Patent ID: 7776532
Filing Date: 2010-08-17
Classification: C12N,C12P

Abstract:
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 3′-5′ exonuclease activity, whereby a single-stranded overhanging portion is generated in each of the dsDNA molecules by 3′-5′ 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).