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

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.

authors

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

doi

10.1073/pnas.79.21.6409

subject

Has Abstract

pub_date

1982-11-01 00:00:00

pages

6409-13

issue

21

eissn

0027-8424

issn

1091-6490

journal_volume

79

pub_type

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