HOMOLOGOUS RECOMBINATION METHOD, CLONING METHOD, AND KIT

摘要

Disclosed is a method which can achieve the homologous recombination of a gene of interest selectively. Also disclosed is a recombinant DNA molecule produced by the method. Specifically disclosed is a homologous recombination method which uses a PCR product and a linearized vector. The PCR product comprises a sequence for a target gene and amplification primer sequences P1 and P2 attached to both termini of the sequence for the target gene. The vector has homologous recombination regions VP1 and VP2 respectively comprising nucleotide sequences homologous to the amplification primer sequences P1 and P2 contained in the PCR product, and also has a homologous recombination region VT1 comprising a nucleotide sequence homologous to a sequence T1 that is a partial internal sequence of P1 on the terminal side of VP1 and/or a homologous recombination region VT2 comprising a nucleotide sequence homologous to a sequence T2 that is a partial internal sequence of P2 on the terminal side of VP2 (provided that at least one of T1 and T2 has a nucleotide sequence specific to the target gene). The method comprises subjecting the PCR product to a homologous recombination reaction to insert the PCR product into the vector, thereby producing a recombinant DNA molecule comprising the vector and the desired PCR product specifically inserted into the vector. Also specifically disclosed is a cloning method for producing the recombinant DNA molecule comprising the vector and the desired PCR product specifically inserted into the vector by the homologous recombination method and amplifying the recombinant DNA molecule.