Nucleic Acids Research Advance Access published online on June 10, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp494
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Methods Online |
Plasmid-based lacZ
assay for DNA polymerase fidelity: application to archaeal family-B DNA polymerase
Institute of Cell and Molecular Biosciences (ICaMB), Newcastle University, Newcastle upon Tyne, NE2 4HH, UK
*To whom correspondence should be addressed. Tel: +44 0191 222 7371; Fax: +44 0191 222 7424; Email: b.a.connolly{at}ncl.ac.uk
Received March 19, 2009. Revised May 20, 2009. Accepted May 21, 2009.
The preparation of a gapped pUC18 derivative, containing the lacZ
reporter gene in the single-stranded region, is described. Gapping is achieved by flanking the lacZ 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 (
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.