alexa Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications.
Genetics & Molecular Biology

Genetics & Molecular Biology

Advances in Molecular Diagnostics

Author(s): Tsiatis AC, NorrisKirby A, Rich RG, Hafez MJ, Gocke CD,

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Abstract Mutations in codons 12 and 13 of the KRAS oncogene are relatively common in colorectal and lung adenocarcinomas. Recent data indicate that these mutations result in resistance to anti-epidermal growth factor receptor therapy. Therefore, we assessed Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS codon 12/13 mutations in formalin-fixed paraffin-embedded samples, including 58 primary and 42 metastatic colorectal adenocarcinomas, 63 primary and 17 metastatic lung adenocarcinomas, and 20 normal colon samples. Of 180 tumor samples, 62.2\% were KRAS mutant positive, and 37.8\% were negative. Melting curve analysis yielded no false positive or false negative results, but had 10\% equivocal calls. Melting curve analysis also resulted in 4 cases with melting curves inconsistent with either wild-type or codon 12/13 mutations. These patterns were generated from samples with double mutants in codons 12/13 and with mutations outside of codons 12/13. Pyrosequencing yielded no false positive or false negative results as well. However, two samples from one patient yielded a pyrogram that was flagged as abnormal, but the mutation subtype could not be determined. Finally, using an electronic cutoff of 10\%, Sanger sequencing showed 11.1\% false positives and 6.1\% false negatives. In our hands, the limit of detection for Sanger sequencing, pyrosequencing, and melting curve analysis was approximately 15 to 20\%, 5\%, and 10\% mutant alleles, respectively.
This article was published in J Mol Diagn and referenced in Advances in Molecular Diagnostics

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