Multisite-Directed Mutagenesis

This protocol, suitable for both general and close-proximity mutagenesis, includes a simple and rapid procedure that combines polymerase chain reaction (PCR), DpnI digestion, and overlap extension. The key point of this approach is the use of overlap extension to form a circular DNA plasmid with mutations without the need for phosphorylated primers or ligase reactions. Essentially, during the first round of PCR, the new DNA is synthesized with nicks between the 3' ends of the synthesized DNA and the 5' ends of the first pair of primers. During successive rounds of PCR, a new pair of mutagenic oligonucleotides leads to the synthesis of two DNA segments that anneal together with the overlap sequence inside the two primers. This new mutated molecule also contains nicks but at different positions compared with those formed in the first round of PCR that had been "repaired" by overlap extension. Mutations can be introduced successfully by this method. Finally, the circular DNA is transformed into E. coli cells, where the nicks are ligated into a circular plasmid. One important requirement is that the parental plasmid carrying the target gene needs to be methylated by Dam methyltransferase or purified from Dam⁺ E. coli (i.e., DH5a).