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A plasmid-based lacZα gene assay for DNA polymerase fidelity measurement

Lookup NU author(s): Brian Keith, Dr Stanislaw Jozwiakowski, Professor Bernard Connolly

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Abstract

A significantly improved DNA polymerase fidelity assay, based on a gapped plasmid containing the lacZ alpha reporter gene in a single-stranded region, is described. Nicking at two sites flanking lacZ alpha, and removing the excised strand by thermocycling in the presence of complementary competitor DNA, is used to generate the gap. Simple methods are presented for preparing the single-stranded competitor. The gapped plasmid can be purified, in high amounts and in a very pure state, using benzoylated-naphthoylated DEAE-cellulose, resulting in a low background mutation frequency (similar to 1 x 10(-4)). Two key parameters, the number of detectable sites and the expression frequency, necessary for measuring polymerase error rates have been determined. DNA polymerase fidelity is measured by gap filling in vitro, followed by transformation into Escherichia coil and scoring of blue/white colonies and converting the ratio to error rate. Several DNA polymerases have been used to fully validate this straightforward and highly sensitive system. (C) 2012 Elsevier Inc. All rights reserved.

Publication metadata

Author(s): Keith BJ, Jozwiakowski SK, Connolly BA

Publication type: Article

Publication status: Published

Journal: Analytical Biochemistry

Year: 2013

Volume: 433

Issue: 2

Pages: 153-161

Print publication date: 01/02/2013

ISSN (print): 0003-2697

ISSN (electronic): 1096-0309

Publisher: Elsevier

URL: http://dx.doi.org/10.1016/j.ab.2012.10.019

DOI: 10.1016/j.ab.2012.10.019

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