METHOD FOR IN VITRO RECOMBINATION
First Claim
1. An in vitro method for joining a first set of double-stranded (ds) DNA molecules, comprising:
- (a) providing two or more dsDNA molecules to be joined in a reaction mixture, wherein, for each pair of dsDNA molecules to be joined, a distal region of a first DNA molecule and a proximal region of a second DNA molecule share a region of sequence homology;
(b) treating the provided dsDNA molecules with a substantially purified enzyme having 5′
-3′
exonuclease activity, whereby a single-stranded overhanging portion is generated in each of the dsDNA molecules by 5′
-3′
exonuclease digestion, wherein each overhanging portion contains the region of homology or a portion thereof sufficient to specifically anneal to the overhanging portion in the other molecule of the pair;
(c) incubating the DNA molecules generated in step (b), under conditions whereby they anneal through the regions of homology or portions thereof; and
(d) treating the annealed molecules with a substantially purified polymerase and a substantially purified compatible ligase, under conditions whereby remaining single-stranded gap(s) are filled in by the polymerase and nicks are sealed by the ligase;
thereby joining the dsDNA molecules, wherein a crowding agent is present in the reaction mixture during each of steps (b), (c), and (d).
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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
29 Claims
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1. An in vitro method for joining a first set of double-stranded (ds) DNA molecules, comprising:
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(a) providing two or more dsDNA molecules to be joined in a reaction mixture, wherein, for each pair of dsDNA molecules to be joined, a distal region of a first DNA molecule and a proximal region of a second DNA molecule share a region of sequence homology; (b) treating the provided dsDNA molecules with a substantially purified enzyme having 5′
-3′
exonuclease activity, whereby a single-stranded overhanging portion is generated in each of the dsDNA molecules by 5′
-3′
exonuclease digestion, wherein each overhanging portion contains the region of homology or a portion thereof sufficient to specifically anneal to the overhanging portion in the other molecule of the pair;(c) incubating the DNA molecules generated in step (b), under conditions whereby they anneal through the regions of homology or portions thereof; and (d) treating the annealed molecules with a substantially purified polymerase and a substantially purified compatible ligase, under conditions whereby remaining single-stranded gap(s) are filled in by the polymerase and nicks are sealed by the ligase; thereby joining the dsDNA molecules, wherein a crowding agent is present in the reaction mixture during each of steps (b), (c), and (d). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
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Specification