An improved PCR-based method for site directed mutagenesis using megaprimers

An improved protocol for site-directed mutagenesis based on the two-step po lymerase chain reaction (PCR) megaprimer method is described. Compared to p reviously published protocols, the protocol described in this article ensur es consistently a success rate of at least 85% with essentially no introduc tion of unwanted secondary mutations. The essential features of this protoc ol include an optimization of the template-primer amounts and ratio that al lows the use of a reduced number of PCR cycles and the use of proof-reading thermostable DNA polymerases. (C) 1998 Academic Press.