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Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines
Published on: October 8, 2015
Mutation detection of epidermal growth factor receptor and KRAS genes using the smart amplification process version 2
Yohei Miyamae1, Kimihiro Shimizu, Yasumasa Mitani
1Department of Thoracic and Visceral Organ Surgery, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi 371-8511, Japan.
Abstract:
Recent evidence indicates that the presence of epidermal growth factor receptor (EGFR) or KRAS mutations in non-small cell lung cancer (NSCLC) can predict the response of the tumor to gefinitib. However, it is difficult to detect these mutations using formalin-fixed, paraffin-embedded (FFPE) tissues because the fixation process and aging can damage the DNA. In this study, we describe our work in adapting the Smart Amplification Process version 2 (SmartAmp2) to detect EGFR or KRAS mutations in DNA extracted from FFPE tissues. We were able to detect these mutations in 37 (97%) of 38 FFPE lung cancer tissue samples within 60 minutes with the SmartAmp2 assay and to confirm the correlation between EGFR mutations in FFPE tissues and gefitinib responsiveness. All mutations had previously been confirmed in the 38 samples using DNA extracted from frozen tissues. Electrophoresis results indicated that PCR analysis was not reliable for DNA extracted from FFPE tissue when primers with a long amplicon (>300 bp) were used. This study confirms that the SmartAmp2 assay is suitable for use with DNA extracted from FFPE as well as frozen tissues.
