KRAS and BRAF Mutation Analysis in Routine Molecular Diagnostics

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KRAS and BRAF Mutation Analysis in Routine Molecular Diagnostics Daniëlle A.M. Heideman, Irene Lurkin, Marije Doeleman, Egbert F. Smit, Henk M. Verheul, Gerrit A. Meijer, Peter J.F. Snijders, Erik Thunnissen, Ellen C. Zwarthoff The Journal of Molecular Diagnostics Volume 14, Issue 3, Pages 247-255 (May 2012) DOI: 10.1016/j.jmoldx.2012.01.011 Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Representative output files of evaluated assays. A: HRM (right panel, normalized and temperature-shifted difference plot), followed by sequencing (left panel). B: SNaPshot readout after multiplex PCR (multiplex mutation assay). C: SNaPshot readout after HRM, for a representative specimen with a KRAS mutation (c.35G>A; pG12D). The Journal of Molecular Diagnostics 2012 14, 247-255DOI: (10.1016/j.jmoldx.2012.01.011) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Single-nucleotide probe extension by the SNaPshot method. Diagram showing the mechanism of SNaPshot single-nucleotide probe extension for KRAS exon 2 using fluorescently labeled ddNTPs complementary to the target for a WT specimen (A), a specimen with two mutations in cis (c.34G>T and c.35G>T) using original SNaPshot probes (T30 and T36, Table 1) (B), and a specimen with two mutations in cis (c.34G>T and c.35G>T) using modified SNaPshot probes (T29 and T36, Table 1) (C). In B, T30 and T36 probes cannot bind the target at positions c.34 and c.35, respectively, because of the nucleotide substitutions and, accordingly, primer extension cannot be accomplished, resulting in nondetection of the mutations. In C, the T29 probe can fully bind the target and, accordingly, primer extension can be accomplished, resulting in detection of c.34G>T. Asterisk, nucleotide alteration; diamond, ddNTP incorporation. The Journal of Molecular Diagnostics 2012 14, 247-255DOI: (10.1016/j.jmoldx.2012.01.011) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Flowchart of laboratory working time and costs for the three methods used to detect KRAS and BRAF mutations. Strategy 1 indicates a multiplex mutation assay consisting of a multiplex PCR, a SNaPshot reaction, and an ABI run for all specimens; strategy 2, HRM-sequencing consisting of three HRM reactions per specimen and, depending on melting curve analysis, a subsequent cycle-sequencing reaction and ABI run for a subset of HRM PCR products; and strategy 3, HRM-SNaPshot consisting of three HRM reactions per specimen and, depending on melting curve analysis, a subsequent SNaPshot reaction and ABI run for a subset of HRM PCR products. The Journal of Molecular Diagnostics 2012 14, 247-255DOI: (10.1016/j.jmoldx.2012.01.011) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions