Multiple-site fragment deletion, insertion and substitution mutagenesis by modified overlap extension PCR

Introducing various mutations at multiple specific sites within a gene requires multiple steps of DNA manipulation, which is the initial, but limiting step of protein structure–function studies. In the present work, we standardized a simple and fast procedure to perform site-directed mutagenesis, multiple-site fragment deletion, insertion and substitution mutagenesis by a modified version of overlap extension polymerase chain reaction (PCR). In this procedure, target genes divided into several fragments based on the site of mutagenesis are amplified and annealed with their complementary overhanging, followed by extension and amplification to full-length gene with expected mutation(s) by PCR. Vectors inserted with the modified target gene are screened by colony PCR. By using the standardized procedure, we have easily generated single-site mutations, replaced/deleted DNA fragment into/from a target gene and engineered a cysteine-free protein. Practically, the standardized procedure provides an efficient choice for almost all kinds of mutagenesis, especially for multiple-site and large DNA fragment modification mutagenesis. Therefore, this method can be utilized to analyze protein structure and function, to optimize codons of genes for protein expression and to assemble genes of interest