Homologous recombination-based DNA cloning methods and compositions

摘要

Methods and compositions for cloning a donor DNA molecule into an acceptor vector at a predetermined location are described. The methods are based on homologous recombination mediated by in vitro treatment of the donor DNA and the acceptor vector with an enzyme cocktail containing an exonuclease and a single-stranded DNA binding protein.

主权项

1. A method of cloning a donor DNA molecule into an acceptor vector at a predetermined location, the method comprising:
a) preparing an extended donor DNA molecule by adding to the 5′-end and the 3′-end of the donor DNA molecule a first sequence and a second sequence, respectively, wherein each of the first and second sequences, independently, is at least 12 nucleotides in length and is at least 90% identical to a first region and a second region of the acceptor vector, respectively; b) providing a reaction mixture comprising:
i) the acceptor vector;ii) the extended donor DNA molecule; andiii) an enzyme cocktail comprising an exonuclease having only 3′- or 5′-exonucleolytic single-strand degradation activity, and a single-stranded DNA binding protein; c) incubating the reaction mixture to obtain an intermediate product; d) transforming a cell with the intermediate product to obtain a transformed cell; and e) culturing the transformed cell under conditions to produce a recombinant DNA molecule comprising the donor DNA located between the first region and the second region.