By Ben Butkus
Fluidigm has licensed from the University of British Columbia a chip-based digital PCR technology with a 100-fold greater reaction chamber density than Fluidigm's current microfluidic valve-based devices, PCR Insider has learned.
The new technology could improve the utility of digital PCR in diagnostic applications, and help drive the technique's adoption as a low-cost alternative to quantitative real-time PCR for applications such as copy number variation, rare allele detection, and single-cell gene expression analysis, according to its inventors.
The innovation may also help change the perception that Fluidigm's digital PCR products are limited by the physical size of its chips and unable to partition a sample into the millions of individual nanoliter-scale-or-less PCR reaction volumes that can be achieved with technology from competitors such as Life Technologies, RainDance Technologies, and QuantaLife.
"It really is the best of both worlds of chip-based and droplet-based digital PCR," Carl Hansen, an assistant professor in UBC's Department of Physics and Astronomy and Centre for High-Throughput Biology and one of the technology's inventors, told PCR Insider this week.
Hansen and colleagues at UBC described their technique for the first time and demonstrated its potential as a research and diagnostic tool in a scientific paper published online last week in Nature Methods.
As described in the paper, megapixel digital PCR uses surface tension-based sample partitioning and dehydration control — a departure from the microfluidic valves featured in Fluidigm's commercial products and the emulsion-based nanodroplets or nanoscale "through-holes" used in other commercial technologies.
The megapixel digital PCR setup is composed of a PDMS device with a 3 µm2 bifurcating channel network that connects to linear arrays of 10-picoliter "dead-end" chambers. The chambers are then partitioned by flushing the device with an immiscible fluorinated oil that displaces the aqueous phase of the sample and creates an "advancing contact line" at the PDMS-oil-water interface.
As the contact line moves past the entrance of each access channel, it becomes pinned at the leading edge, causing the aqueous phase at the chamber inlet to thin and ultimately separate from the bulk reagents, the researchers wrote. The result is uniform and defect-free partitioning of molecules in an array of 1,000,000 chambers with densities exceeding valve-based digital PCR, such as that currently marketed by Fluidigm, by a factor of 100, according to the paper.
According to Hansen, who is also a longstanding member of Fluidigm's scientific advisory board, motivation for developing the technique stemmed from work he and others conducted in the laboratory of Steven Quake, a Fluidigm co-founder, while at the California Institute of Technology.
"I was aware of digital PCR and realized that, in principle, it offers some new measurement capabilities that should be of interest to areas like diagnostics and other high-value applications where real-time PCR just doesn't have the required precision, resolution, or sensitivity," Hansen said.
As an example, Hansen pointed to the Quake group's work in the area of non-invasive detection of fetal aneuploidies, which requires a technique sensitive enough to detect a minute enrichment of a specific chromosome from a small amount of fetal or placental DNA circulating in a mother's bloodstream.
"It's a clinical diagnostic example of a very small allelic imbalance," Hansen said. "It's essentially a copy number variation, but not in the sense that a chromosome has a duplication of a gene. It's a CNV in the sense that the total load of DNA is enriched for one of the chromosomes because the fetus has aneuploidy. But it's a similar example."
Using this concrete problem as a starting point, Hansen and colleagues began exploring other ways to "enable high-value applications that require performance that was not met by qPCR." In addition, as the group began to more frequently use digital PCR in its lab, "it became our go-to technique … anytime we want to know what the concentration of a template is, or how much RNA is in a sample," Hansen said. "And the reason is, for us, it's inexpensive, it's fast, and it's absolute in quantification."
Having helped develop the microfluidic valve technology core to Fluidigm's Digital Array chips, Hansen came to realize that "if you were able to greatly increase the density of digital PCR on a chip format, then you could split that number of reactions — in this case it's a million over an inch — you could split those into hundreds of samples. So it's a cost-competitive surrogate for qPCR."
In their Nature Methods paper, Hansen and colleagues demonstrated that their device could perform high-fidelity single DNA molecule amplification in 1,000,000 picoliter-volume reactors with densities of up to 44,000 reactors per cm2. In addition, they showed that the device achieved a dynamic range of 107 (as opposed to 104 for tube-based qPCR); single-nucleotide-variant detection of less than one copy per 100,000 wild-type sequences; and discrimination of a 1 percent difference in chromosome copy number in a proof-of-principle experiment that mimicked the aneuploidy-detection problem.
Hansen conceded that other approaches to digital PCR, such as oil emulsion-based nanodroplets, are just beginning to achieve the same types of benchmarks, but that such setups have been plagued by poor amplification efficiency or difficult-to-work-with chemistries.
However, the new technique certainly improves upon Fluidigm's current offerings in terms of the number of individual reactions that can be performed on a chip format with physical size constraints. The company's most advanced Digital Array chip partitions each of 48 samples into 770 PCR reactions for 36,960 individual reactions in volumes as small as 4 µL — good enough for many digital PCR applications, but perhaps not for diagnostics or extreme research applications like the single-cell gene expression analysis experiments currently underway in Hansen's lab at UBC.
In an interview with PCR Insider last August, Fluidigm President and CEO Gajus Worthington noted that the company was working with undisclosed academic collaborators on methods that would allow Fluidigm chips, or some version of them, to perform millions of digital PCR reactions in parallel (PCR Insider, 8/19/10).
This week, Fluidigm spokesperson Howard High confirmed that the company has licensed the megapixel digital PCR technology from UBC, although he declined to disclose additional details about the license or speculate on how the technology might be incorporated into future Fluidigm products, citing the early nature of the agreement.
Hansen said that his group has been working on the technology for several years and has applied for patents surrounding it, but did not say whether any of those patents have issued. Besides licensing the technology to Fluidigm, his group is also working with an undisclosed commercial entity to explore using megapixel digital PCR in various applications.
Hansen said that regardless of its commercial path, megapixel digital PCR bodes well for the future of digital PCR in general as a laboratory technique and diagnostic method.
"As this comes out, and as other similar technologies come out, I'm pretty certain that, more times than not, researchers who are currently doing qPCR to measure gene expression or copy number variation …. will use digital PCR on a regular basis instead."
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