By Ben Butkus
Part one of a two-part series. Part two can be found here.
Digital PCR, a version of quantitative real-time PCR that allows for much more sensitive and precise nucleic acid measurements, may be on the verge of becoming nearly as ubiquitous as its predecessor thanks to the integration of empowering nanoscale and microfluidic technologies.
For the past few years, just one company — South San Francisco-based Fluidigm — has offered a commercial digital PCR platform, and the company has enjoyed a relative monopoly on the technique even while working with its customers to find suitable applications for the technology.
However, Fluidigm may soon have competition as no fewer than four other companies are either developing digital PCR research platforms or diagnostics, or are actively surveying the landscape.
But even while digital PCR holds promise for a host of applications such as copy number variation, rare allele detection, validation of next-generation sequencing, and diagnostics, it remains to be seen whether the method flourishes as a next-generation variation of the laboratory workhorse that is qPCR, or whether it becomes a one-off technique, relegated to a few specialty molecular biology applications.
In addition, as with any technology on the cusp of a potential breakthrough, the intellectual property landscape surrounding dPCR will undoubtedly become a hurdle for potential developers — or a boon for those who believe that their patent position predates the competition.
First conceptualized in the early 1990s in a BioTechniques paper, digital PCR, or dPCR, has been much slower to develop than qPCR. Although qPCR's development predated dPCR by just a few years, by the late 1990s Applied Biosystems had introduced a commercial system, which was followed soon thereafter by scores of commercial platforms. Now, qPCR has become a nearly indispensable laboratory technique.
Meantime, dPCR enjoyed some additional incremental development in the mid 1990s, then stalled somewhat until the mid 2000s, when Fluidigm introduced the first commercially viable dPCR platform based on its patented integrated fluidic circuit technology.
At its core, the amplification reaction in dPCR and qPCR are essentially the same, and amplification is performed on a single sample. Where dPCR differs, however, is that a sample is partitioned into much smaller reaction volumes, which allows the amplification and quantification of increasingly smaller amounts of genetic material — even single transcripts or nucleic acids from single cells.
The quantification can be performed by counting how many wells or partitions or individual reactions generate a signal — a large amount of "yes-or-no" answers; or zeroes and ones, hence the "digital" moniker.
Therefore, the true innovation in dPCR lies in the ability of the technology to partition a sample into hundreds or thousands or even millions of uniform reactions, although other aspects such as maintaining consistent chemistry and statistical analysis are not trivial.
To be sure, Fluidigm's integrated fluidic chip technology has allowed researchers to partition a large enough number of qPCR reactions to allow a host of new applications previously unachievable with standard qPCR on well plates. For instance, the most recent iteration of Fluidigm's technology, a 96-by-96 Dynamic Array, is capable of producing 9,216 real-time qPCR data points in the same amount of time as a single 384-well plate, while using approximately 1/200th the amount of reagents, according to the company.
But a number of life science tool vendors are now pushing the envelope by applying technologies such as nanowells, microdroplets, and emulsions in an effort to increase the number of individual reactions to the hundreds of thousands or even millions. Following is an overview of some of those companies.
That's Life
Of the companies currently eyeing the digital PCR space, qPCR market stalwart Life Technologies may be first to market with a product to compete with the Fluidigm platform.
In the past year, Life Tech has made three key acquisitions — microfluidics firm Cytonix in July 2009; BioTrove in December; and Irish microfluidics startup Stokes Bio in April — with an eye toward building out technology and intellectual property around the qPCR and digital PCR space.
Indeed, at the time of the Cytonix acquisition Life Tech said that it acquired the firm for IP surrounding microfluidics-based digital PCR technology; and at an investor day presentation in early June, company officials openly stated that BioTrove's 3,072-well OpenArray platform and Stokes Bio's microfluidics technology were both compatible with digital PCR applications.
In a recent interview with PCR Insider, Gordon Janaway, product manager for real-time PCR reagents at Life Tech, confirmed that the Cytonix acquisition "provided some foundational IP around dPCR," and that both the BioTrove and Stokes Bio platforms "are very attractive for dPCR, which requires a large number of reactions to give you an answer. Both of them, by offering a much higher density than conventional 96- or 384-well formats, open the window for digital PCR to be commercially viable."
Of these technologies, the BioTrove OpenArray system is most immediately translatable to digital PCR applications. Life Tech had already been working with BioTrove prior to the acquisition to integrate OpenArray with TaqMan-based qPCR, and it currently offers what it calls a "mid-density" solution for PCR-based SNP analysis of hundreds to thousands of samples and candidate SNPs using OpenArray's 3,072 nanoliter-scale "through-holes."
Janaway told PCR Insider that for digital PCR applications, OpenArray can be thought of as "an extension of the plate-based … batch processing that we do on our conventional PCR systems, just a higher density."
