A Multiplex SNaPshot Assay as a Rapid Method for Detecting KRAS and BRAF Mutations in Advanced Colorectal Cancers Sandrine Magnin, Erika Viel, Alice.

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A Multiplex SNaPshot Assay as a Rapid Method for Detecting KRAS and BRAF Mutations in Advanced Colorectal Cancers Sandrine Magnin, Erika Viel, Alice Baraquin, Severine Valmary-Degano, Bernadette Kantelip, Jean-Luc Pretet, Christiane Mougin, Marthe Bigand, Benoît Girardo, Christophe Borg, Christophe Ferrand The Journal of Molecular Diagnostics Volume 13, Issue 5, Pages 485-492 (September 2011) DOI: 10.1016/j.jmoldx.2011.05.010 Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Assay validation of SNaPshot genotyping. Each peak corresponds to a specific extended primer. Positions of nucleotides are indicated at the bottom of the figure. Arrows indicate the location of each mutation. Bases are represented by the following colors: A = green; C = black; G = blue; T = red. A–C, E, and F show the results obtained using DNA extracted from FFPE colorectal tumor tissue. D: Genotyping result obtained using DNA extracted from lung adenocarcinoma. G: An example of no-DNA negative control. A wild-type case (A) and cases harboring a p.G12R (B), a p.G12D (C), a p.G13C (D), and a p.G13D (E) mutation of the KRAS gene and a mutated case (p.V600E) of the BRAF gene (F) are shown. The Journal of Molecular Diagnostics 2011 13, 485-492DOI: (10.1016/j.jmoldx.2011.05.010) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Strategies used to compare time and cost per sample for the three methods used to detect KRAS and BRAF mutations. Strategy 1: The 110 DNA samples were submitted to SNaPshot after KRAS exon 2 and BRAF exon 15 amplification detecting KRAS and BRAF mutations simultaneously. Strategy 2: Sequencing analysis was performed on the 110 cases for KRAS mutation identification using the same multiplex PCR products as for the SNaPshot strategy. Strategy 3: The 110 DNA samples were used for HRM KRAS exon 2 genotyping. Only samples without KRAS mutation were tested by HRM for BRAF mutation detection. Each BRAF mutation was confirmed by direct sequencing. The Journal of Molecular Diagnostics 2011 13, 485-492DOI: (10.1016/j.jmoldx.2011.05.010) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 High-resolution melting analysis of BRAF exon 15 and KRAS exon 2. A: Difference plots of one colorectal cancer sample carrying the p.V600E mutation. BRAF mutated sample was normalized against wild-type DNA. Products of the positive control (HT29 cell line) and the mutated sample are shown in red curves. Products of wild-type templates are shown in blue. B: Difference plots showing the melting curve profiles of three cases revealing the presence of KRAS exon 2 mutation, namely p.G12D, p.G12V, and p.G13D. Mutated samples normalized against wild-type sample. Each sample was analyzed in triplicate. The Journal of Molecular Diagnostics 2011 13, 485-492DOI: (10.1016/j.jmoldx.2011.05.010) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions