SITE-DIRECTED MUTAGENESIS IN CIRCULAR METHYLATED DNA

摘要

The invention provides a new method for conducting site-specific mutation in methylated circular stranded DNA molecules conferred by means of mutagenic primer pairs and methylase deficient Escherichia coli. The mutagenic primer pairs are complementary at 5' end or 3' end, or completely complementary to each other. Firstly, mutagenic primer pair is annealed to opposite strands of the methylated circular double-stranded parent DNA molecules. Then, polymerase chain reaction with DNA polymerase is performed by using unmethylated dNTPs to create unmethylated mutagenized double-stranded daughter DNA molecules. Finally, the reaction mixture of the methylated parent DNA molecules and unmethylated mutagenized daughter DNA molecules is transformed into a methylase deficient competent E.coli. The replication of methylated parent DNA is inhibited in methylase deficient host cell. In contrast, the unmethylated daughter DNA, which contains the desired mutation, are efficiently replicated in methylase deficient host cell and recovered thereafter. The invention also provides a kit for introducing site-specific mutagenesis in accordance with the method of the present invention.