Abstract 1574: KRAS genotyping by digital PCR combining melting curve analysis
Authors: Tanaka Junko , Shimazaki Yuzuru , Nakagawa Tatsuo , Shiratori Akiko , Kamahori Masao , Yokoi Takahide , Harada Kunio , Kohara Yoshinobu 
Published: August 2018

Abstract
Background
ctDNA is a remarkable liquid biopsy for cancer diagnosis. Highly sensitive quantification method is required to measure a tiny amount of ctDNA. Digital PCR has been developed as a method that can quantify nucleic acids more sensitively than real-time PCR does. However, the digital PCR has large fluctuation in the fluorescence intensity of the droplets or chambers resulting in lower accuracy. Main cause is probably due to the insufficient PCR in the small partitions. In this study, we have proposed a new measurement method combined a digital PCR with melting curve analysis using molecular beacons to solve above mentioned problems, and applied it to KRAS genotyping.
Methods
Molecular beacons, which have hydrophobic moiety in the stem, were designed for detecting KRAS mutation. The digital PCR with combination of asymmetric PCR was performed using the molecular beacons in the droplets. After the PCR, the fluorescence of the droplets was observed with a microscope while changing the temperature. A melting curve was prepared from the change in fluorescence intensity of the droplet, and the melting temperature (Tm) was calculated from the differential melting curve.
Results
The melting curve analysis for the KRAS mutation was performed in the droplets where the asymmetric PCR was performed using molecular beacons with hydrophobic stem, which improved signal-to-noise ratio of melting curves. The use of molecular beacons with hydrophobic stem can keep a background fluorescence at a constant value even at high temperature. The change in fluorescence intensity of PCR solution using molecular beacons with hydrophobic stem during the measurement was one-tenth of that using molecular beacon without hydrophobic stem. The asymmetric PCR enabled us to increase the amount of the PCR products hybridized with molecular beacons, resulting in the increase in the fluorescence intensity. The KRAS genotyping of wild-type (WT) and G12D mutant was conducted by the melting curve analysis with a combination of the asymmetric PCR with molecular beacons. The results showed that the peaks of the distributions of the Tm values of DNA in the droplets were 77.9°C for WT and 74.0°C for G12D mutant, which indicates that the WT and the mutant could be successfully discriminated by the proposed method.
Conclusion
We have proposed a new measurement method combining digital PCR with asymmetric PCR using molecular beacons and melting curve analysis. The genotyping of KRAS mutation was successfully performed by the proposed method. We are planning to prove the concept of this method for the clinical specimens in the future.
Citation Format: Junko Tanaka, Yuzuru Shimazaki, Tatsuo Nakagawa, Akiko Shiratori, Masao Kamahori, Takahide Yokoi, Kunio Harada, Yoshinobu Kohara. KRAS genotyping by digital PCR combining melting curve analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1574.