High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number

TLDR

Digital PCR enables absolute quantitation of nucleic acids, yet scalable, practical technologies have limited its widespread adoption. The study presents a high‑throughput droplet digital PCR system designed to process approximately 2 million PCR reactions. It achieves this throughput by employing conventional TaqMan assays within a 96‑well plate workflow. The ddPCR system delivers orders‑of‑magnitude greater precision and sensitivity than real‑time PCR, accurately measuring germline copy number variation, detecting rare mutant alleles amid a 100,000‑fold excess of wildtype, and quantifying fetal and maternal DNA in plasma, thereby opening new avenues for genetic research and diagnostics.

Abstract

Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ∼2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100 000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.