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Site-Directed Mutagenesis Using Altered /gb-Lactamase Specificity

  • Christine A. Andrews
  • Scott A. Lesley
Part of the Methods in Molecular Biology™ book series (MIMB, volume 182)

Abstract

Site-directed mutagenesis (SDM) is a powerful tool for the study of gene expression/regulation and protein structure and function. Hutchinson et al. (1) developed a general method for the introduction of specific changes in DNA sequence, which involves hybridization of a synthetic oligonucleotide (ON) containing the desired mutation to a single-stranded DNA (ssDNA) target template. Following hybridization, the oligonucleotide is extended with a DNA polymerase to create a double-stranded structure. The heteroduplex DNA is then transformed into an Escherichia coli, in which where both wild type and mutant strands are replicated. In the absence of any selection this method is very inefficient, often resulting in only a few percent of mutants obtained. Various strategies of selection have since been developed, which can increase mutagenesis efficiencies well above the theoretical yield of 50%. The methods of Kunkel (2), Eckstein (3), and Deng (4,5) employ negative selection against the wild-type DNA strand, in which the parental DNA is selectively degraded, either by growth in an alternate host strain, or by digestion with a nuclease or restriction enzyme. The methods of Lewis and Thompson (6) and Bonsack (7) utilize antibiotic resistance to positively select for the mutant DNA strand. This chapter describes a method for the positive selection of mutant strand DNA, which relies on the altered activity of the enzyme β-lactamase against extended spectrum cephalosporins (8).

Keywords

Isoamyl Alcohol Transformation Reaction JM109 Cell Annealing Reaction Extend Spectrum Cephalosporin 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Christine A. Andrews
    • 1
  • Scott A. Lesley
    • 2
  1. 1.Protein Expression/Enzymology, Research and DevelopmentPromega CorporationMadison
  2. 2.Protein Expression and AnalysisGenomics Institute of the Novartis Research FoundationSan Diego

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