PCR-Based Site-Directed Mutagenesis

Protocols for site-directed mutagenesis are widely used in molecular biology and include many polymerase chain reaction (PCR)-based methods that have been developed in order to achieve efficient mutagenesis of a target DNA sequence (1 –4 ). However, some of these methods described require two or more specific-oligonucleotides for each round of mutagenesis, making the cost of such procedures expensive. This chapter describes an efficient and economic PCR-based site-directed mutagenesis method, which is designed to introduce a series of mutations into DNA cloned into pUC vectors (pUC 18, 19, 118, 119). The protocol uses a combination of a primer designed for introducing a mutation at the target sequence, with primers that may be reused for each mutagenesis reaction (Fig. 1 ). By using this method, a series of site-directed mutations may be undertaken that only require a single primer for each desired change, and furthermore, no reiterative transformation steps are necessary.