The effect of replication errors on the mismatch analysis of PCR-amplified DNA

Reiss, Jochen; Krawczak, Michael; Schloesser, Manfred; Wagner, Michael J.; Cooper, David Neil

doi:10.1093/nar/18.4.973

Cited by 42 publications

(27 citation statements)

References 11 publications

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“…However, such errors can compromise genetic diagnostic methods based on mismatch detection with amplified DNA (8,9) and render results obtained with cloned PCR products problematic (10), with the severity of this problem increasing with DNA chain length and the number of doublings. By permitting removal of most mutant sequences generated during amplification, the method described here should prove useful in circumventing these difficulties.…”

Section: Discussionmentioning

confidence: 99%

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Removal of polymerase-produced mutant sequences from PCR products

Smith

Modrich

1997

Proc. Natl. Acad. Sci. U.S.A.

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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 A⅐T 3 T⅐A 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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Section: Discussionmentioning

confidence: 99%

“…However, inasmuch as the mutant frequency is a linear function of the size of the DNA segment, mutations produced by the latter polymerases become a problem when larger sequences are amplified. The significance of polymerase errors for PCR applications in genetic diagnostics and cDNA cloning has been documented in the literature (8)(9)(10).…”

mentioning

confidence: 99%

Removal of polymerase-produced mutant sequences from PCR products

Smith

Modrich

1997

Proc. Natl. Acad. Sci. U.S.A.

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“…This is not unexpected, because the Taq polymerase used i:l the PCR reaction lacks a proofreading exonuclease activity, and therefore exhibits a much higher nucleotide mis-incorporation rate (about 1/ 10000) than the Klenow polymerase (about 10-7 ) (Tindall and Kunkel 1988). From the theoretical study of Reiss et al (1990), it can be assumed that, after 50 cycles, each PCR copy of the parent 1339-bp cDNA would probably contain one or more PCR errors, which will be present in individual clones.…”

Section: Discussionmentioning

confidence: 99%

Molecular characterization of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: identification of a lys329 to glu mutation in the MCAD gene, and expression of inactive mutant enzyme protein in E. coli

et al. 1991

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Summary. A series of experiments has established the molecular defect in the medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) gene in a family with MCAD deficiency. Demonstration of intra-mitochon drial mature MCAD indistinguishable in size (42.5-kDa) from control MCAD, and of mRNA with the correct size of 2.4 kb, indicated a point-mutation in the coding region of the MCAD gene to be disease-causing. Con sequently, cloning and DNA sequencing of polymerase chain reaction (PCR) amplified complementary DNA (cDNA) from messenger RNA of fibroblasts from the patient and family members were performed. All clones sequenced from the patient exhibited a single base sub _titution from adenine (A) to guanine (G) at position ~85 in the MCAD cDNA as the only consistent base-var iation compared with control cDNA. In contrast, the parents contained cDNA with the normal and the mu tated sequence, revealing their obligate carrier status. Allelic homozygosity in the patient and heterozygosity for the mutation in the parents were established by a modified PCR reaction, introducing a cleavage site for the restriction endonuclease NcoI into amplified geno mic DNA containing G 985 . The same assay consistently revealed A 985 in genomic DNA from 26 control individu als. The A to G mutation was introduced into an E. coli expression vector producing mutant MCAD, which was demonstrated to be inactive, probably because of the in ability to form active tetrameric MCAD. All the experi ments are consistent with the contention that the G 985 mutation, resulting in a lysine to glutamate shift at posi tion 329 in the MCAD polypeptide chain, is the genetic cause of MCAD deficiency in this family. We found the Offprint requests to: N. Gregersen.

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“…Base substitutions are verified by DNA sequencing. Assuming an error rate of the Pfu DNA polymerase of 1.3 ϫ 10 Ϫ6 (Cline et al, 1996), twenty cycles in the first PCR would result in a fraction of 1.3 ϫ 10 Ϫ5 alleles in which a base is substituted at a defined position (Reiss et al, 1990). The subtraction of wild-type alleles reduces the number of fragments with polymerase errors in the subsequent PCR reactions.…”

Section: Discussionmentioning

confidence: 99%

Enrichment of Mutant KRAS Alleles in Pancreatic Juice by Subtractive Iterative Polymerase Chain Reaction

Fischer

Büthe

Nollau

et al. 2001

Laboratory Investigation

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SUMMARY:The detection of mutant tumor genes holds great promise for an early diagnosis of primary tumors and residual malignant disease. When few tumor cells are present with an excess of nonmalignant cells of the same lineage, the excess of wild-type alleles over mutant tumor alleles presents an analytical problem. The subtractive iterative PCR (siPCR) assay presents a new approach to solving this problem. To achieve an enrichment of mutant alleles, wild-type alleles are removed by differential hybridization to complementary oligonucleotides spanning the region of the gene in which point mutations are expected. The nonbound fraction is reamplified by PCR. By iterating this process, mutant alleles can be detected in the presence of an excess of wild-type alleles with high sensitivity. To prove the feasibility of siPCR, pancreatic juice samples were analyzed for KRAS mutations. Pancreatic juice obtained from patients with pancreatic carcinoma or chronic pancreatitis during endoscopic retrograde cholangiopancreatography was analyzed for point mutations in codons 12 and 13 of the KRAS gene. In each of six samples from tumor patients, mutations in codon 12 were detected. One of nine samples from patients with chronic pancreatitis scored positive. (Lab Invest 2001, 81:827-831).

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The effect of replication errors on the mismatch analysis of PCR-amplified DNA

Cited by 42 publications

References 11 publications

Removal of polymerase-produced mutant sequences from PCR products

Removal of polymerase-produced mutant sequences from PCR products

Molecular characterization of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: identification of a lys329 to glu mutation in the MCAD gene, and expression of inactive mutant enzyme protein in E. coli

Enrichment of Mutant KRAS Alleles in Pancreatic Juice by Subtractive Iterative Polymerase Chain Reaction

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