Method for in vitro recombination
First Claim
1. An in vitro method, using isolated proteins, for joining two or more double-stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a region of sequence identity, comprising (a) treating the DNA molecules with an enzyme having an exonuclease activity, under conditions effective to yield single-stranded overhanging portions of each DNA molecule which contain a sufficient length of the region of sequence homology to hybridize specifically to the region of sequence homology of its pair;
- (b) incubating the treated DNA molecules of (a) under conditions effective to achieve specific annealing of the single-stranded overhanging portions; and
(c) treating the incubated DNA molecules in (b) under conditions effective to fill in remaining single-stranded gaps and to seal the nicks thus formed, wherein the region of sequence identity comprises at least 20 non-palindromic nucleotides (nt).
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Abstract
The present invention relates, e.g., to an in vitro method, using isolated protein reagents, for joining two double-stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising (a) chewing back the DNA molecules with an enzyme having an exonuclease activity, to yield single-stranded overhanging portions of each DNA molecule which contain a sufficient length of the region of sequence identity to hybridize specifically to each other; (b) specifically annealing the single-stranded overhangs; and (c) repairing single-stranded gaps in the annealed DNA molecules and sealing the nicks thus formed (ligating the nicked DNA molecules). The region of sequence identity generally comprises at least 20 non-palindromic nucleotides (nt), e.g., at least about 40 non-palindromic nt. In some embodiments of the invention, about 5% PEG is present during all steps of the reaction, and/or the repair reaction is achieved with Taq DNA polymerase and a compatible ligase, such as Taq DNA ligase. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest.
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Citations
63 Claims
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1. An in vitro method, using isolated proteins, for joining two or more double-stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a region of sequence identity, comprising
(a) treating the DNA molecules with an enzyme having an exonuclease activity, under conditions effective to yield single-stranded overhanging portions of each DNA molecule which contain a sufficient length of the region of sequence homology to hybridize specifically to the region of sequence homology of its pair; -
(b) incubating the treated DNA molecules of (a) under conditions effective to achieve specific annealing of the single-stranded overhanging portions; and
(c) treating the incubated DNA molecules in (b) under conditions effective to fill in remaining single-stranded gaps and to seal the nicks thus formed, wherein the region of sequence identity comprises at least 20 non-palindromic nucleotides (nt). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 28, 30, 31, 33, 34, 36, 38, 41, 46, 52, 61, 62)
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12. (canceled)
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14. (canceled)
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24. An in vitro method, using isolated proteins, for joining at least two ds DNA molecules of interest, each of about 5-6 kilobases (kb), wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a unique region of sequence identity, comprising
(a) treating approximately equimolar amounts of the DNA molecules with T4 DNA polymerase at about 37° - C., in a solution comprising about 0.2 M Tris at about pH 7.5, in the absence of added dNTPs, under conditions effective to chew-back at least the regions of sequence identity in each molecule, thereby forming single-stranded overhanging ends of sufficient length to hybridize specifically to overhangs having the complement of the shared region of sequence identity;
(b) annealing the treated DNA molecules in (a) by incubating them at 75°
C. plus or minus about 5°
C. for about 20 minutes, and slow cooling them to about 24°
C. or less, under conditions effective to anneal the single-stranded DNA regions which were generated during (a); and
(c) incubating the cooled DNA molecules in (b) with Taq DNA polymerase and Taq DNA ligase at about 45°
C., in the presence of added dNTPs, under conditions effective to fill in the gaps and seal the nicks,wherein about 5% PEG is present throughout the joining procedure.
- C., in a solution comprising about 0.2 M Tris at about pH 7.5, in the absence of added dNTPs, under conditions effective to chew-back at least the regions of sequence identity in each molecule, thereby forming single-stranded overhanging ends of sufficient length to hybridize specifically to overhangs having the complement of the shared region of sequence identity;
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25. An in vitro method, using isolated proteins, for joining at least two ds DNA molecules of interest, each of about 5-6 kilobases (kb), wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a unique region of sequence identity, comprising
(a) incubating approximately equimolar amounts of the DNA molecules with: - T4 DNA polymerase;
a protein that enhances annealing of single-stranded DNAs; and
a ligase that is compatible with the polymerase, at about 37°
C., in the absence of added dNTPs, under conditions effective to chew-back at least the regions of sequence identity in each molecule, thereby forming single-stranded overhanging ends of sufficient length to hybridize specifically to overhangs having the complement of the shared region of sequence identity, and to allow hybridization of the single-stranded overhangs, thereby forming gapped molecules; and
(b) incubating the incubated DNA molecules in (a) with a sufficient amount of dNTPs, under conditions effective to allow filling in of the gaps, generation of nicks, and sealing of the nicks, wherein the method is carried out in a single vessel. - View Dependent Claims (27)
- T4 DNA polymerase;
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26. (canceled)
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29. (canceled)
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32. (canceled)
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35. (canceled)
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37. (canceled)
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39-40. -40. (canceled)
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42-45. -45. (canceled)
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47-50. -50. (canceled)
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51. An in vitro method, using isolated protein reagents, for joining a plurality of double-strand (ds) DNA molecules of interest, wherein for each pair of molecules to be joined, the distal region of one DNA molecule comprises a region of sequence identity to the proximal region of the other DNA molecule, and each set of distal and proximal regions of identity is unique for each pair of DNA molecules to be joined, comprising generating 5′
- single-strand overhangs at both ends of the DNA molecules;
annealing the regions of sequence identity in the single-stranded overhangs, filling in the gaps formed; and
sealing the nicks,wherein each region of sequence identity comprises at least 20 non-palindromic nucleotides (nt).
- single-strand overhangs at both ends of the DNA molecules;
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53. (canceled)
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54. A kit for in vitro joining a plurality of dsDNA molecules, comprising
(a) an isolated enzyme having a 3′ - or 5′
exonuclease activity;
(b) an isolated non strand-displacing DNA polymerase;
(c) a ligase which is compatible with the polymerase; and
, optionally,(d) solution, or components for making the solution which, when combined with the exonuclease and the dsDNA molecules to be joined, comprises about 5% PEG and/or about 0.2 M Tris, at about pH7.5.
- or 5′
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55-57. -57. (canceled)
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58. A kit for in vitro joining a plurality of dsDNA molecules, comprising
(a) a vessel containing purified T4 DNA polymerase; - a protein that enhances annealing of single-stranded DNAs; and
a ligase that is compatible with the polymerase; and
, optionally(b) a solution, or components for making the solution, that, when combined with an aliquot of the protein mixture in (a) and a plurality of suitable DNA molecules containing regions of sequence identity at their termini, is effective to allow chew-back of regions of sequence identity of the DNA molecules, the formation of single-stranded overhangs containing the regions of sequence identity, and hybridization of the single-stranded overhangs, thereby forming gapped molecules; and
, optionally(c) a concentrated solution of dNTPs that, when added in a suitable volume to the solution in (b) which contains gapped molecules, and incubated with that solution under suitable conditions, is effective to allow filling in of the gaps.
- a protein that enhances annealing of single-stranded DNAs; and
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59-60. -60. (canceled)
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63. (canceled)
Specification