ePrints

Plasmid-based lacZ alpha assay for DNA polymerase fidelity: application to archaeal family-B DNA polymerase

Lookup NU author(s): Dr Stanislaw Jozwiakowski, Professor Bernard Connolly

Downloads

• Published version [.pdf]

Abstract

The preparation of a gapped pUC18 derivative, containing the lacZ alpha reporter gene in the single-stranded region, is described. Gapping is achieved by flanking the lacZ alpha 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 (similar to 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.

Publication metadata

Author(s): Jozwiakowski SK, Connolly BA

Publication type: Article

Publication status: Published

Journal: Nucleic Acids Research

Year: 2009

Volume: 37

Issue: 15

Date deposited: 06/04/2010

ISSN (print): 0305-1048

ISSN (electronic):

Publisher: Oxford University Press

URL: http://dx.doi.org/10.1093/nar/gkp494

DOI: 10.1093/nar/gkp494

Altmetrics

Funding

Share