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Anal Biochem. 2013 Feb 15;433(2):153-61. doi: 10.1016/j.ab.2012.10.019. Epub 2012 Oct 22.

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

Author information

1
Institute of Cell and Molecular Biosciences (ICaMB), Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.

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.

PMID:
23098700
PMCID:
PMC3552156
DOI:
10.1016/j.ab.2012.10.019
[Indexed for MEDLINE]
Free PMC Article

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