Method for in vitro recombination
First Claim
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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 overlapping 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); and
wherein the overhanging portions are created without the use of restriction enzymes.
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Abstract
The present invention relates 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. 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
26 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 overlapping 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); and wherein the overhanging portions are created without the use of restriction enzymes. - 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)
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Specification