Whether it’s an alien concept or a familiar technology, researchers often find themselves asking these questions about digital PCR – What is it? When and why to use it? How does it compare to qPCR?
We have compiled answers to some of the most frequently asked questions, from principle to workflow and applications.
What is digital PCR?
Digital PCR (dPCR) is a highly precise approach to nucleic acid quantification. It estimates the absolute number of target molecules through statistical methods rather than relying on the number of amplification cycles to determine the initial amount of template molecule in each sample.
How does it work?
You’ll be relieved to know that the initial dPCR reaction is assembled using familiar assay components as those used in qPCR. By discretizing or partitioning each sample into a large number of individual and parallel reactions, you’re left with one or more target molecules in some partitions whereas others may contain none. Partitioning can be achieved either by dividing the sample into microplates containing capillaries or channels, arrays of miniaturized chambers, or water-oil emulsions as droplets. Each partition undergoes PCR amplification to the endpoint. Partitions with and without amplified product are individually counted. Those containing amplified product and showing a fluorescent signal are designated as positives and scored as “1”; those with no amplified product and showing only a background signal are designated as negatives and scored as “0”. Poisson statistical analysis is then applied to determine the absolute concentration of the target present in the initial sample, without relying on references or standards.
What are the major advantages over qPCR?
Quantitative PCR (qPCR) is the well-established and preferred method of choice for relative measurement in routine applications requiring a broad dynamic range, high throughput, and rapid time to results in screening large numbers of samples. Absolute quantification with dPCR offers significant advantages over qPCR when it comes to quantifying rare targets in complex backgrounds and detecting small fold change differences with high sensitivity, superior precision, better reproducibility, and high multiplexing capabilities. Moreover, increased tolerance to inhibitors owing to partitioning and non-reliance on amplification efficiency or standard curves of qPCR makes it a simple and affordable next-gen technology.
Which applications or assays can be performed using dPCR?
As mentioned above, applications such as rare mutation detection, copy number variation, NGS library quantification, low-level pathogen detection, viral load detection, and GMO detection can leverage the tremendous precision and high sensitivity that digital PCR offers compared to qPCR. For most researchers, dPCR represents a complementary approach to qPCR.
How will QIAGEN’s digital PCR instrument prove to be any different from commercially available systems?
Our systems are developed on nanoplate-based technology, offering significant benefits over droplet digital PCR (ddPCR) technology. Fixed partitions integrated into the dPCR nanoplate prevent variation in size and coalescence as seen in ddPCR method. Besides, simultaneous reading of all partitions of sample results in a faster readout. Nanoplates are not only user-friendly and easy to pipette just like qPCR but are also amenable to front-end automation. Most importantly, correctly sealed nanoplates prevent sample evaporation and well to well contamination.
Partitioning, thermocycling, imaging, and analysis are all integrated into a single fully automated instrument, delivering results in about 90 minutes, vs. more than four hours for currently available digital PCR systems.