Site-Directed Mutagenesis Using Overlap Extension PCR

Site-directed mutagenesis and the polymerase chain reaction (PCR) represent two powerful techniques that have led to rapid advances in our understanding of gene expression and function. Early protocols for site-directed mutagenesis depended on the production of single-stranded DNA containing the gene of interest (11 ), using M13 phage, or phagemids such as pBluescript. One limitation with this method is the presence of inverted repeat sequences and other complementary regions, which form extensive secondary structures within single-stranded DNA, which often severely decrease the ability to extend annealed primers containing the mutation of interest.