Patent ID: 7723077
Filing Date: 2010-05-25
Classification: C12N,C12P,C12Q,C12Y

Abstract:
1. An in vitro method, using isolated proteins, for joining two double strand (ds) DNA molecules of interest, comprising: providing a first DNA molecule and a second dsDNA molecule which share a region of sequence identity at a terminal end on each DNA molecule, wherein the region is less than about 150 base pairs in length; and contacting the two dsDNA molecules with: (a) a purified 5′ exonuclease; (b) a purified single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing; (c) a purified non strand-displacing DNA polymerase; and (d) a purified ligase, under conditions whereby: a 3′ single-stranded overhang is generated in each molecule by the exonuclease without the use of a restriction enzyme; the two single-stranded overhangs anneal to form a gapped molecule; the gaps are filled in by the polymerase; and nicks are sealed by the ligase, thereby joining the molecules and forming a substantially intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained, wherein none of the enzymatic reactions is actively terminated prior to beginning another of the reactions.