We present a rapid, cheap and highly efficient method for site-directed mutagenesis using the polymerase chain reaction (PCR). This method is applicable to every DNA fragment which has to be cloned into the multiple cloning site of any vector, or vector pair, in two different orientations. It requires only two primers, one new and specific mutagenic 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. In contrast, wild-type sequences are only linearly amplified resulting in an efficiency of mutagenesis of nearly 100%.
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