Digital PCR has no doubt made a breakthrough in research and development, but it’s easy to ask; how exactly is PCR done? So far, only two types of platforms exist. One platform is droplet technology which uses microfluidics to emulsify samples in oil, creating reproducible droplets to be processed and analyzed. The goal is to have one molecule of DNA per droplet, and each droplet is subject to many thousands or even millions of PCR reactions.
Fluorescent detection is used to analyze samples, and the number of molecules containing the target DNA is simply counted. This technology is currently utilized by RainDance Technologies and Bio-Rad Laboratories, where, respectively, one system creates picoliter droplets and the other creates nanoliter droplets.
The second platform is a chip-based technology, which is used by Life Technologies and Fluidigm. In this platform, samples are partitioned evenly across a disposable chip with the same idea in mind as droplet technology: to ideally have one molecule of DNA per partition and detect specific sequences using fluorescence.
Unlike droplet platforms, chip-based platforms are limited to synthesizing only a few thousand partitions at once (usually up to 20,000), creating a narrower dynamic range. However, an advantage to the system is that each partition is structured to hold a fixed volume, while in the droplet systems there is a slight chance of coalescence occurring among the samples.
When using both the droplet and chip-based platforms, there’s very little noise due to wild-type DNA detected because of the concentration of each droplet or partition. Rare sequences that once “blended in” with wild-type DNA now stand out because of the higher specificity and sensitivity of the technology. Both systems can even detect sequences among fragments of DNA, including formalin-fixed, paraffin-embedded DNA and cell-free circulating DNA. As long as the sequence of interest is still intact, it will be detected.
This advancement in technology enables researchers to study, among others, rare mutant alleles, viruses, and prenatal defects, and publish their work sooner than with other methods.
Although the technology is still on the rise, there are a few institutions that have fully adopted the technology and converted over to the digital methods. In contrast, there are also institutions that are unfamiliar with what the technology has to offer, and still others aren’t interested in learning more because the methods don’t apply to their line of research.