Sensitive Detection of KRAS Mutations in Archived Formalin-Fixed Paraffin-Embedded Tissue Using Mutant-Enriched PCR and Reverse-Hybridization Christoph.

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Presentation transcript:

Sensitive Detection of KRAS Mutations in Archived Formalin-Fixed Paraffin-Embedded Tissue Using Mutant-Enriched PCR and Reverse-Hybridization Christoph Ausch, Veronika Buxhofer-Ausch, Christian Oberkanins, Barbara Holzer, Michael Minai-Pour, Stephan Jahn, Nadia Dandachi, Robert Zeillinger, Gernot Kriegshäuser The Journal of Molecular Diagnostics Volume 11, Issue 6, Pages 508-513 (November 2009) DOI: 10.2353/jmoldx.2009.090022 Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Schematic pictorial of our RH protocol. After mutant-enriched duplex PCR, RH of biotinylated amplification products to test strips was carried out manually (left) or, for increased sample throughput, essentially automated using commercially available instrumentation (right). The Journal of Molecular Diagnostics 2009 11, 508-513DOI: (10.2353/jmoldx.2009.090022) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Identification of KRAS mutations by RH. Test trips obtained after hybridization and enzymatic color development are shown. Strips 1–10: Mutant-enriched PCR was perfomed using a mix of 50 ng wild-type and 0.5 ng genomic DNA derived from tumor cell lines (strips 1, 2, 4, 5, 7–10) and mutant PCR product generated by site-directed mutagenesis (strips 3, 6), respectively. Strips 11–18: Mutant-enriched PCR was performed on genomic DNA isolated from selected FFPE tissue samples (strips 11–16). The following genotypes were identified: Val12 (strip 11), Cys12/Cys13 (strip 12), Asp13 (strip 13), Asp12 (strip 14), Cys12 (strip 15), Ala12 (strip 16), 50 ng Colo320 (strip 17), and negative PCR control (strip 18). The Journal of Molecular Diagnostics 2009 11, 508-513DOI: (10.2353/jmoldx.2009.090022) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions