Nucl._Acids_Res.-2009-Jozwiakowski-e102.pdf (552.56 kB)
Plasmid-based lacZalpha assay for DNA polymerase fidelity: application to archaeal family-B DNA polymerase
journal contribution
posted on 2023-06-08, 00:22 authored by Stanislaw Jozwiakowski, BA ConnollyThe preparation of a gapped pUC18 derivative, containing the lacZalpha reporter gene in the single-stranded region, is described. Gapping is achieved by flanking the lacZalpha gene with sites for two related nicking endonucleases, enabling the excision of either the coding or non-coding strand. However, the excised strand remains annealed to the plasmid through non-covalent Watson-Crick base-pairing; its removal, therefore, requires a heat-cool cycle in the presence of an exactly complementary competitor DNA. The gapped plasmids can be used to assess DNA polymerase fidelity using in vitro replication, followed by transformation into Escherichia coli and scoring the blue/white colony ratio. Results found with plasmids are similar to the well established method based on gapped M13, in terms of background ( approximately 0.08% in both cases) and the mutation frequencies observed with a number of DNA polymerases, providing validation for this straightforward and technically uncomplicated approach. Several error prone variants of the archaeal family-B DNA polymerase from Pyrococcus furiosus have been investigated, illuminating the potential of the method.
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- Published
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- Published version
Journal
Nucleic Acids ResearchISSN
0305-1048Publisher
Oxford University PressExternal DOI
Issue
15Volume
37Article number
e102Department affiliated with
- Sussex Centre for Genome Damage Stability Publications
Full text available
- Yes
Peer reviewed?
- Yes
Legacy Posted Date
2012-02-06First Open Access (FOA) Date
2016-03-22First Compliant Deposit (FCD) Date
2016-11-18Usage metrics
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