Multiplex digital PCR combined with melting curve analysis to detect KRAS mutations

Background

Digital PCR (dPCR) is a promising method for liquid biopsies and 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 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 probes. Fluorescence images were captured while controlling the temperature of the chip, and melting curve was measured for each well. Then, genotyping was performed using the fluorescence intensity, dye color of the probe, and melting temperature (Tm). Because the Tm 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 wildtype KRAS and its mutants, G12D, G12R, G12V, and G13D. The results showed that the peaks of the Tm distributions of the DNA groups were 69.2°C and 62.8°C for the wildtype and G13D with the first color dye, in addition to 66.9°C, 63.5°C, and 59.1°C for G12R, G12D, and G12V, respectively, with the second color dye. The wildtype and mutant groups were clearly identified by fluorescence intensities and Tm values, and able to be quantified. Furthermore, genotyping of cfDNA of pancreatic cancer patients was successfully performed.

Conclusion

We have proposed a new method that combines dPCR with melting curve analysis. To our knowledge, this is the first demonstration of genotyping of five DNA groups with a single mutation of cancer-related genes by combining dPCR with melting curve analysis.