A new method for multi-site-directed mutagenesis.

A modified method for multi-site-directed mutagenesis was developed here based on polymerase chain reaction (PCR), DpnI digestion, and overlap extension. It needs only methylated plasmids obtained by Dam methyltransferase or plasmids from dam(+)Escherichia coli containing target gene. The procedure consists of PCR, DpnI digestion, overlap extension PCR, and plasmid transformation. The method was developed for multi-site-directed mutagenesis, including close proximity of mutation sites. It does not require 5'-phosphorylated primers and ligation and, thus, significantly simplifies the routine work and reduces the experimental cost for multi-site-directed mutagenesis.