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Site-directed mutagenesis (SDM) is used to introduce a defined mutation into target DNA of known sequence to study, for example, gene expression or protein structure–function relationship. A number of polymerase chain reaction (PCR)-based mutagenesis methods have been developed. Among them, the “megaprimer” method is probably the simplest and most flexible. The megaprimer method utilizes two external oligonucleotide primers and one internal mutagenic primer in two rounds of PCR with a DNA template that contains the sequence to be mutated. The first round of PCR is carried out using one of the external primers and the mutagenic primer containing the desired mutation. This amplifies an intermediate PCR product that is purified and used as a “megaprimer” for the second round of PCR, along with the other external primer. The final PCR product is cloned into appropriate vectors and used in downstream applications.