucleotides of the target DNA; etc. DNA mutagenesis, e.g.,
introducing DNA mutations including point mutations, additions and
deletions, is traditionally performed in the same way as DNA
cloning using conventional methods. The method of the present
invention can also be used for quick DNA mutagenesis, thereby
significantly reducing the time and associated costs for DNA
mutagenesis.
[0072] In an embodiment of the present invention, the donor DNA is
extended by adding to each of its two ends a first sequence or a
second sequence that shares sufficient homology with the first
region or the second region of the acceptor vector described supra,
so that efficient homologous recombination occurs precisely between
the first sequence and first region, and the second sequence and
the second region, respectively.
[0073] In another embodiment of the present invention, each of the
first and second sequences, independently, is at least 12
nucleotides in length and shares at least about 90% sequence
identity to the first and second regions, respectively. For
example, each of the first and second sequences, independently, can
be 12, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length and
share about 90%, 95% or 100% sequence identity with the first and
second regions, respectively.
[0074] In an embodiment of the present invention, each of the first
and second sequences, independently, is at least 12 nucleotides in
length and shares about 100% sequence identity to the first and
second regions, respectively.
[0075] The extended donor DNA molecule can be prepared using any
method known in the art in view of the present disclosure. For
example, the extended donor DNA can be synthesized by chemical
synthesis. The extended DNA can also be recombinantly produced
followed by appropriate restriction enzyme cleavage and
purification.
[0076] In a preferred embodiment, the extended donor DNA molecule
is produced by PCR. Each of the PCR primers is composed of two
parts: a 5'-end add-on sequence and 3'-end donor DNA specific
sequence. One of the PCR primers contains the first sequence as the
5'-end add-on sequence and the other primer contains the complement
of the second sequence. Methods known in the art can be used to
design the 3'-end donor DNA specific sequences in the PCR primers
in view of the present disclosure. For example, the donor DNA
specific sequences must be specific to the targeted regions on the
donor DNA, can be 10-25 bases in length and have a GC content of
about 35-65%. Preferably, the two PCR primers have similar melting
temperature (T.sub.m) in the range of 55-70.degree. C., etc.
[0077] Any of the PCR applications known in the art can be used in
view of the present disclosure. PCR conditions, such as the amounts
of template and primers, the concentrations of Mg.sup.2+ and dNTPs,
the annealing and thermocycling conditions, etc., can be chosen or
optimized by common knowledge or routine experimentation.
[0078] In an embodiment of the present invention, more than one
donor DNA molecules can be cloned in a desired order at a
predetermined location on an acceptor vector. The add-on sequences
at the ends of the extended donor DNA molecules are designed such
to allow homologous recombination between the donor DNA molecules
in the desired order, as well as to allow homologous recombination
between the donor DNA molecules and the acceptor vector at the
predetermined location.
[0079] Alternatively, more than one donor DNA molecules can first
be joined together using methods known in the art, such as by DNA
ligation or by fusion PCR. The joint product containing multiple
donor DNA molecules can then be inserted into the acceptor vector
using methods of the present invention.
[0080] In another embodiment of the present invention, a group of
donor DNA molecules of heterogeneous sequences, e.g. from a DNA
library or cDNA library, can be cloned at a predetermined location
on an acceptor vector. For example, a pair of universal PCR primers
can be used to add the first and the second sequences to the ends
of each of the donor DNA molecules via PCR. The extended
heterogeneous donor DNA sequences are then cloned onto the acceptor
vector through homologous recombination using methods described
herein.
[0081] Enzyme Cocktail
[0082] Unlike the In-Fusion.TM. Cloning System from Clontech, which
uses a proprietary In-Fusion Enzyme, a protein that fuses
PCR-generated donor sequence to linearized vectors by recognizing a
15 by overlap at their ends, methods of the present invention
utilize an enzyme cocktail in an in vitro treatment of the extended
donor DNA and acceptor vector to initiate and mediate the
homologous recombination, which is completed by the recombination
system of the transformed cell in vivo.
[0083] The enzyme cocktail according to embodiments of the present
invention comprises an exonuclease and a single-stranded DNA
binding protein. Each of the proteins can be substituted with
similar proteins known in the art that function in substantially
the same way.
