Inverse PCR for Site-Directed Mutagenesis

Inverse PCR is a powerful tool for the rapid introduction of desired mutations at desired positions in a circular double-stranded DNA sequence. In this technique, custom-designed mutant primers oriented in the inverse direction are used to amplify the entire circular template with incorporation of the required mutation(s). By careful primer design, it can be used to perform such diverse modifications as the introduction of point or multiple mutations, the insertion of new sequences, and even sequence deletions. Three primer formats are commonly used, nonoverlapping, partially overlapping, and fully overlapping primers, and here we describe the use of nonoverlapping primers for introduction of a point mutation. Use of such a primer setup in the PCR, with one of the primers containing the desired mismatch mutation, results in the amplification of a linear, double-stranded, mutated product. Methylated template DNA is removed from the non-methylated PCR product by DpnI digestion, and the PCR product is then phosphorylated by polynucleotide kinase treatment before being recircularized by ligation and transformed to E. coli. This relatively simple site-directed mutagenesis procedure is of major importance in biology and biotechnology where it is commonly employed for the study and engineering of DNA, RNA, and proteins.