RAPID AND EFFICIENT SITE-DIRECTED MUTAGENESIS BY SINGLE-TUBE MEGAPRIMER PCR METHOD

We describe a rapid and efficient megaprimer PCR procedure for site-di rected mutagenesis that does not require any intermediate purification of DNA between the two rounds of PCR. This protocol is based on the d esign of forward and reverse flanking primers with significantly diffe rent melting temperatures (T-m). A megaprimer is synthesized in the fi rst PCR reaction using a mutagenic primer, the low T-m flanking primer and a low annealing temperature. The second PCR reaction is performed in the same tube as the first PCR and utilizes the high T-m flanking primer, the megaprimer product of the first PCR and a high annealing t emperature, which prevents priming by the low T-m primer from the firs t PCR reaction. We have used this protocol with two different plasmids to produce cDNAs encoding seven distinct mutated proteins. We have ob served an average mutagenesis efficiency of 82% in these experiments.