Enhanced Ratio of Signals Enables Digital Mutation Scanning for Rare Allele Detection

Publication date: Available online 13 March 2015 Source:The Journal of Molecular Diagnostics Author(s): Elena Castellanos-Rizaldos , Cloud Paweletz , Chen Song , Geoffrey R. Oxnard , Harvey Mamon , Pasi A. Jänne , G. Mike Makrigiorgos The use of droplet digital PCR (ddPCR) for low-level DNA mutation detection in cancer, prenatal diagnosis, and infectious diseases is growing rapidly. However, although ddPCR has been implemented successfully for detection of rare mutations at pre-determined positions, no ddPCR adaptation for mutation scanning exists. Yet, frequently, clinically relevant mutations reside on multiple sequence positions in tumor suppressor genes or complex hotspot mutations in oncogenes. Here, we describe a combination of coamplification at lower denaturation temperature PCR (COLD-PCR) with ddPCR that enables digital mutation scanning within approximately 50-bp sections of a target amplicon. Two FAM/HEX-labeled hydrolysis probes matching the wild-type sequence are used during ddPCR. The ratio of FAM/HEX-positive droplets is constant when wild-type amplicons are amplified but deviates when mutations anywhere under the FAM or HEX probes are present. To enhance the change in FAM/HEX ratio, we employed COLD-PCR cycling conditions that enrich mutation-containing amplicons anywhere on the sequence. We validated COLD-ddPCR on multiple mutations in TP53 and in EGFR using serial mutation dilutions and cell-free circulating DNA samples, and demonstrate detection...

Source: The Journal of Molecular Diagnostics - March 13, 2015 Category: Pathology Source Type: research