A Do-it-Yourself Protocol for Making Desired Mutant Proteins

Abstract

The generation of a mutant protein is often crucial to the elucidation of the pathogenesis of a genetic disease. This requires site-directed mutagenesis of an expression vector containing the gene of interest. Current site-directed mutagenesis procedures are derivatives of either a restriction site elimination method or a PCR-based mutagenesis method, which involve a commercial kit manufactured by any one of a number of biotechnology companies. A new efficient economical site-directed mutagenesis procedure is described here to provide researchers an additional choice that does not to rely on a commercial kit. This novel method has been validated, and requires only basic molecular biological techniques and reagents available in most laboratories even in developing countries. The mutation efficiency of this new method is usually greater than 80%. In case several different mutants of a protein are required for elucidation of the protein structure-function relationships, this method has distinct advantages since multiple mutations can be derived from the initial single strand DNA template.