1. T-blocker: a simple and robust probe-free quantitative PCR assay to detect somatic mutations down to 0.1% frequency
- Author
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Hanyoup Kim, Aaron E Ruby, Harini G Shandilya, Arvind K Virmani, Nandita Rahman, Christina M Strange, and Jarkko Huuskonen
- Subjects
intercalating dye ,qPCR ,somatic mutation detection ,T-blocker ,Biology (General) ,QH301-705.5 - Abstract
We have developed a simple and robust probe-free quantitative PCR (qPCR) assay method that can detect minor mutant alleles with a frequency as low as 0.1% in a heterogeneous sample by introducing a novel T-blocker concept to the allele-specific PCR method. Four new KRAS and BRAF mutation detection assays were developed and their performance was demonstrated by testing a large number of replicates, utilizing a customized PCR protocol. Highly efficient and specific mutant amplification in conjunction with selective wild-type suppression by the T-blocker concept enabled 0.1% detection sensitivity using the intercalating dye-based qPCR chemistry instead of more complex target-specific dye-labeled probes. Excellent consistency in sensitivity and specificity of the T-blocker assay concept was demonstrated.
- Published
- 2018
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