Life in the next-generation world moves quickly. And I’m talking not about next-generation sequencing, but something termed ‘next-generation PCR’ by internal folks at Life Technology, and can be described as ‘third-generation PCR’ if one thinks about the Polymerase Chain Reaction in terms of measurement.
By “first generation”, I mean end-point PCR, a presence or absence of an expected PCR product on an ethidium-bromide stained gel. (Other measurement technologies have been developed for the detection or absence of a particular PCR product.) For the “second generation” PCR (otherwise known as ‘quantitative PCR‘ or ‘qPCR’), I refer to the monitoring of the amplification reaction while it occurs, measuring fluorescence in real-time, either of which is the incorporation of a SYBR-green dye, or the cleaving of a fluor-quencher probe as the amplification process occurs (otherwise known by its trademarked TaqMan name).
Based upon a principle of limiting dilution and partitioning the original sample, the idea is to dilute a sample so that there is less than one copy of a single molecule in a single well (out of many wells); for example, for a 96-well plate, the dilution would be calculated in such a way that about 48 of the wells would have a single copy of your PCR target, and 48 of the wells would be empty. After amplification and detection, it is a simple matter to back-calculate from the number of PCR positive wells and the dilution factor, and obtain the exact quantitation of the number of molecules of that target in your original sample.
The advantages of such an approach is absolute concentration of your target; as you may already know, for qPCR you need to have a standard curve of set
dilutions of a known target to know what Ct value equals what concentration. With digital PCR, you don’t need that standard curve to calibrate against. There are
occassions where qPCR the results of the number of copies in the sample is ambiguous, and a different method is needed to determine quantitation. This is where
digital PCR would come in.
As a concept the idea has been around for some time, but until only recently has there been the technical advancements for the vendors to make commercial
products out of it.
And the developments have come fast and furious. In October of 2011, BioRad acquired a startup called QuantaLife for $162M as QuantaLife was poised to launch
their modestly-priced (~80K) digital droplet PCR product into the marketplace. Just a few weeks later in October 2011, the QuantStudio 12K Flex™ was launched
by Life Technologies which incorporated the ability to do digital PCR in an instrument that had a lot of flexibility for qPCR, whether in a 96-well, 384-well,
microfluidic TaqMan Array Card™, or BioTrove-derived OpenArray™ plates. (It is a more expensive instrument than the single-purpose QuantaLife system, but also has a lot of qPCR capability to offer simultaneously.) And just this past April, RainDance Technologies launched their RainDrop™ digital PCR system as well. (Fluidigm has had a digital PCR offering for a few years now, but with limited traction and market penetration, mainly due to technical considerations.)
Each of the three offerings have their pluses and minuses – but my point is not to make those distinctions here. Rather, it is to point out with an attractive market
future (some projections I’ve seen place the digital PCR market to expand from it’s current $5M – $10M size now, to some $200M in 5 years), digital PCR may start to disrupt the qPCR market with its absolute quantitation and unambiguous precision. Of course there needs to be a lot of education that needs to take place, as customers understand what digital PCR has to offer their work, and three or four major competitors battling it out in the marketplace at the beginning of this
fast-growing market you can be sure that the local sales reps will be busy educating the prospective customer-base.