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Lookup NU author(s): Dr Benjamin Biles, Professor Bernard Connolly
Random mutagenesis constitutes an important approach for identifying critical regions of proteins, studying structure-function relations and developing novel proteins with desired properties. Perhaps, the most popular method is the error-prone PCR, in which mistakes are introduced into a gene, and hence a protein, during DNA polymerase-catalysed amplification cycles. Unfortunately, the relatively high fidelities of the thermostable DNA polymerases commonly used for PCR result in too few mistakes in the amplified DNA for efficient mutagenesis. In this paper, we describe mutants of the family B DNA polymerase from Pyrococcus furiosus (Pfu-Pol), with superb performance in error-prone PCR. The key amino acid changes occur in a short loop linking two long alpha-helices that comprise the 'fingers' sub-domain of the protein. This region is responsible for binding the incoming dNTPs and ensuring that only correct bases are inserted opposite the complementary base in the template strand. Mutations in the short loop, when combined with an additional mutation that abolishes the 3'-5' proof-reading exonuclease activity, convert the extremely accurate wild-type polymerase into a variant with low fidelity. The mutant Pfu-Pols can be applied in error-prone PCR, under exactly the same conditions used for standard, high-fidelity PCR with the wild-type enzyme. Large quantities of amplified product, with a high frequency of nearly indiscriminate mutations, are produced. It is anticipated that the Pfu-Pol variants will be extremely useful for the randomization of gene, and hence protein, sequences.
Author(s): Biles BD, Connolly BA
Publication type: Article
Publication status: Published
Journal: Nucleic Acids Research
Year: 2004
Volume: 32
Issue: 22
Print publication date: 01/01/2004
Date deposited: 07/02/2012
ISSN (print): 0305-1048
ISSN (electronic): 1362-4962
Publisher: Oxford University Press
URL: http://dx.doi.org/10.1093/nar/gnh174
DOI: 10.1093/nar/gnh174
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