Next generation digital PCR: high dynamic range single molecule DNA counting via ultra-partitioning

Abstract

Digital PCR (dPCR) was first conceived for single molecule quantitation. However, current dPCR systems often require DNA templates to share partitions due to limited partitioning capacities. Here, we introduce Ultra-dPCR, a next generation dPCR system where DNA counting is performed in a single-molecule regimen through a 6-log dynamic range using a swift and parallelized workflow. Each Ultra-dPCR reaction is divided into >30 million partitions without microfluidics to achieve single template occupancy. Combined with a unique emulsion chemistry, partitions are optically clear and enable the use of a 3D imaging technique to rapidly detect DNA-positive partitions. Single molecule occupancy also allows for more straightforward multiplex assay development due to the absence of partition-specific competition. As a proof-of-concept, we developed a 222-plex Ultra-dPCR assay and demonstrated its potential use as a rapid, low-cost screening assay for non-invasive prenatal testing (NIPT) for as low as 4% trisomy fraction samples with high precision, accuracy, and reproducibility.