A simple and economical site-directed mutagenesis method for large plasmids by direct transformation of two overlapping PCR fragments.

Abstract

Despite the development of various methods and commercial kits, site-directed mutagenesis of large plasmids remains a challenge in many laboratories. A site-directed mutagenesis method was developedfor large plasmids by directly transforming two overlapping PCR fragments intoEscherichia coli. This method successfully generated mutations for plasmids of 8.3kb and 11.0kb with high efficiencies. The method only requires Q5 DNA polymerase and DpnI, which greatly reduces costs. The procedureis simple, including PCR reaction, DpnI treatmentand transformation. This simple, efficient andeconomical site-directed mutagenesis method for large plasmids is likely to bewidely applied in the future.