Abstract 579: Multiplex digital PCR combined with melting curve analysis for detecting KRAS mutations towards pancreatic cancer diagnosis

Abstract

Abstract 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 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 simultaneously identifying wild-type KRAS, BRAF and, eight mutants of these genes (G12D, G12R, G12V, G13D, G12A, G12C, G12S, and V600E) through four-color melting curve analysis. 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, G12S and G12C-mutant KRAS alleles were detected with Cy5-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 3.5 °C on average, while the standard deviation of the Tm distributions was 0.23 °C on average. The difference in Tm was 15 times larger than the standard deviation, enabling more accurate genotyping than when using the fluorescence intensity. Conclusion We have proposed a measurement method that combines dPCR with melting curve analysis, and successfully performed the multiplexed genotyping assay. We plan to evaluate the effect of this method on measurement using clinical specimens of pancreatic cancer patients in the future. Citation Format: Junko Tanaka, Tatsuo Nakagawa, Takeshi Ishida, Yoshio Kamura, Akiko Shiratori, Kunio Harada, Chihiro Uematsu. Multiplex digital PCR combined with melting curve analysis for detecting KRAS mutations towards pancreatic cancer diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 579.