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Removal of polymerase-produced mutant sequences from PCR products.

Publication ,  Journal Article
Smith, J; Modrich, P
Published in: Proc Natl Acad Sci U S A
June 24, 1997

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.

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Published In

Proc Natl Acad Sci U S A

DOI

ISSN

0027-8424

Publication Date

June 24, 1997

Volume

94

Issue

13

Start / End Page

6847 / 6850

Location

United States

Related Subject Headings

  • Sequence Analysis, DNA
  • Polymerase Chain Reaction
  • Mutation
  • Lactose Factors
  • Escherichia coli
 

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Smith, J., & Modrich, P. (1997). Removal of polymerase-produced mutant sequences from PCR products. Proc Natl Acad Sci U S A, 94(13), 6847–6850. https://doi.org/10.1073/pnas.94.13.6847
Smith, J., and P. Modrich. “Removal of polymerase-produced mutant sequences from PCR products.Proc Natl Acad Sci U S A 94, no. 13 (June 24, 1997): 6847–50. https://doi.org/10.1073/pnas.94.13.6847.
Smith J, Modrich P. Removal of polymerase-produced mutant sequences from PCR products. Proc Natl Acad Sci U S A. 1997 Jun 24;94(13):6847–50.
Smith, J., and P. Modrich. “Removal of polymerase-produced mutant sequences from PCR products.Proc Natl Acad Sci U S A, vol. 94, no. 13, June 1997, pp. 6847–50. Pubmed, doi:10.1073/pnas.94.13.6847.
Smith J, Modrich P. Removal of polymerase-produced mutant sequences from PCR products. Proc Natl Acad Sci U S A. 1997 Jun 24;94(13):6847–6850.
Journal cover image

Published In

Proc Natl Acad Sci U S A

DOI

ISSN

0027-8424

Publication Date

June 24, 1997

Volume

94

Issue

13

Start / End Page

6847 / 6850

Location

United States

Related Subject Headings

  • Sequence Analysis, DNA
  • Polymerase Chain Reaction
  • Mutation
  • Lactose Factors
  • Escherichia coli