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

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posted on 2023-06-08, 13:39 authored by Brian J Keith, Stanislaw K Jozwiakowski, Bernard A Connolly
A significantly improved DNA polymerase fidelity assay, based on a gapped plasmid containing the lacZa reporter gene in a single-stranded region, is described. Nicking at two sites flanking lacZa, 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.

History

Publication status

  • Published

File Version

  • Accepted version

Journal

Analytical Biochemistry

ISSN

0003-2697

Publisher

Elsevier Masson

Issue

2

Volume

433

Page range

153-161

Department affiliated with

  • Sussex Centre for Genome Damage Stability Publications

Full text available

  • Yes

Peer reviewed?

  • Yes

Legacy Posted Date

2013-11-05

First Open Access (FOA) Date

2013-11-05

First Compliant Deposit (FCD) Date

2013-11-05

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