Mutation Detection of Epidermal Growth Factor Receptor and KRAS Genes Using the Smart Amplification Process Version 2 from Formalin-Fixed, Paraffin-Embedded.

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Presentation transcript:

Mutation Detection of Epidermal Growth Factor Receptor and KRAS Genes Using the Smart Amplification Process Version 2 from Formalin-Fixed, Paraffin-Embedded Lung Cancer Tissue Yohei Miyamae, Kimihiro Shimizu, Yasumasa Mitani, Takuya Araki, Yuki Kawai, Masaru Baba, Seiichi Kakegawa, Masayuki Sugano, Kyoichi Kaira, Alexander Lezhava, Yoshihide Hayashizaki, Koujirou Yamamoto, Izumi Takeyoshi The Journal of Molecular Diagnostics Volume 12, Issue 2, Pages 257-264 (March 2010) DOI: 10.2353/jmoldx.2010.090105 Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 A: Amplification by the SmartAmp2 assay requires five unique primers: turn-back primer (TP), folding primer (FP), boost primer (BP), and two outer primers (OP1 and OP2). The genomic sequence between and including the TP and FP primers is the target region to be amplified in the SmartAmp2 assay. B: SmartAmp2 assay PNA clamp. The PNA clamp competitive probe is designed for the wild-type allele sequence. The greater stability of the PNA probe in hybridization inhibits SmartAmp2 amplification and suppresses wild-type allele amplification. Amplification of the mutant allele is not inhibited by PNA regardless of the point mutation (KRAS) or deletion (EGFR exon 19). The Journal of Molecular Diagnostics 2010 12, 257-264DOI: (10.2353/jmoldx.2010.090105) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 A typical SmartAmp2 assay result. The graphs represent amplification curves of the L858R mutation detection in wild-type and mutant templates. If the amplification started within 40 minutes, it was defined as a positive reaction. The Journal of Molecular Diagnostics 2010 12, 257-264DOI: (10.2353/jmoldx.2010.090105) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Electrophoresis results of samples 7, 23, and 33. The PCR products of DNA extracted from blood samples were used as a positive control. S, short; M, medium; L, long amplicon of each primer. For example, S lane of EGFR exon 19 represents the result of electrophoresis using the PCR product of the 192-bp amplicon of EGFR exon 19. The left lane is used as the 100-bp ladder marker. The Journal of Molecular Diagnostics 2010 12, 257-264DOI: (10.2353/jmoldx.2010.090105) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Microscopic examination of sample number 31. A typical case of a tumor with severe fibrotic changes. Most of the tumor had undergone fibrotic changes, and true tumor cells were found in only a small portion of the tumor, as indicated by the arrows. In such cases, mutations were difficult to detect by direct sequencing. The Journal of Molecular Diagnostics 2010 12, 257-264DOI: (10.2353/jmoldx.2010.090105) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions