PCR Method for Generating Multiple Mutations at Adjacent Sites

Site-directed mutagenesis is a commonly used tool for identifying the role of specific amino acids in the structure and function of proteins. Various methods of in vitro mutagenesis have been described and are widely used for introducing modified coding sequences (1 -7 ). In comparison, polymerase chain reaction (PCR)-based methods (1 -6 ) are generally faster and more efficient than non-PCR-based methods (7 ). On the other hand, some PCR-based methods require two or more primers for each round of mutagenesis, whereas others need a single very long oligonucleotide or two round of PCR for introducing one mutation (5 -7 ). These all can increase the cost of mutagenesis.