Abstract 2455: Single-color accurate and precise quantification of wild-type versus mutant allele using a novel integrated digital PCR platform

Abstract

In this study we present a single-step, one-color quantification of mutant versus wild-type allele using a novel integrated, clinic-optimized digital PCR (dPCR) platform. Allelic variants in DNA or RNA can be distinguished and quantified using Polymerase Chain Reaction (PCR) in concert with intelligent primer and probe design. Most commonly, a researcher or clinician will use unlabeled forward and reverse primers that bind to conserved regions outside of the allelic variation in addition to two differently labeled hydrolysis probes that bind at the region of allelic variation. Here we describe a novel method that leverages the precision of digital PCR (dPCR) technology and requires only one-color fluorescence utilizing probe-free, intercalating dye chemistry. The expense in developing or purchasing a complete PCR assay is normally embedded in the cost of the fluorescently labeled probe. The method described here eliminates the need for the fluorescently labeled probe, thereby reducing chemistry cost. Furthermore, technical complexity is reduced by requiring only one-color fluorescence. In our assay, we use a common unlabeled reverse primer that binds downstream of the allelic variation. Two unlabeled, allele-specific forward primers bind preferentially to either the wild-type or mutant allele at the site of allelic variation. The mutant forward primer contains a non-annealing tail (ie: stretch of the DNA primer that does not anneal to the initial DNA template) that will cause the ultimate mutant PCR product to be longer than the wild-type product. Digital PCR is characterized by dividing up a bulk PCR reaction volume into thousands of fluidically isolated partitions or droplets. In the presence of an intercalating dye, a longer amplicon will result in a higher endpoint fluorescence in that specific dPCR partition. As such, using our novel assay, dPCR partitions containing mutant versus wild-type amplicons can be distinguished by differing levels of end-point partition fluorescence. In this study we used the BRAF gene and the clinically relevant BRAF V600E mutation to benchmark this assay using a novel integrated one-step dPCR platform. This dPCR platform consists of a single, fully-integrated instrument along with a novel micro-injection molded plastic consumable providing a simple, single-step workflow. The instrument is dry and contamination-free, making it an attractive instrument for easy transition to the clinic. Parameters including thermal-cycling time and primer concentration were serially optimized for the novel integrated dPCR platform. This work highlights that we can accurately and precisely quantify samples containing varying ratios of wild-type versus V600E BRAF down to 0.1% mutant/wild-type ratio using one color of fluorescence and a novel integrated single-step walkaway dPCR workflow. Citation Format: Megan E. Dueck, Christina Wood-Bouwens, Andrew Zayac, Robert Lin, Steve Gallagher, Hanlee Ji, Paul Hung. Single-color accurate and precise quantification of wild-type versus mutant allele using a novel integrated digital PCR platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2455.