Patent ID: 8685676

Claim:
A method for producing a DNA product comprising two or more mutations by introducing said two or more mutations to a double-stranded target DNA molecule, said method comprising: a) annealing one or more primers to the same strand of said target DNA molecule in a solution, wherein said one or more primers comprise two or more mutation sites with respect to said target DNA molecule; b) using said target DNA molecule as a template, synthesizing a mutagenized single strand DNA comprising said primers and a flap structure in said solution by an amplification reaction in the presence of a DNA polymerase, a DNA ligase, and a flap endonuclease; and c) adding a selection enzyme to the solution after step (b) to perform a digestion reaction, wherein said selection enzyme digests said target DNA molecule but not significantly digests said mutagenized single strand, thereby producing said DNA product.