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Comparison of Sanger Sequencing, Pyrosequencing, and Melting Curve Analysis for the Detection of KRAS Mutations  Athanasios C. Tsiatis, Alexis Norris-Kirby,

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Presentation on theme: "Comparison of Sanger Sequencing, Pyrosequencing, and Melting Curve Analysis for the Detection of KRAS Mutations  Athanasios C. Tsiatis, Alexis Norris-Kirby,"— Presentation transcript:

1 Comparison of Sanger Sequencing, Pyrosequencing, and Melting Curve Analysis for the Detection of KRAS Mutations  Athanasios C. Tsiatis, Alexis Norris-Kirby, Roy G. Rich, Michael J. Hafez, Christopher D. Gocke, James R. Eshleman, Kathleen M. Murphy  The Journal of Molecular Diagnostics  Volume 12, Issue 4, Pages (July 2010) DOI: /jmoldx Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

2 Figure 1 Melting curve, Sanger sequencing, and pyrosequencing data from a wild-type KRAS specimen and mutant KRAS specimen (G12D) are shown. Arrows indicate the mutation. The Journal of Molecular Diagnostics  , DOI: ( /jmoldx ) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

3 Figure 2 Results of SS, MC, and PS for a G12F mutation. A: SS demonstrated G to T transversions at positions 1 and 2 of codon 12. Arrows indicate the mutation. B: MC showed a Tm significantly lower than expected for a single mutation. Arrows indicate the mutation. C: PS generated a pyrogram that was flagged as abnormal, but the pattern could not be definitively interpreted with regard to the specific mutation subtype. D: A hypothetical PS pyrogram that would have resulted had the G to T transversions at positions 1 and 2 of codon 12 been on separate alleles. The Journal of Molecular Diagnostics  , DOI: ( /jmoldx ) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

4 Figure 3 Dilution series data presented here include 100%, 20%, 10%, 5%, and 0% KRAS mutant cell mixtures and the corresponding predicted percentage of mutant alleles in each mixture. The dilutions representing the limit of detection for a KRAS mutation at codons 12/13 by SS, MC, and PS are designated by the arrow. The Journal of Molecular Diagnostics  , DOI: ( /jmoldx ) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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