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PMID: 6983070 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

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 ·Vol. 79 ·No. 21 ·1982-11-00 ·Pages 6409-13

Dalbadie-McFarland G, Cohen LW, Riggs AD, Morin C, Itakura K, Richards JH

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.

MeSH Terms
Amino Acid Sequence Ampicillin/pharmacology Base Sequence Genetic Engineering/methods Mutation Penicillin Resistance Plasmids Structure-Activity Relationship beta-Lactamases/genetics
Chemicals
Ampicillin beta-Lactamases
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Dalbadie-McFarland G
Cohen L W
Riggs A D
Morin C
Itakura K
Richards J H
References (34)
34 references, click to expand
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.79.21.6409
Published
1982-11-00
Pages
6409-13
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC347135
Subset
IM
Grants
NIGMS NIH HHS · GM16424 · United States
NIGMS NIH HHS · GM25825 · United States
NIGMS NIH HHS · GM30395 · United States
External Links
PubMed source DOI: 10.1073/pnas.79.21.6409 PMC Full Text View journal details
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