Abstract 1980: Multiplex digital PCR combined with melting curve analysis for detecting KRAS and BRAF mutations

Background Digital PCR (dPCR) is a promising method for liquid biopsies and is able to quantify nucleic acids more sensitively than real-time PCR. However, dPCR has a large fluctuation in the fluorescence intensity of the droplets or wells due to insufficient PCR in the small partitions, limiting the multiplexing capability of using the fluorescence intensity. In this study, we propose a new measurement method that combines dPCR with melting curve analysis for highly multiplexed genotyping. Methods A sample is digitized into a silicon chip with up to ~104 wells in which asymmetric PCR was performed to obtain more single-stranded amplicons that are complimentary to molecular beacon probes. Fluorescence images were captured while controlling the temperature of the chip, and melting curve was measured for each well. Then, genotyping was performed by using the fluorescence intensity, dye color of the probe, and melting temperature (Tm). Because the Tm of the PCR products does not considerably depend on the amplification efficiency of PCR, genotyping accuracy is improved by using Tm values, enabling highly multiplexed genotyping. Results The concept was confirmed by measuring four common KRAS mutations (G12D, G12V, G12R, and G13D) and one common BRAF mutation (V600E). G12D, G12V and G12R-mutant KRAS alleles were detected with FAM-labeled probes, G13D-mutant KRAS allele and wildtype KRAS allele were detected with HEX-labeled probes, and V600E-mutant BRAF allele and wildtype BRAF allele were detected with CAL Fluor Red 610-labeled probes. The results showed that the peaks of the Tm distributions of the DNA groups were separated by more than 2°C. By combining the fluorescence intensities and the Tm values, the wildtype and mutant groups were clearly identified and able to be quantified. Conclusion We have proposed a new measurement method that combines dPCR with melting curve analysis, and successfully performed the multiplexed genotyping assay. To our knowledge, this is the first demonstration of genotyping of seven DNA groups with a single mutation of cancer-related genes by combining dPCR with melting curve analysis. Citation Format: Junko Tanaka, Tatsuo Nakagawa, Akiko Shiratori, Kunio Harada, Chihiro Uematsu, Erina Takai, Shinichi Yachida. Multiplex digital PCR combined with melting curve analysis for detecting KRAS and BRAF mutations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1980.