Multiple Site-Directed Mutagenesis In Vitro

Polymerase chain reaction (PCR)-based site-directed mutagenesis (SDM) methods are widely used in molecular biology to selectively alter gene sequences (1 ). However, common protocols allow for the introduction of only one (1 –3 ) or two (4 ) independent point mutations at a time, followed by the time-consuming phenotypic selection in bacteria cells and isolation of plasmid DNA, to identify the correctly targeted clones. Here is described an improved protocol for mutagenesis that allows introduction of multiple independent mutations with high efficiency, involving only one cloning step (5 ). The authors’ procedure is based on the QuikChange™ SDM method (2 ), which combines PCR mutagenesis using Pfu DNA-polymerase and mutagenic primers, with the subsequent elimination of the template DNA by DpnI digestion (2 ,3 ,6 ). The high fidelity of Pfu DNA polymerase minimizes undesirable mutations on newly amplified DNA during PCR and the selectivity of Dpn I for fully or hemimethylated 5′-G^m6 ATC-3′ sequences quantitatively degrades the parental DNA, resulting in high mutagenesis efficiencies (2 ,3 ,6 ).