Patent ID: 9347057
Filing Date: 2016-05-24
CPC Classification: C07H,C12N,C12P

Claim Text:
1. A method for introducing one or more mutations to a single-stranded target nucleic acid molecule, said method comprising: a) providing a single-stranded uracil-containing template comprising a target nucleic acid molecule in a circular DNA vector; b) annealing at least one mutagenic oligonucleotide comprising at least one mutation to the target nucleic acid molecule at a first elevated temperature; c) conducting a first amplification reaction in the presence of a thermostable DNA polymerase and a thermostable DNA ligase at a second elevated temperature to synthesize a mutagenized strand of DNA comprising the at least one mutagenic oligonucleotide; d) denaturing the mutagenized strand of DNA at a third elevated temperature; e) annealing a reverse primer to the mutagenized strand of DNA at a fourth elevated temperature; f) conducting a second amplification reaction in the presence of a thermostable DNA polymerase and a thermostable DNA ligase at a fifth elevated temperature to synthesize a complementary mutant strand of DNA; and g) degrading the uracil-containing DNA and non-covalently closed circular nucleic acid molecules at a sixth elevated temperature to obtain a mutation-containing double-stranded DNA product.