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Method for in vitro recombination

  • US 20070037196A1
  • Filed: 08/11/2006
  • Published: 02/15/2007
  • Est. Priority Date: 08/11/2005
  • Status: Active Grant
First Claim
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1. An in vitro method, using isolated proteins, for joining two or more 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 of each pair share a region of sequence identity, comprising (a) treating the DNA molecules with an enzyme having an exonuclease activity, under conditions effective to yield single-stranded overhanging portions of each DNA molecule which contain a sufficient length of the region of sequence homology to hybridize specifically to the region of sequence homology of its pair;

  • (b) incubating the treated DNA molecules of (a) under conditions effective to achieve specific annealing of the single-stranded overhanging portions; and

    (c) treating the incubated DNA molecules in (b) under conditions effective to fill in remaining single-stranded gaps and to seal the nicks thus formed, wherein the region of sequence identity comprises at least 20 non-palindromic nucleotides (nt).

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