As such, an OpenArray digital PCR platform would be most comparable to the Fluidigm platform in that they are both two-dimensional reaction templates with a physical footprint, although OpenArray has a potentially higher throughput.
Janaway said that the increased throughput "for the first time offers this new capability for absolute quantitation," an area that Life Tech is targeting for its first digital PCR product. This would be a research-only product and would include, for example, applications like measuring viral load or detecting copies of rare alleles.
"For rare allele detection, we're building on the power of dPCR to partition … a mixture into its components and enrich in each replicate. You start with a few copies of your rare allele against a large background of wild-type DNA; and then when you partition your sample, most but not all of the replicates will have the background. Open Array will provide an excellent open platform for researchers to develop rare allele detection applications," Janaway said.
Janaway added that Life Tech is currently in the "pre-release phase" and that undisclosed customers are currently testing an early version of the product, which will be launched by the end of this year.
Meantime, Janaway described the Stokes platform as "a flow system where individual droplets pass through thermal cycling and detector regions in a continuous flow. It is very flexible and allows us to adjust the volumes of droplets we're producing, and the frequency and flow rate of measuring these droplets. So we think it can be extended quite a bit in the digital space."
In this way, a digital PCR platform based on the Stokes Bio technology may greatly increase the number of individual reactions that can be partitioned, opening up applications such as routine gene expression research.
Janaway was less forthcoming about the development timeline of a digital PCR product based on the Stokes Bio technology, saying only that a gene expression application is in "the distant future" because it would require "hundreds of thousands or millions of replicates. So this is not the target application for the OpenArray or the current embodiment of Stokes."
And while Life Tech is firmly on the digital PCR boat, it may not be convinced that creating such a large number of replicate reactions isn't overkill in many cases.
"It's an oversimplification to say that for every digital application you need an enormous number of replicates," Janaway said. "With absolute quantitation in particular, even a few hundred or thousand can give you an answer that's much better than you can get with other methods."
In addition, Janaway said that he doesn't see digital PCR applications cannibalizing sales of Life Tech's lucrative qPCR product estate or replacing qPCR altogether — at least for the foreseeable future.
"Digital PCR needs to be joined with dramatically reduced cost per answer," Janaway said. "If digital can be offered only at a high cost per answer, then clearly it will only be useful for a small number of applications that can justify that cost. qPCR is so mature and inexpensive, that in the near term there is no expected transition. But as the cost per answer drops with digital, there is the potential for disruptive movement."
Rainmaker
In a recent interview, RainDance Technologies president and CEO Roopom Banerjee told PCR Insider that the company is "actively looking" at the digital PCR space, and has demonstrated in house that its microdroplet technology could seriously up the ante in terms of dPCR throughput.
Considering that the crux of dPCR is the ability to partition single PCR reactions into individual volumes, it appears to be a no-brainer application area for RainDance's "RainStorm" technology, which produces picoliter-volume droplets at a rate of 10 million per hour.
Indeed, according to Banerjee, a dPCR platform incorporating RainDance's microdroplet technology can produce the equivalent of 10,000 PCR reactions per second, which has the potential to increase throughput between 1,000- and 5,000-fold over current dPCR platforms while retaining a sensitivity of greater than 99 percent. Although he didn't specifically name Fluidigm as the comparator, it currently offers the only real dPCR platform on the market.
Banerjee was also reluctant to make a direct comparison between the potential throughput of a RainStorm-driven dPCR platform and that of a chip-based platform such as Fluidigm's (RainDance and Fluidigm share a common major investor in Alloy Ventures); but he did point out that chip-based dPCR "can't ever interrogate more" PCR reactions than the physical footprint of the device will allow – 384 by 384 wells, in Fluidigm's case.
A RainDance platform would also likely be about one-third to one-tenth cheaper in terms of reagent usage over current gold standard quantitative real-time PCR, according to the company.
To be sure, were RainDance to throw a dPCR platform into the burgeoning market, Banerjee said that the company's goal would be to "price it at a point that makes digital PCR ubiquitous" and the preferred PCR method over qRT-PCR.
Banerjee said that the company could foresee a microdroplet-based dPCR platform having initial applications in immune monitoring and histocompatibilty leukocyte antigen genotyping, which generally requires a large number of multiplex PCR reactions; and digital gene expression research in which scientists could examine single transcripts in single droplets.
Longer term applications might include diagnostics – "anything that requires higher sensitivity … or where there is a low limit of detection or rare variant," Banerjee said; as well as stem cell research, infectious disease screening, and prenatal or neonatal screening. As an example of what a RainDance dPCR platform might enable, Banerjee said that it could interrogate as many as 100 million individual reactions per milliliter of blood.
Part two of this story, to be published in the next issue of PCR Insider, will examine other companies exploring the digital PCR space and will survey the dPCR IP landscape.