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

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.

Evaluation of droplet digital PCR for characterizing plasmid reference material used for quantifying ammonia oxidizers and denitrifiers

“…The ddPCR response was linear over the dynamic range of 2.3-215 982 copies 20 μL −1 of ddPCR, covering five orders of magnitude. This is consistent with a previous study [19], in which the linearity range of ddPCR for quantifying lambda genomic DNA covered more than four orders of magnitude. The linearity regression of the ddPCR for quantifying pNIM-003 plasmid DNA is shown in Fig.…”

“…The ddPCR workflow and data analysis were performed as described by Pinheiro [19]. Amplification conditions consisted of a 10 min activation period at 95°C, followed by 40 cycles of a three-step thermal profile of 15 s at 94°C for denaturation, 60 s at 55°C for annealing, and 60 s at 72°C extension, and a final 10 min inactivation step at 98°C.…”

“…The relative standard uncertainty for M was estimated by Eq. (3) [19]. The relative standard uncertainty of the droplet volume was determined from analysis of an individual droplet volume measured using a Zeiss Observer Z1 microscope (please refer the Electronic Supplementary Material Fig.…”

“…S4), and the calibration of that microscope. The digital image was analyzed using ImageJ v1.34 s, and the process used for determining the equivalent circular diameter and the equivalent spherical volume of a sphere using ImageJ was as described in a previous report [19].…”

“…Droplet digital PCR (ddPCR) is a droplet-based form of dPCR that has recently been commercialized. Pinheiro [19] reported it has high accuracy and precision for quantifying genomic DNA concentration. However, little information is available regarding use of ddPCR for quantifying plasmid DNA.…”