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Oligonucleotide-Directed Mutagenesis as a General and Powerful Method for Studies of Protein Function

Dalbadie-McFarland, G. and Cohen, L. W. and Riggs, A. D. and Morin, C. and Itakura, K. and Richards, J. H. (1982) 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, 79 (21). pp. 6409-6413. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:DALpnas82

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Abstract

We have used oligonucleotide-directed mutagenesis to make a specific change in the ß -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.


Item Type:Article
Additional Information:Copyright © 1982 by the National Academy of Sciences Communicated by Norman Davidson, June 25, 1982 We acknowledge many helpful discussions with Norman Davidson, R. Bruce Wallace, and John Rossi. This work was supported by National Institutes of Health Grants GM16424, GM25825, and GM30395. K.I. and A.D.R. are members of the Cancer Research Center (CA16434) at the City of Hope Research Institute. C.M. 's stay was made possible by a grant from the North Atlantic Treaty Organization. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Subject Keywords:β-lactamase; plasmid pBR322; enzyme mechanisms; colony screening; protein structure-function
Record Number:CaltechAUTHORS:DALpnas82
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:DALpnas82
Alternative URL:http://www.pnas.org/cgi/content/abstract/79/21/6409
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:6988
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:04 Jan 2007
Last Modified:26 Dec 2012 09:27

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