[0084] As used herein, the term "exonuclease" refers to an enzyme
that cleaves nucleotides one at a time from the end of a
polynucleotide chain via a hydrolyzing reaction that breaks
phosphodiester bonds at either the 3' or 5' end. The "exonuclease"
can be a 3' to 5' exonuclease or a 5' to 3' exonuclease. E. coli
exonuclease I and exonuclease III are two commonly used
3'-exonucleases that have 3'-exonucleolytic single-strand
degradation activity. E. coli exonuclease VII and T7-exonuclease
Gene 6 are two commonly used 5'-3' exonucleases that have 5%
exonucleolytic single-strand degradation activity.
[0085] The exonuclease can be originated from prokaryotes, such as
E. coli exonucleases, or eukaryotes, such as yeast, worm, murine,
or human exonucleases.
[0086] Examples of exonuclease that can be used in the present
invention include, but are not limited to, E. coli exonuclease I,
E. coli exonuclease III, E. coli exonuclease VII, bacteriophage
lambda exonuclease, and bacteriophage T7-exonuclease Gene 6, or a
combination thereof.
[0087] As used herein, a "single-stranded DNA binding protein,"
also known as SSB or SSBP, refers to a protein that binds single
stranded regions of DNA. "SSBs" can be originated from viruses to
humans. "SSBs" can be monomeric, such as many identified in phage
and virus, or multimeric, such as the tetrameric bacterial SSBs or
the heterotrimeric eukaryotic Replication Protein A (RPA).
[0088] Examples of single-stranded DNA binding proteins that can be
used in the present invention include, but are not limited to,
extreme thermostable single-stranded DNA binding protein (ET SSB),
RecA (such as E. coli RecA, any RecA recombinantly expressed by E.
coli, or derivatives thereof), T4 Gene 32 Protein, Thermus
thermophilus RecA (Tth RecA), and E. coli single-strand DNA binding
protein, or a combination thereof.
[0089] The enzyme cocktail according to embodiments of the present
invention can comprise any of the combination of an exonuclease and
an SSB. Examples of such combination, include, but are not limited
to, a combination selected from the group consisting of Escherichia
coli exonuclease I and RecA; Escherichia coli exonuclease III and
RecA; Escherichia coli exonuclease VII and RecA; bacteriophage
lambda exonuclease and RecA; bacteriophage T7-exonuclease Gene 6
III and RecA; Escherichia coli exonuclease I and Tth RecA;
Escherichia coli exonuclease III and Tth RecA; Escherichia coli
exonuclease VII and Tth RecA; bacteriophage lambda exonuclease and
Tth RecA; bacteriophage T7-exonuclease Gene 6 III and Tth RecA;
Escherichia coli exonuclease I and ET SSB; Escherichia coli
exonuclease I and T4 Gene 32 Protein; Escherichia coli exonuclease
I and Escherichia coli SSB; Escherichia coli exonuclease III and ET
SSB; Escherichia coli exonuclease III and T4 Gene 32 Protein;
Escherichia coli exonuclease III and Escherichia coli SSB;
Escherichia coli exonuclease VII and ET SSB; Escherichia coli
exonuclease VII and T4 Gene 32 Protein; and Escherichia coli
exonuclease VII and Escherichia coli SSB.
[0090] In Vitro Treatment
[0091] According to embodiments of the present invention, the
homologous recombination between the extended donor DNA and the
acceptor vector is initiated, mediated or facilitated by incubating
the DNA molecules and the enzyme cocktail in a reaction mixture in
vitro.
[0092] The reaction mixture comprises an acceptor vector, an
extended donor molecule, an enzyme cocktail, and a reaction
buffer.
[0093] The reaction buffer comprises buffering agents, salts and
adenosine-5'-triphosphate (ATP), having a pH of about 5.0 to about
9.0. In an embodiment of the invention, the reaction buffer
comprises tris(hydroxymethyl)aminomethane (Tris), NaCl, EDTA,
MgCl.sub.2, glycerol, bovine serum albumin (BSA), ATP, and
dithiothreitol (DTT), at a pH of about 6.8 to about 7.4. Each of
these elements can be substituted with similar elements known in
the art that function in solution in substantially the same way.
BSA, for example can be substituted with casein and/or other agents
known in the art. NaCl can also be replaced by KCl.
