High-Fidelity DNA Polymerase Enhances the Sensitivity of a Peptide Nucleic Acid Clamp PCR Assay for K-ras Mutations Bjørnar Gilje, Reino Heikkilä, Satu.

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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, Oddmund Nordgård The Journal of Molecular Diagnostics Volume 10, Issue 4, Pages 325-331 (July 2008) DOI: 10.2353/jmoldx.2008.070183 Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Melting curve analysis of PCR products amplified by a PNA clamp PCR assay adapted from Dabritz et al.17 Melting curves for PCR products generated from LS174T (heterozygous codon 12 mutant) DNA, wt DNA template (isolated from normal peripheral blood), and no template (NTC) in the absence (−PNA) or presence (+PNA) of PNA are shown. Mutant (mut.)- and wt-specific peaks are indicated. The Journal of Molecular Diagnostics 2008 10, 325-331DOI: (10.2353/jmoldx.2008.070183) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Schematic pictorial of our PNA clamp assay. In our design, the primer and the PNA bind competitively to part of the same sequence. With wild-type K-ras as template, the PNA binds to DNA and blocks primer annealing and elongation (A). When a mutation is present, there is a mismatch between PNA and DNA leading to much weaker binding, allowing the primer to bind DNA, and elongation can take place (B). The Journal of Molecular Diagnostics 2008 10, 325-331DOI: (10.2353/jmoldx.2008.070183) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Sensitivity of the three PNA clamp PCR assays for detection of K-ras mutations in large excess of wild-type DNA. Mean ΔCt values from four independent dilution series of DNA from the colon carcinoma cell line LS174T (K-ras codon 12 mutation) in DNA from the colon carcinoma cell line HT29 (wild-type K-ras) are plotted. The error bars show 95% confidence intervals. The ΔCt values were calculated as ΔCt = Ct+PNA − Ct−PNA, where Ct+PNA and Ct−PNA denote the Ct values for the same reactions with and without PNA, respectively. The Journal of Molecular Diagnostics 2008 10, 325-331DOI: (10.2353/jmoldx.2008.070183) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Sensitivity of the Phusion HS-based PNA clamp PCR assay for colon carcinoma cells in large excess of normal lymphocytes. Mean ΔCt values from three independent dilution series of LS174T cells (colon carcinoma cell line with codon 12 mutation) in normal lymphocytes (L) are plotted. The error bars show 95% confidence intervals. The Journal of Molecular Diagnostics 2008 10, 325-331DOI: (10.2353/jmoldx.2008.070183) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 Sequencing of PCR products amplified from wild-type template by Taq-based PNA clamp PCR. A: Chromatograms showing the sequencing of a representative PCR product amplified from wild-type template with PNA present (bottom) compared with a PCR product generated from wild-type template without PNA (top). The DNA sequences are shown above the chromatograms, with codons 12 and 13 underlined. B: The DNA sequence of the PNA-binding site in 30 PCR products amplified from wild-type DNA by the Platinum Taq and HotGoldStar PNA clamp PCR assays, compared with the wild-type K-ras sequence. Only the introduced mutations are shown explicitly. Peaks higher than 10% of the mean peak height of the wild-type bases in the sequencing chromatograms were judged as introduced mutations. The Journal of Molecular Diagnostics 2008 10, 325-331DOI: (10.2353/jmoldx.2008.070183) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions