A_plasmid-based_lacZa_gene_assay_for_DNA_polymerase_fidelity_measurement.pdf (1.22 MB)
A plasmid-based lacZa gene assay for DNA polymerase fidelity measurement
journal contribution
posted on 2023-06-08, 13:39 authored by Brian J Keith, Stanislaw K Jozwiakowski, Bernard A ConnollyA 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 BiochemistryISSN
0003-2697Publisher
Elsevier MassonExternal DOI
Issue
2Volume
433Page range
153-161Department affiliated with
- Sussex Centre for Genome Damage Stability Publications
Full text available
- Yes
Peer reviewed?
- Yes
Legacy Posted Date
2013-11-05First Open Access (FOA) Date
2013-11-05First Compliant Deposit (FCD) Date
2013-11-05Usage metrics
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