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Biotechniques. 1998 Jun;24(6):972-4, 976, 978 passim.

Selection strategy for site-directed mutagenesis based on altered beta-lactamase specificity.

Author information

1
Promega Corporation, Madison, WI 53711-5399, USA. candrews@promega.com

Abstract

Conventional approaches to oligonucleotide-directed mutagenesis rely upon the application of a selection strategy to maximize mutagenesis efficiencies. We have developed a mutagenesis procedure that incorporates a novel antibiotic resistance for selection. The selection involves altering the substrate specificity of TEM-1 beta-lactamase, the enzyme responsible for bacterial resistance to beta-lactam antibiotics such as ampicillin. The gene encoding beta-lactamase is commonly found on cloning and shuttle vectors used in molecular biology. Amino acid substitutions in several active site residues of beta-lactamase result in increased hydrolytic activity against extended-spectrum penicillins and cephalosporins. This increased activity confers a novel resistance specific to the mutant and thus provides the basis of the selection strategy. We describe a simple and efficient mutagenesis procedure and its application to creating a range of oligonucleotide-directed mutants.

PMID:
9631188
DOI:
10.2144/98246st03
[Indexed for MEDLINE]
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