One-Step site-directed mutagenesis of ATM cDNA in large (20kb) plasmid constructs

Abstract

In vitro mutagenesis of large genes has proven to be difficult for a number of reasons, including the number of steps involved and the instability of large inserts. We describe here a single-step PCR method to introduce mutations into an ataxia-telangiectasia mutated (ATM) gene cDNA construct (20 kb). This involved several modifications of the Stratagene QuikChange Site-Directed Mutagenesis Kit. Four sites implicated in the function of ATM were mutagenised in a 20 kb plasmid, improving upon existing methods capable of mutagenesis in DNA constructs up to 13 kb, while maintaining a high efficiency of mutagenesis (85-100%). This approach makes it possible to introduce multiple mutations into large cDNA for structural/functional studies.