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.

We have developed a real-time polymerase chain reaction (PCR) method that can quantify hydrocarbon-degrading bacteria in sediment samples based on a catabolic gene associated with the first step of anaerobic toluene and xylene degradation. The target gene, bssA, codes for the alpha-subunit of benzylsuccinate synthase. The primer-probe set for real-time PCR was based on consensus regions of bssA from four denitrifying bacterial strains; bssA sequences for two of these strains were determined during this study. The method proved to be sensitive (detection limit ca. 5 gene copies) and had a linear range of >7 orders of magnitude. We used the method to investigate how gasohol releases from leaking underground storage tanks could affect indigenous toluene-degrading bacteria. Microcosms inoculated with aquifer sediments from four different sites were incubated anaerobically with BTEX (benzene, toluene, ethylbenzene, and xylenes) and nitrate in the presence and absence of ethanol. Overall, population trends were consistent with observed toluene degradation activity: the microcosms with the most rapid toluene degradation also had the largest numbers of bssA copies. In the microcosms with the most rapid toluene degradation, numbers of bssA copies increased 100-to 1000-fold over the first 4 days of incubation, during which time most of the toluene had been consumed. These results were supported by slot blot analyses with unamplified DNA and by cloning and sequencing of putative bssA amplicons, which confirmed the real-time PCR method's specificity for bssA. Use of a companion real-time PCR method for estimating total eubacterial populations (based on 16S rDNA) indicated that, in some cases, ethanol disproportionately supported the growth of bacteria that did not contain bssA. The real-time PCR method for bssA could be a powerful tool for monitored natural attenuation of BTEX in fuel-contaminated groundwater. To our knowledge, this is the first reported molecular method that targets anaerobic, hydrocarbon-degrading bacteria based on a catabolic gene.

Two years ago, we described the first droplet digital PCR (ddPCR) system aimed at empowering all researchers with a tool that removes the substantial uncertainties associated with using the analogue standard, quantitative real-time PCR (qPCR). This system enabled TaqMan hydrolysis probe-based assays for the absolute quantification of nucleic acids. Due to significant advancements in droplet chemistry and buoyed by the multiple benefits associated with dye-based target detection, we have created a "second generation" ddPCR system compatible with both TaqMan-probe and DNA-binding dye detection chemistries. Herein, we describe the operating characteristics of DNA-binding dye based ddPCR and offer a side-by-side comparison to TaqMan probe detection. By partitioning each sample prior to thermal cycling, we demonstrate that it is now possible to use a DNA-binding dye for the quantification of multiple target species from a single reaction. The increased resolution associated with partitioning also made it possible to visualize and account for signals arising from nonspecific amplification products. We expect that the ability to combine the precision of ddPCR with both DNA-binding dye and TaqMan probe detection chemistries will further enable the research community to answer complex and diverse genetic questions.

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