High-fidelity DNA polymerase enhances the sensitivity of a peptide nucleic acid clamp PCR assay for K-ras mutations.

2.50
Hdl Handle:
http://hdl.handle.net/10143/77384
Title:
High-fidelity DNA polymerase enhances the sensitivity of a peptide nucleic acid clamp PCR assay for K-ras mutations.
Authors:
Gilje, Bjørnar; Heikkilä, Reino; Oltedal, Satu; Tjensvoll, Kjersti; Nordgård, Oddmund
Citation:
The Journal of molecular diagnostics : JMD 2008, 10 (4):325-31

Full metadata record

DC FieldValue Language
dc.contributor.authorGilje, Bjørnar-
dc.contributor.authorHeikkilä, Reino-
dc.contributor.authorOltedal, Satu-
dc.contributor.authorTjensvoll, Kjersti-
dc.contributor.authorNordgård, Oddmund-
dc.date.accessioned2009-08-14T11:55:01Z-
dc.date.available2009-08-14T11:55:01Z-
dc.date.issued2009-08-14T11:55:01Z-
dc.identifier.citationThe Journal of molecular diagnostics : JMD 2008, 10 (4):325-31en
dc.identifier.issn1525-1578-
dc.identifier.pmid18556764-
dc.identifier.doi10.2353/jmoldx.2008.070183-
dc.identifier.urihttp://hdl.handle.net/10143/77384-
dc.description.abstractSensitive detection of tumor-specific point mutations is of interest in both the early detection of cancer and the monitoring of treatment at a molecular level. Recently, peptide nucleic acid (PNA) clamp real-time PCR has provided a time-sparing and sensitive method for the detection of mutations in the presence of a large excess of wild-type DNA. We present the first report that the sensitivity of PNA clamp PCR is limited by the low fidelity of TaqDNA polymerase. Replication errors introduced by Taq polymerase in the PNA-binding site were amplified during PCR due to the resulting mismatches between PNA and DNA. To reduce the frequency of polymerase-induced errors, we developed a PNA clamp PCR assay for the detection of mutations in codons 12 and 13 of the K-ras gene based on a high-fidelity DNA polymerase. The sensitivity of our assay increased approximately 10-fold, significantly detecting mutant DNA diluted 20,000-fold in wild-type DNA (P = 0.025), compared with its detection at 2000-fold dilution (P = 0.039) when Taq polymerase was used. Our data suggest that the replication errors caused by Taq polymerase must be taken into consideration for PNA clamp PCR and for other methods based on selective PCR amplification, and that these assays can be enhanced by high-fidelity DNA polymerases.en
dc.language.isoenen
dc.subjectVDP::Medisinske Fag: 700::Basale medisinske, odontologiske og veterinærmedisinske fag: 710::Medisinsk genetikk: 714en
dc.subjectVDP::Medisinske Fag: 700::Basale medisinske, odontologiske og veterinærmedisinske fag: 710::Generell patologi, patologisk anatomi: 719en
dc.subject.meshBase Sequenceen
dc.subject.meshCell Line, Tumoren
dc.subject.meshDNA Mutational Analysisen
dc.subject.meshDNA-Directed DNA Polymeraseen
dc.subject.meshGenes, rasen
dc.subject.meshHT29 Cellsen
dc.subject.meshHumansen
dc.subject.meshModels, Geneticen
dc.subject.meshMutationen
dc.subject.meshPeptide Nucleic Acidsen
dc.subject.meshPolymerase Chain Reactionen
dc.subject.meshReproducibility of Resultsen
dc.subject.meshTaq Polymeraseen
dc.titleHigh-fidelity DNA polymerase enhances the sensitivity of a peptide nucleic acid clamp PCR assay for K-ras mutations.en
dc.typeJournal articleen
dc.typepeer revieweden
dc.contributor.departmentDepartment of Hematology and Oncology, Stavanger University Hospital, Stavanger, Norway. gibj@sus.noen
dc.identifier.journalThe Journal of molecular diagnostics : JMDen
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