Digital PCR is a nucleic acid molecule counting method with unprecedented resolution, relative sensitivity and accuracy. The template is diluted to such an extent that on average 1 template molecule is present in a PCR reaction, and many different small reactions are run in parallel. By counting the number of positive and negative reactions (hence the term ‘digital’) at the end of the PCR, it is possible to determine the starting number of template molecules in the sample under investigation. In other words, the question of how much nucleic acid target molecules are present in a particular sample is answered by measuring how many miniaturized reactions are positive if template is extremely diluted. Importantly, a Poisson correction needs to be applied on the counts, to correct for the fact that some reactions may contain more than 1 template molecule.
While digital PCR is often perceived as a 2nd generation quantitative PCR technology, it was conceived around the same time as real-time quantitative PCR. Only recently, it has come of age due to the availability of dedicated instruments. Being still a relatively young technology, it is important that guidelines have already been proposed to setup and validate a dPCR experiment, and to report the results from such a study ( ). These dMIQE guidelines address known requirements for dPCR that have been identified during the early stage of its development and commercial implementation.
While quantitative PCR has been the gold standard lab method for nucleic acid quantification in many different application areas (most importantly gene expression analysis and SNP genotyping), some applications are more demanding. In principle, any study that requires higher resolution (ability to measure smaller differences) or higher relative sensitivity (ability to accurately quantify a minority variant in a background of wild-type sequences) could benefit from digital PCR.
Therefore, I see two important application areas for digital PCR, namely gene copy number determination and rare event detection. Digital PCR certainly has advantages for accurate discrimination between higher copy numbers, e.g. 6 from 7 copies, due to its higher resolution, but also for regular gene copy number quantification (1 vs. 2 vs. 3). The great advantage of dPCR is that no specific calibrator samples are required to determine the actual copy number.
Rare event detection is useful in the field of cancer gene mutations (diagnosis, monitoring therapy response, early relapse detection), infectious diseases (pathogen detection), genetically modified organism detection in food, and prenatal testing of genetic diseases. In addition to these main application areas, digital PCR can be used in various niche applications where researchers apply creative thinking to come to clever solutions. Once you realize the power of miniaturized reactions with highly diluted template, you can imagine various novel applications.
While digital PCR certainly has great assets, I believe that quantitative PCR will remain mainstay technology for many more years as the user community is large and as its most important application domain (gene expression analysis) does not necessarily require the precision and resolution that digital PCR offers.
I believe the trend of cost reduction and higher throughput will continue to occur in the near future. This will allow the technology to be used by more people and in application domains where the advantages compared to e.g. qPCR are not necessarily prominent (e.g. regular gene expression analysis). Another development may be the introduction of higher multiplexing capacities. This is relevant as digital PCR in principle lends itself to high multiplexing. Ugozzoli and colleagues were the first to demonstrate true quantitative multiplexing up to 4 targets in a qPCR reaction (Analytical Biochemistry, 2005). But, as this is really the limit for qPCR, most users never try more than 2 targets. As the template is much less complex in a digital PCR reaction, one can image to go way beyond 4 targets, provided the instrumentation is developed to enable this level of multiplexing.
To conclude, I want to emphasize that digital PCR holds great promises for clinical applications:
- data interpretation is quite straightforward, no extra-ordinary assumptions are needed.
- results can be shared and interpreted among different users because absolute concentrations are determined
- the inherently high precision, accuracy and relative sensitivity are relevant attributes for laboratory tests that are used in the clinic.