Patent ID: 8841094

Claim:
A method for homologous recombination comprising: amplifying nucleic acid molecules in a sample, which sample includes a nucleic acid molecule comprising a target gene sequence, by polymerase chain reaction (“PCR”) using an amplification primer containing P1 sequence and an amplification primer containing P2 sequence to obtain a PCR product having primer sequence P1 on one end of the PCR product and primer sequence P2 on the other end of the PCR product and which sample further includes a nucleic acid molecule comprising a non-target gene sequence; providing a linearized vector, wherein the linearized vector has homologous recombination regions on its terminal ends, one end comprising VP1 and VT1 and the other end comprising VP2, or VP2 and VT2, wherein (i) VP1, comprising a nucleotide sequence homologous to the amplification primer sequence P1 and (ii) VP2 comprising a nucleotide sequence homologous to the amplification primer sequence P2 and (iii) a homologous recombination region VT1 comprising a nucleotide sequence homologous to a sequence T1, which T1 is a part of the target gene sequence in the PCR product internal to P1, wherein T1 comprises a sequence that is not part of P1, wherein VT1 is on the terminal side of VP1 and/or (iv) a homologous recombination region VT2 comprising a nucleotide sequence homologous to a sequence T2, which T2 is a part of sequence in the PCR product internal to P2 wherein T2 comprises a sequence that is not part of P2, wherein VT2 is on the terminal side of VP2, combining the PCR product, without purification, and the linearized vector under conditions suitable for homologous recombination, whereby the amplified nucleic acid molecule comprising the target gene sequence may be inserted into the linearized vector by homologous recombination (a) between the P1 and T1 sequences of the amplified nucleic acid molecule comprising the target gene sequence and the VP1 and T1 sequences on the linearized vector, and (b) between (i) the P2 sequence on the amplified nucleic acid molecule comprising the target gene sequence and the VP2 sequence on the linearized vector or (ii) the P2 and T2 sequences on the amplified nucleic acid molecule comprising the target gene sequence with the VP2 and VT2 sequences on the linearized vector; and obtaining a recombinant DNA molecule in which the amplified nucleic acid molecule comprising the target gene sequence is specifically inserted into the vector.