Site-directed Mutagenesis Using DNA Polymerase

Site-directed mutagenesis is a powerful tool for protein engineering and the study of protein structure and function. Numerous methods of site-directed mutagenesis that exploit the primer extension technique have been developed. These methods generate heteroduplex species containing one mutated and one non-mutated strand; subsequent replication and segregation of these molecules inevitably results in a population heavily contaminated with wild-type DNA. Various methods that circumvent the replication of non-mutated DNA or that separate the two species have been developed, but these methods tend to be more difficult and lengthy. This application note describes a fast, simple, and efficient system for the introduction of specific mutations, insertions and/or deletions into DNA cloned in a double-stranded plasmid.