Detection of Low-Level KRAS Mutations Using PNA-Mediated Asymmetric PCR Clamping and Melting Curve Analysis with Unlabeled Probes Ji Eun Oh, Hee Sun.

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Detection of Low-Level KRAS Mutations Using PNA-Mediated Asymmetric PCR Clamping and Melting Curve Analysis with Unlabeled Probes Ji Eun Oh, Hee Sun Lim, Chang Hyeok An, Eun Goo Jeong, Ji Youn Han, Sug Hyung Lee, Nam Jin Yoo The Journal of Molecular Diagnostics Volume 12, Issue 4, Pages 418-424 (July 2010) DOI: 10.2353/jmoldx.2010.090146 Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 PNA-mediated asymmetric PCR clamping system. A: Primer and probe positions. B: Point mutation: Both the sense mutant detection probe and antisense clamp probe are located on the mutation site of KRAS (c.35G>T). The sense mutant detection probes are designed to fail to bind to the antisense clamp probes because of the mismatches. C: Schematic presentation of the reactions to detect point mutations. In the amplification step, the clamp probe cannot bind to the mutation types but binds to the wild-type, resulting in preferential amplification of single-strand mutant sequences by an asymmetric PCR. In the detection step, the mutant probes bind to the single-strand mutant sequences. The signals are detected by a melting curve analysis. The Journal of Molecular Diagnostics 2010 12, 418-424DOI: (10.2353/jmoldx.2010.090146) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Melting curve assay under the nonclamping condition. Melting curve analysis with DCM2 detection probe was performed under nonclamping condition with 100 ng genomic DNA of SW480 cells with KRAS mutation and K562 cells without KRAS mutation by varying the ratio of the 2 types (A, 100% wild-type, 0% mutant; B, 70% wild-type, 30% mutant; C, 80% wild-type, 20% mutant; D, 90% wild-type, 10% mutant; E, 95% wild-type, 5% mutant). WT indicates wild-type melting peak; MT, mutant melting peak. The Journal of Molecular Diagnostics 2010 12, 418-424DOI: (10.2353/jmoldx.2010.090146) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Comparison of sensitivity of KRAS mutation detection between clamping and nonclamping conditions. Melting curve analysis with the DCM2 detection probe under nonclamping (blue line) and clamping (red line) conditions using 100-ng genomic DNAs, which consist of 100% wild-type DNA (A), 50% mutant DNA from SW480 cells (B), 1% mutant DNA from SW480 cells (C), and 0.1% mutant DNA from SW480 cells (D). WT indicates wild-type melting peak; MT, mutant melting peak. The Journal of Molecular Diagnostics 2010 12, 418-424DOI: (10.2353/jmoldx.2010.090146) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Application of the clamping assay to cell lines and paraffin-embedded tumor tissues with different KRAS mutations. Melting curve analysis (A–E) with the DCM2 detection probe under clamping condition using 100 ng genomic DNAs with varying ratio of the mutant DNA (100% wild-type DNA, 50% mutant DNA, 1% mutant DNA, and 0.1% mutant DNA). Melting curve analysis (F–H) under clamping and nonclamping conditions with paraffin-embedded tumor tissue DNA. SW480 cells (A), A549 cells (B), MIA PaCa-2 cells (C), LS174T cells (D), SW1116 cells (E), wild-type tumor tissue (F), c.35G>T mutant tumor tissue (G), and c.35G>A mutant tumor tissue (H). Mut indicates mutant DNA; MT, mutant melting peak; WT, wild-type melting peak. The Journal of Molecular Diagnostics 2010 12, 418-424DOI: (10.2353/jmoldx.2010.090146) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions