Removal of polymerase-produced mutant sequences from PCR products.

Published

Journal Article

Heteroduplex DNA lacking d(GATC) methylation is subject to mismatch-provoked double-strand cleavage at d(GATC) sites in a reaction dependent on MutH, MutL, MutS, and ATP. We have exploited this reaction to develop a method for removal of polymerase-produced mutant sequences that arise during sequence amplification by PCR. After denaturation and reannealing, the PCR product pool is subjected to MutH, MutL, and MutS mismatch repair proteins under double-strand cleavage conditions, followed by isolation of uncleaved product by size selection. Use of an Escherichia coli lac forward mutation assay has shown that this procedure reduces the incidence of polymerase-induced mutant sequences by an order of magnitude. Twenty mutants that originated from three independent PCR amplification reactions and survived MutHLS treatment all were found to contain an infrequently occurring A.T --> T.A transversion mutation at a unique position within the product. By contrast, the majority of mutations in untreated PCR products were transitions occurring throughout the amplified region, although frameshifts and transversions also were observed. The MutHLS method thus can be used to effectively remove the majority of mutant sequences produced by polymerase errors during PCR amplification.

Full Text

Duke Authors

Cited Authors

  • Smith, J; Modrich, P

Published Date

  • June 24, 1997

Published In

Volume / Issue

  • 94 / 13

Start / End Page

  • 6847 - 6850

PubMed ID

  • 9192654

Pubmed Central ID

  • 9192654

International Standard Serial Number (ISSN)

  • 0027-8424

Digital Object Identifier (DOI)

  • 10.1073/pnas.94.13.6847

Language

  • eng

Conference Location

  • United States