[0094] In another general aspect, embodiments of the present
invention relate to a composition for use in cloning a donor DNA
molecule into an acceptor vector at a predetermined location. The
composition comprises: a) an enzyme cocktail comprising an
exonuclease and a single-stranded DNA binding protein; and b) a
reaction buffer.
[0095] The enzyme cocktail and the reaction buffer can be provided
in a kit for use in cloning a donor DNA molecule into an acceptor
vector at a predetermined location, which also includes
instructions on using the enzyme cocktail and reaction buffer in
the cloning. The kit may further contain a competent cell for use
in transformation in the cloning. The kit may also include control
vectors or DNA molecules for the cloning.
[0096] The enzyme cocktail and reaction buffer can be provided in
various forms suitable for their applications. For example, the
enzyme cocktail can be provided in a concentrated liquid form or
lyophilized form, with or without the accompanying dilution or
reconstitution buffer. The reaction buffer can be provided in a
single container in a concentrated or lyophilized form.
Alternatively, one or more ingredients of the reaction buffer can
be provided in separate containers that can be combined together
upon use.
[0097] In an embodiment of the present invention, the reaction
mixture comprises about 0.5 to about 10 ng/.mu.l of the acceptor
vector. The reaction mixture can contain, for example, about 0.5,
1, 5 or 10 ng/.mu.l of the acceptor vector.
[0098] In an embodiment of the present invention, the reaction
mixture comprises about 1 to about 30 ng/.mu.l of the extended DNA
molecules. The reaction mixture can contain, for example, about 1,
5, 10, 15, 20, 25 or 30 ng/.mu.l of the extended DNA molecules.
[0099] In an embodiment of the present invention, the reaction
mixture comprises about 1 to about 100 mg/l of the single-stranded
DNA binding protein. The reaction mixture can contain, for example,
about 1, 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, or 100 mg/l of the
single-stranded DNA binding protein.
[0100] In an embodiment of the present invention, the reaction
mixture comprises about 1 to about 100 mg/l of the exonuclease. The
reaction mixture can contain, for example, about 1, 5, 15, 25, 35,
45, 55, 65, 75, 85, 95, or 100 mg/l of the exonuclease.
[0101] In one embodiment of the present invention, 1 liter of the
reaction mixture contains about 1 gram to 10 grams, such as about 5
grams, of Tris; about 0.1 gram to 10 grams, such as about 5 grams,
of NaCl; about 0.1 grams to about 10 grams, such as about 2 grams,
of EDTA; about 0.1 grams to about 10 grams, such as about 1 gram,
of MgCl.sub.2; about 10 grams to about 200 grams, such as about 50
grams, of glycerol; about 10 grams to 50 grams, such as about 20
grams, of BSA; about 0.1 gram to about 10 grams, such as about 1
gram, of ATP; about 0.1 gram to about 10 grams, such as about 1
gram, of DDT; about 1 miligram to about 100 miligrams, such as 75
miligrams, of RecA (e.g., E. coli RecA, any RecA recombinantly
expressed by E. coli, or derivatives thereof); about 1 miligram to
about 100 miligrams, such as about 35 miligrams of E. coli
Exonuclease VII. The reaction mixture further comprises about 0.5
ng/.mu.l to about 10 ng/.mu.l, such as 5 ng/.mu.l, of the
linearized vector; and about 1 ng/.mu.l to about 30 ng/.mu.l, such
as about 5 to about 15 ng/.mu.l, of the extended donor DNA. The pH
of the reaction mixture can be about 5.0 to about 9.0, such as
about 6.8 to about 7.4, or about 7.6.
[0102] In an embodiment of the present invention, the reaction
mixture is incubated at a temperature of about 10 to 38.degree. C.
for about 10 to 60 minutes.
[0103] In another embodiment of the present invention, the reaction
mixture is incubated at room temperature for about 30 minutes.
[0104] It is noted that the present invention is not limited to the
concentrations, ingredients, or assay conditions described herein.
Equivalent concentrations, ingredients, or assay conditions can
also be used.
[0105] Transformation
[0106] An intermediate product is formed after the in vitro
incubation of the reaction mixture. The intermediate product is
transformed into a host cell using methods known in the art in view
of the present disclosure.
