Oligonucleotide-Directed Mutagenesis Using an Improved Phosphorothioate Approach

Oligonucleotide-directed in vitro mutagenesis is an established tool for the investigation of protein structure and function, and increasingly for studying the role of DNA sequences in gene expression. The method described here is based on the phosphorothioate technique developed by Eckstein’s group (1 ,2 ) that has been improved to make it quicker and more convenient (3 ). Selection for the desired mutation takes place in vitro, allowing a high efficiency of mutagenesis to be achieved. Typically, an efficiency of greater than 85% mutants can be obtained with a range of types of mutation.