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

Keith, Brian J, Jozwiakowski, Stanislaw K and Connolly, Bernard A (2013) A plasmid-based lacZα gene assay for DNA polymerase fidelity measurement. Analytical Biochemistry, 433 (2). pp. 153-161. ISSN 0003-2697

[img]
Preview
PDF - Accepted Version
Download (1MB) | Preview

Abstract

A significantly improved DNA polymerase fidelity assay, based on a gapped plasmid containing the lacZα reporter gene in a single-stranded region, is described. Nicking at two sites flanking lacZα, 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 (~1 × 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 coli 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.

Item Type: Article
Schools and Departments: School of Life Sciences > Sussex Centre for Genome Damage and Stability
Subjects: Q Science > QD Chemistry > QD0241 Organic chemistry > QD0415 Biochemistry
Depositing User: Stanislaw Jozwiakowski
Date Deposited: 05 Nov 2013 13:11
Last Modified: 14 Mar 2017 02:22
URI: http://sro.sussex.ac.uk/id/eprint/42462

View download statistics for this item

📧 Request an update