The digital polymerase chain reaction (dPCR) consists of many discrete, small-volume reactions to represent genetic information from a single sample.
If a DNA template is sufficiently diluted so that some of these reactions contain no target molecules, while others contain discrete amounts, statistical analysis can then be used to quantify the exact number of target molecules in the sample. Therefore, dPCR can be used to accurately determine the number of nucleic acid molecules in a sample without comparison to a reference standard, provided that many reactions are performed in parallel.
Because the reaction volumes for dPCR are generally very small, and the template must be sufficiently dilute to achieve single-molecule resolution, nucleic acids can be isolated from minimal quantities of specimen. The dilution of template also removes many of the inhibitors of PCR that are present in the original sample, further improving accuracy and efficiency.
These properties make dPCR a superior diagnostic tool for the clinic, as well as any application where extreme sensitivity or precise quantification is essential, such as identifying mutations or copy number variations in tumor cells, or examining gene expression at the single-cell level.
While dPCR uses established thermal cycling technologies and enzymes, and fluorescent probes are used to read the amplification signal, as they are in qPCR, its essence lies in the partitioning of the sample into many small reactions. The number of reactions that can be run depends on the equipment and the process it uses for partitioning.
There are two types of dPCR machines currently available: chip-based and digital droplet PCR (ddPCR). For chip-based dPCR systems, such as the LifeTechnologies QuantStudio™, the sample is mixed with reagents and loaded into individual reaction chambers in plates that are about the size of a microscope slide. The reaction mixture is loaded into these small partitions by hydrophobic/hydrophilic interactions and capillary forces and the machine monitors the reactions as they occur.
In ddPCR, employed by Bio-Rad and RainDance machines, the samples are first mixed with the reagents and dispersed into nanoliter-sized droplets. The droplets for each sample are then placed in a tube and PCR is performed in a thermocycler. A droplet reader then detects a fluorescent signal to determine whether or not reactions have occurred. Unlike chip-based systems, ddPCR does not use physical partitions to separate the reactions, but the properties of the droplets themselves. For further discussion on dPCR technologies, please see Nature Methods 9, 541-544 (2012).