Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function.

Abstract

Oligonucleotide-directed mutagenesis was used to make a specific change in the .beta.-lactamase (EC 3.5.2.6) (ampicillin resistance) gene of the plasmid pBR322. Evidence suggests that the active site for this enzyme may include a serine-threonine dyad (residues 70 and 71). By priming in vitro DNA synthesis with a chemically synthesized 16-base oligodeoxyribonucleotide, the Ser-Thr dyad was inverted to Thr-Ser and a mutant with an ampicillin-sensitive phenotype was generated. This double-mismatch method is relatively simple and also very general because detection of mutants is at the level of DNA and involves only colony hybridizatoin. Accordingly, the procedure can be applied to any DNA sequence and does not depend on the phenotype of the mutant.