A general method for introducing a series of mutations into cloned DNA using the polymerase chain reaction

A simple and fast method for introducing a series of mutations in cloned DNA has been developed. The polymerase chain reaction (PCR) has been used for site-directed mutagenesis. Because mutations can be introduced only within the primer sequences used for PCR, a suitable restriction site in the vicinity of the mutated nucleotide is required to permit recloning. Several methods have been devised to overcome this limitation. Our present method is a modification of the overlap extension method [Ho et al., Gene 77 (1989) 51-57], and has some advantages over this and other published methods. In our method, three common primers and a series of primers specific for various mutations are chemically synthesized. Once the proper oligodeoxyribonucleotides are selected as common primers, each mutation requires only one additional primer. Therefore, this method is very useful for introducing many mutations in various sites of the target DNA. We describe our protocol for the site-directed mutagenesis and an example of the introduction of several mutations in the hen egg-white lysozyme-encoding gene.