Patent ID: 6291213
Filing Date: 2001-09-18
Classification: C12N,C12Q,Y10S

Abstract:
A method for generating a directed, recombinant fusion nucleic acid molecule capable of cross-over recombination which comprises:(A) contacting(i) a first pair of single-stranded primers with a first strand and a second strand of a first nucleic acid molecule;(ii) a second and a third pair of primers with a second nucleic acid molecule having a first strand and a second strand, wherein the primers are suitable for use in a polymerase chain reaction, and(a) the first primer of the first pair comprises a 3' sequence that is homologous to the first strand of the first nucleic acid molecule and a 5' sequence;(b) the second primer of the first pair of primers comprises a 3' sequence that is homologous to the second strand of the first nucleic acid molecule and a 5' sequence;(c) the first primer of the second pair of primers comprises a sequence that is homologous to the second strand of the second nucleic acid molecule;(d) the second primer of the second pair of primers comprises a 3' sequence that is homologous to the first strand of the second nucleic acid molecule and a 5' sequence that is complementary to the 5' sequence of the first primer of the first pair of primers;(e) the first primer of the third pair of primers comprises a 5' sequence complementary to the 5' sequence of the second primer of the first pair of primers and a 3' sequence homologous to the second strand of the second nucleic acid molecule, and(f) the second primer of the third pair of primers comprises a sequence that is homologous to the first strand of the second nucleic acid molecule;(B) amplifying (1) the first nucleic acid molecules with the first pair of primers and (2) the second nucleic acid molecule with the second and third pairs of primers, so as to generate at least one linear double-stranded nucleic acid product from each reaction;(C) denaturing the products from step (B) so as to obtain single-stranded products;(D) contacting the single-stranded products from step (C) with the first primer of the second set of primers and the second primer from the third set of primers, under suitable hybridization conditions, and(E) amplifying the the single-stranded products from step (D) under suitable amplification conditions, so as to generate a fusion nucleic acid molecule capable of cross-over recombination.