Highly Sensitive and Highly Multiplexed Cell-Free DNA Genotyping Using 7.7x104-Well Digital PCR with Melting Curve Analysis

In this paper, we propose a combination of digital polymerase chain reaction (dPCR) and melting curve analysis for the highly multiplexed genotyping of cell-free DNA (cfDNA) with high sensitivity. A silicon chip with 7.7x10
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high-aspect-ratio wells with controlled surface hydrophobicity/hydrophilicity was developed. A sample containing cfDNA was divided into this large number of wells and amplified by PCR to enable sensitive detection. In addition, high multiplexing was achieved by capturing fluorescence images while controlling the temperature of the chip, measuring the melting curve for each well, and genotyping using fluorescence intensity, probe dye color, and melting temperature (Tm). The simultaneous dPCR genotyping of 10 types of DNA including single-nucleotide mutations of cancer-related genes was demonstrated for the first time with up to 7.7x10
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wells.