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. 2018 Oct;65(4):205-210.
doi: 10.2144/btn-2018-0111.

T-blocker: a simple and robust probe-free quantitative PCR assay to detect somatic mutations down to 0.1% frequency

Hanyoup Kim  1 , Aaron E Ruby  1 , Harini G Shandilya  1 , Arvind K Virmani  1 , Nandita Rahman  1 , Christina M Strange  1 , Jarkko Huuskonen  1
Affiliations
Free article

T-blocker: a simple and robust probe-free quantitative PCR assay to detect somatic mutations down to 0.1% frequency

Hanyoup Kim et al. Biotechniques. 2018 Oct.
Free article

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.

Keywords: T-blocker; intercalating dye; qPCR; somatic mutation detection.

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