[0107] The intermediate product can be introduced into prokaryotic
or eukaryotic cells via conventional transformation or transfection
techniques, including, but not limited to, calcium phosphate or
calcium chloride co-precipitation, electroporation,
DEAE-dextran-mediated transfection, lipofection, protoplast fusion,
and viral infection. Suitable methods for transforming or
transfecting host cells can be found in Sambrook et al. (supra),
and other laboratory manuals.
[0108] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
for resistance to antibiotics) is generally introduced into the
host cells along with the gene of interest. Preferred selectable
markers include those which confer resistance to drugs, such as
G418, hygromycin and methotrexate. Cells stably transfected with
the introduced nucleic acid can be identified by drug selection
(e.g., cells that have incorporated the selectable marker gene will
survive, while the other cells die).
[0109] Cells suitable for the transformation of the present
invention include, but are not limited to, a bacterial cell, e.g. a
Gram-negative bacterial cell, or a Gram-positive bacterial cell, a
yeast cell, a mammalian cell, etc.
[0110] In an embodiment of the present invention, the cell used for
transformation is a competent E. coli cell. For example, about 2-10
.mu.l of the reaction mixture after the incubation was used to
transform 50 .mu.l of competent cells following standard
transformation procedure.
[0111] The transformed cells can be selected on a selective medium.
Cells containing the recombinant DNA that comprises the donor DNA
inserted at the predetermined location of the acceptor vector can
be screened using a PCR screening method.
[0112] While not wishing to be bound by theory, it is believed that
in a method according to an embodiment of the present invention,
the homologous recombination is initiated and mediated during in
vitro treatment of the acceptor vector and the donor DNA, and is
completed in vivo in a transformed cell. It is believed that during
the in vitro treatment, the exonuclease acts on the ends on the
linear extended donor DNA molecule to produce single-stranded DNA
(ssDNA) overhangs at both ends. The SSB binds to the ssDNA
overhangs to protect them from degradation, particularly upon
introduction into a host cell, such as E. coli. It is believed that
in the intermediate product formed after the in vitro treatment,
the ssDNA overhangs form base pair interactions with their
complementary sequences in the first or second regions of the
acceptor vector. Upon introduction of the intermediate product into
a host cell, the homologous recombination occurs between the first
region and first sequence and the second region and the second
sequence, respectively, as a result of the recombination functions
of the host cell.
[0113] Regardless of the underline mechanism, it was found that
using methods according to the present invention, donor DNA
molecules of different sizes were cloned into vectors at
predetermined locations at high efficiency.
[0114] Table 1 summarizes the cloning results of donor DNA
molecules of the sizes of 1 kb, 2 kb, and 3 kb into UC57
vector.
TABLE-US-00001 TABLE 1 PCR DNA Size 1 kb 2 kb 4 kb Number of
Colonies ~1200 ~1000 221 Positive Ratio 8/8 7/8 7/8
In each recombination test, 8 colonies were screened for the
correct insertion of the PCR DNA.
[0115] In another general aspect, embodiments of the present
invention relate to a system for use in cloning a donor DNA
molecule into an acceptor vector at a predetermined location. The
system comprises:
[0116] a) the acceptor vector;
[0117] b) an extended donor DNA molecule comprising a first
sequence and a second sequence at the 5'-end and the 3'-end of the
donor DNA molecule, respectively, wherein each of the first and
second sequences, independently, is at least 12 nucleotides in
length and is at least 90% identical to a first region and a second
region of the acceptor vector, respectively;
[0118] c) an enzyme cocktail comprising an exonuclease and a
single-stranded DNA binding protein; and
[0119] d) a cell transformable with an intermediate product formed
after incubating a reaction mixture comprising (a), (b) and (c),
the transformed cell producing a recombinant DNA molecule that
comprises the donor DNA located between the first and the second
regions.
[0120] Various embodiments of the invention have now been
described. It is to be noted, however, that this description of
these specific embodiments is merely illustrative of the principles
underlying the inventive concept. It will be appreciated by those
skilled in the art that changes could be made to the embodiments
described above without departing from the broad inventive concept
thereof. It is understood, therefore, that this invention is not
limited to the particular embodiments disclosed, but it is intended
to cover modifications within the spirit and scope of the present
invention as defined by the appended claims.
REFERENCES
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