PMID: 6983070Nov 1, 1982Paper

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

Proceedings of the National Academy of Sciences of the United States of America
G Dalbadie-McFarlandJ H Richards

Abstract

We have used oligonucleotide-directed mutagenesis 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, we have inverted the Ser-Thr dyad to Thr-Ser and thereby generated a mutant with an ampicillin-sensitive phenotype. 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 hybridization. Accordingly, the procedure can be applied to any DNA sequence and does not depend on the phenotype of the mutant.

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Citations

Dec 16, 2004·Angewandte Chemie·Lei Wang, Peter G Schultz
Sep 1, 1987·Molecular & General Genetics : MGG·G SchmitzG F Ames
Oct 1, 1983·Applied Biochemistry and Biotechnology·W H Rastetter
Oct 1, 1988·Applied Biochemistry and Biotechnology·G Tripathi
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Jan 1, 1987·Gene·K Foss, W H McClain
Feb 1, 1991·Epidemiology and Infection·P PohlM Couturier
Dec 1, 1982·Proceedings of the National Academy of Sciences of the United States of America·I S SigalR Arentzen
Nov 1, 1983·Proceedings of the National Academy of Sciences of the United States of America·J C WilliamsG Feher
Apr 1, 1984·Proceedings of the National Academy of Sciences of the United States of America·K M LoH G Khorana
Jun 1, 1985·Proceedings of the National Academy of Sciences of the United States of America·M BodnerI Gozes
Jun 1, 1985·Proceedings of the National Academy of Sciences of the United States of America·F Barany
Apr 1, 1985·Proceedings of the National Academy of Sciences of the United States of America·Y TakahashiK Matsubara
Apr 1, 1985·Proceedings of the National Academy of Sciences of the United States of America·D StrausW Gilbert
Jun 1, 1986·Proceedings of the National Academy of Sciences of the United States of America·A Warshel, F Sussman
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Mar 1, 1986·Proceedings of the National Academy of Sciences of the United States of America·S C Schultz, J H Richards
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Jan 1, 1984·Biotechnology & Genetic Engineering Reviews·S L Neidleman
Jan 1, 1985·Biotechnology & Genetic Engineering Reviews·A Coulson
Jan 1, 1990·DNA and Cell Biology·A V MazinR I Salganik

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