Digital droplet PCR is an accurate and precise method to measure DNA copy number

DNA copy number research is impeded by limited methodology to determine true DNA copy numbers accurately and precisely. Human alpha defensin 1-3 (DEFA1A3) is a multiallelic gene with DNA copy numbers generally ranging from 2 to 12 copies per diploid genome. In this study, we developed a digital droplet PCR (ddPCR) protocol using DEFA1A3 as a model locus. We compared these results to DNA copy numbers determined by pulsed field gel electrophoresis (PFGE), which is considered a gold standard in CNV identification, on 40 DNA samples from a clinical study cohort. Taqman real-time quantitative PCR (qPCR) was also compared, being the other major available low cost, high-throughput system. The copy number measurements of 40 genomic samples were highly concordant between ddPCR and PFGE, while copy number by qPCR correlated only weakly with PFGE copy number. In conclusion, ddPCR is a low-cost, high-throughput technique with accurate resolution of CNV at both low and high DNA copy numbers. This makes it an ideal model to adapt for CNV testing in clinical practice.