The invention provides improved methods of introducing site-directed mutationsinto circular DNA molecules of interest by means ofmutagenic primer pairs. The mutagenic primer pairs are also selected so as tobe either completely complementary or partially complementaryto each other, wherein the mutation site (or sites) is located within theregion of complementarity. A mutagenic primer pair is annealed toopposite strands of a circular DNA molecule containing the DNA sequence to bemutagenized. After annealing, first and second mutagenizedDNA strands, each incorporating a member of the mutagenic oligonucleotideprimer pair is synthesized by a linear cyclic amplificationreaction. After the linear cyclic amplification mediated synthesis step iscompleted, the reaction mixture is treated with a selection enzymethat digests the parental template strands. After the digesting step, a double-stranded circular DNA intermediate is formed. The double--strandedcircular DNA intermediates are transformed in suitable competent host cellsand closed circular double-stranded DNA correspondingto the parental template molecules, but containing the desired mutation ormutations of interest, may be conveniently recovered from thetransformed cells. The invention also provides kits for site-directedmutagenesis in accordance with methods of the present invention.
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