SITE-DIRECTED MUTAGENESIS IN CIRCULAR METHYLATED DNA

摘要

Site-specific mutation in methylated circular stranded DNA molecules is conferred by 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 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 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 mutations in accordance with the described method.