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Direct cloning

  • US 10,443,051 B2
  • Filed: 06/10/2011
  • Issued: 10/15/2019
  • Est. Priority Date: 06/10/2010
  • Status: Active Grant
First Claim
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1. A method for performing homologous recombination between at least a first linear nucleic acid molecule and a second linear nucleic acid molecule which share at least one region of sequence homology, wherein the method comprises bringing the first nucleic acid molecule into contact with the second nucleic acid molecule in a first step of linear to linear homologous recombination, in the presence of a 5′

  • to 3′

    exonuclease, RecT, and Red gamma;

    wherein the 5′

    to 3′

    exonuclease is an N-terminally extended RecE expressed from heterologous DNA that comprises or consists of amino acids 1-866 of SEQ ID NO;

    1, or a variant of this sequence having at least 95% sequence identity to SEQ ID NO;

    1 over the entire length of the 866 amino acid sequence,wherein said first step is carried out in the absence of truncated RecE, Red alpha and Red beta,and wherein the method further comprises bringing the product of the linear to linear homologous recombination reaction into contact with a further nucleic acid molecule in a second step of linear to circular homologous recombination in the presence of Red alpha, Red beta, and Red gamma,wherein said second step is carried out in the absence of the N-terminally extended RecE.

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