AN EFFICIENT AND OPTIMIZED PCR METHOD WITH HIGH-FIDELITY FOR SITE-DIRECTED MUTAGENESIS

We have developed an efficient method for site-directed mutagenesis us ing two subsequential rounds of PCR. In this method, PCR conditions ar e optimized to favor high fidelity of fag DNA polymerase in the presen ce of equimolar concentrations of MgCl2 and dNTP in the reaction mixtu re (pH 5.5-6.2). This method makes use of a pair of universal primers and the multiple cloning site of pUC/M13 vectors. Only one mutagenic p rimer is required per target site. In the second round of PCR, the 3' extension of the wild-type DNA strand is blocked by the presence of a segment of nonhomologous sequence at its 3' end, and as a consequence, the amplified, full-length DNA fragment is chiefly from the mutant st rand. Furthermore, because mutated DNA fragment has flanking restricti on sites different from those of the wild-type DNA fragment, the wild- type DNA fragment is totally excluded in the step involving selective cloning of the mutant DNA fragment. This method was successfully used to introduce four, nonadjacent mutations in the 5' regulatory region o f the cytochrome P450(BM-3) gene. All 20 analyzed clones from these fo ur cases of mutagenesis carried the desired mutations, and no undesire d mutations were observed. We observed that the larger the number of m ismatched nucleotide residues in the mutagenic primer, the higher the concentration of MgCl2 was necessary for successful PCR amplification. Our experimental results indicate that this method offers improvement s in efficiency, flexibility, and fidelity.