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In vitro recombination method

  • US 7,723,077 B2
  • Filed: 08/11/2006
  • Issued: 05/25/2010
  • 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 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.

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