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METHOD FOR IN VITRO RECOMBINATION

  • US 20100311126A1
  • Filed: 06/22/2010
  • Published: 12/09/2010
  • Est. Priority Date: 08/11/2005
  • Status: Active Grant
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 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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