Patent ID: 7220548
Filing Date: 2007-05-22
Classification: C12N,C12Q

Abstract:
1. A method of constructing a DNA library having an increased content of a first dsDNA by removing a second dsDNA, which is different from the first dsDNA, from a DNA library containing the first dsDNA whose content is to be increased and the second dsDNA, comprising: (1) adding a third ss nucleic acid, which contains a homologous sequence to the DNA library, said third ss nucleic acid containing a sequence that is homologous to a 3′ terminal portion of a first strand of the second dsDNA, the homologous sequence being located at a position other than the 3′ terminal portion of the third ss nucleic acid, said third ss nucleic acid having a 3′ terminal sequence that is different from that of the second dsDNA; (2) adding a RecA protein to the DNA library, thereby catalyzing homologous recombination between the 3′ terminal portion of the first strand of the second dsDNA and the third ss nucleic acid to form a triple stranded portion at the 3′ terminal portion of the second dsDNA consisting of the first strand of the second dsDNA, the third ss nucleic acid, and a second strand of the second dsDNA; (3) adding Exonuclease I to the DNA library containing a homologous recombinant (triple stranded portion) to digest the first strand of the second dsDNA of the triple stranded portion; (4) ligating a DNA fragment to circularize the first dsDNA; and (5) removing linear DNA not reacted in the ligation treatment of (4), thereby constructing the DNA library having an increased content of the first dsDNA.