A RAPID AND HIGHLY EFFICIENT METHOD FOR PCR-BASED SITE-DIRECTED MUTAGENESIS USING ONLY ONE NEW PRIMER

We present a rapid, cheap and highly efficient method for site-directe d mutagenesis using the polymerase chain reaction (PCR). This method i s applicable to every DNA fragment which has to be cloned into the mul tiple cloning site of any vector, or vector pair, in two different ori entations. It requires only two primers, one new and specific mutageni c primer and one of the usual sequencing primers. In the first PCR, a mutagenic DNA fragment is synthesized which is amplified exponentially in the second PCR. Tn contrast, wild-type sequences are only linearly amplified resulting in an efficiency of mutagenesis of nearly 100%.