Multiple Site-Directed Mutagenesis

Site-directed mutagenesis techniques allow selective engineering of gene sequences. In in vitro site-directed mutagenesis, base alterations are introduced into a target sequence by incorporating DNA base changes within a primer utilized in the DNA synthesis step. Several site-directed mutagenesis procedures based on this general principle have been described (1 ,2 ). However, application of these methodologies for multiple site-directed mutagenesis requires several cloning steps. I have developed a polymerase chain reaction (PCR)-based multiple site-directed mutagenesis procedure (3 ). This technique utilizes a combination of DNA amplification and chain extension to alter specific bases, starting from one end of a target DNA molecule and progressing to the other end. This multiple site-directed mutagenesis procedure is outlined in Fig. 1 . Each mutagenesis cycle involves three steps: PCR, restriction digestion, and DNA chain extension. The PCR step introduces base changes into the DNA and then amplifies the mutated fragment. Restriction digestion, followed by gel electrophoresis is then used to select for this mutagenized DNA fragment. Finally, the chain extension reaction step extends the mutagenized DNA fragment along the target DNA, generating template for the next mutagenic primer to bind. This mutagenesis cycle is repeated until all the desired site-specific mutations are completed in the target DNA sequence.