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Methods and compositions for synthesis of nucleic acid molecules using multiple recognition sites

  • US 7,198,924 B2
  • Filed: 03/04/2004
  • Issued: 04/03/2007
  • Est. Priority Date: 12/11/2000
  • Status: Expired due to Term
First Claim
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1. An in vitro method of cloning a nucleic acid molecule comprising:

  • (a) obtaining a first nucleic acid molecule to be cloned;

    (b) mixing said first nucleic acid molecule to be cloned in vitro with a second nucleic acid molecule comprising at least a first topoisomerase recognition site flanked by at least a first recombination site, and at least a second topoisomerase recognition site flanked by at least a second recombation site, wherein said first and second recombination sites do not recombine with each other, and at least one topoisomerase; and

    (c) incubating said mixture under conditions such that said first nucleic acid molecule to be cloned is inserted into said second nucleic acid molecule between said first and second topoisomerase recognition sites, thereby producing a first product molecule comprising said first nucleic acid molecule to be cloned between said first and second recombination sites.

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