SNaPshot and StripAssay as Valuable Alternatives to Direct Sequencing for KRAS Mutation Detection in Colon Cancer Routine Diagnostics Arantza Fariña.

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Presentation transcript:

SNaPshot and StripAssay as Valuable Alternatives to Direct Sequencing for KRAS Mutation Detection in Colon Cancer Routine Diagnostics Arantza Fariña Sarasqueta, Elna Moerland, Hanneke de Bruyne, Henk de Graaf, Tamara Vrancken, Gesina van Lijnschoten, Adriaan J.C. van den Brule The Journal of Molecular Diagnostics Volume 13, Issue 2, Pages 199-205 (March 2011) DOI: 10.1016/j.jmoldx.2010.10.006 Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Schematic representation of the SNaP shot technique. The primers have a determined length, are specific for the genomic region where the mutant is, and end at a nucleotide preceding the mutation. Subsequently, one fluorochrome-labeled dideoxynucleotide is added. Using capillary electrophoresis, products are separated according to size. Depending on the nucleotide build up after primer extension, either one or two of the fluorochromes will be detected depending on whether the patient is homozygous or heterozygous for the mutant allele, respectively. The Journal of Molecular Diagnostics 2011 13, 199-205DOI: (10.1016/j.jmoldx.2010.10.006) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 KRAS mutations present on StripAssay. 1: Wild type; 2: p.Gly12Ala; 3: p.Gly12Arg; 4: p.Gly12Asp; 5: p.Gly12Cys; 6: p.Gly12Ser; 7: p.Gly12Val; 8: p.Gly13Asp; and 9: p.Gly13Cys. p.Gly12Ile and p.Gly12Leu are not present in our series but are present on the StripAssay at position 5 and 6. p.Gly13Val and p.Gly13Arg are not present on the StripAssay. The Journal of Molecular Diagnostics 2011 13, 199-205DOI: (10.1016/j.jmoldx.2010.10.006) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Sensitivity comparison between SNaPshot and dideoxy sequencing. Sensitivity comparison between SNaPshot and dideoxy sequencing. Arrows show the mutation. The Journal of Molecular Diagnostics 2011 13, 199-205DOI: (10.1016/j.jmoldx.2010.10.006) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Sensitivity comparison between StripAssay (A) and SNaPshot (B) using a tumor DNA dilution series with a known mutation codon 12 Val. (1 = 80%, 2 = 40%, 3 = 20%, 4 = 10%, 5 = 5%, and 6 = 1% tumor cells). Sensitivity comparison between StripAssay and SNaPshot using a tumor DNA dilution series with a known mutation codon 12 Val. (1 = 80%, 2 = 40%, 3 = 20%, 4 = 10%, 5 = 5% and 6 = 1% tumor cells). Arrows show the mutation. The Journal of Molecular Diagnostics 2011 13, 199-205DOI: (10.1016/j.jmoldx.2010.10.006) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions