Site-Directed Mutagenesis with LA-PCR Technology

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 –8 ). This chapter describes an efficient and economic PCR-based site-directed mutagenesis method, which is designed to introduce a series of mutations into long DNA cloned in pUC vectors (pUC 18, 19, 118, 119), pBluescript� vectors (Stratagene, Cambridge, UK) (pBluescript� II SK(�), KS (�)) and pGEM� vectors (Promega, Madison, WI) (pGEM�-3Zf,4Z) by adding the advantage of LA-PCR technology™ (TaKaRa Shuzo Co., Kyoto, Japan). 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 (9 ). As based on the Long and Accurate (LA)-PCR-technology with the improved enzyme, TaKaRa LA Taq , this method provides the high fidelity and introduction of site-directed mutation into longer DNA can be achieved.