Published online before print June 13, 2008

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Journal of Molecular Diagnostics 2008, Vol. 10, No. 4
Copyright © 2008 American Society for Investigative Pathology & Association for Molecular Pathology
DOI: 10.2353/jmoldx.2008.070183

Technical Advances

High-Fidelity DNA Polymerase Enhances the Sensitivity of a Peptide Nucleic Acid Clamp PCR Assay for K-ras Mutations

Bjørnar Gilje^*, Reino Heikkilä^*, Satu Oltedal^*, Kjersti Tjensvoll^* and Oddmund Nordgård^*

From the Laboratory for Molecular Biology, * Department of Hematology and Oncology; and the Department of Mathematics and Natural Sciences, University of Stavanger, Stavanger, Norway

Abstract

Sensitive 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.