Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA Copy Number Quantification

TLDR

Droplet digital PCR (ddPCR) is a recently commercialized technology that quantifies nucleic acids by counting molecules in discrete water‑in‑oil droplets, offering a simple workflow and highly stable DNA partitioning. This study evaluated the key performance parameters of the ddPCR system. ddPCR produced a linear response across more than four orders of magnitude, matched simplex and duplex Lambda DNA assays within expanded uncertainties, achieved a relative expanded uncertainty below 5%, and outperformed existing real‑time and digital cdPCR technologies in precision.

Abstract

Droplet digital polymerase chain reaction (ddPCR) is a new technology that was recently commercialized to enable the precise quantification of target nucleic acids in a sample. ddPCR measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions. This novel ddPCR format offers a simple workflow capable of generating highly stable partitioning of DNA molecules. In this study, we assessed key performance parameters of the ddPCR system. A linear ddPCR response to DNA concentration was obtained from 0.16% through to 99.6% saturation in a 20,000 droplet assay corresponding to more than 4 orders of magnitude of target DNA copy number per ddPCR. Analysis of simplex and duplex assays targeting two distinct loci in the Lambda DNA genome using the ddPCR platform agreed, within their expanded uncertainties, with values obtained using a lower density microfluidic chamber based digital PCR (cdPCR). A relative expanded uncertainty under 5% was achieved for copy number concentration using ddPCR. This level of uncertainty is much lower than values typically observed for quantification of specific DNA target sequences using currently commercially available real-time and digital cdPCR technologies.