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Team with UC-Berkeley Ties Looks to Make Bead-based Digital PCR Tech More Widely Available

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A team led by researchers from the University of California, Berkeley, has developed a bead-based, microfluidic digital PCR technology and demonstrated its ability to quantitatively measure cancer-related translocation mutations at extremely low levels and subsequently sequence single mutated clones.

The scientists believe that their technology has advantages over commercial emulsion-based droplet digital PCR platforms, such as those offered by Bio-Rad and RainDance Technologies, because it enables downstream sequencing analysis following the digital PCR analysis step.

This capability, they claim, makes their technology promising for applications such as high-throughput, single-cell analysis of multiple mutations, which could in turn provide a window into tumor heterogeneity.

However, the technology in its current form is complicated to use and requires extensive training, the researchers told PCR Insider, and as such they are exploring routes to develop it into an easy-to-use, integrated instrument that could be made more widely available to the research community.

The researchers described their technology and demonstrated its application to studying a model translocation mutation prevalent in many cancers in a paper published last month in Nucleic Acids Research.

The technology combines hemi-nested microfluidic digital PCR reactions in discrete reaction volumes generated by so-called microfabricated emulsion generator array, or MEGA, devices — a technology the researchers previously described in a paper published in 2010 in Analytical Chemistry.

However, the major difference between the researchers' technology and other emulsion-based droplet digital PCR systems is the former's use of single functionalized microbeads in each reaction volume. This key component, the researchers said, enables them to recover any mutated clones from the surface of individual beads for additional downstream analysis.

"There are a few commercial platforms out there for digital PCR as well as emulsion PCR," said Yong Zeng, a joint first author on the paper and former UC-Berkeley postdoc who is currently an assistant professor of chemistry at the University of Kansas. "I think the difference with ours is the [ability to] expand the emulsion-based PCR into sequencing applications.

Current droplet-based commercial platforms from Bio-Rad and RainDance Technologies, he noted, "don't have the capability to do subsequent genetic analysis out of those droplets. By using the beads … we think we will be able to do the quantitative measurement as well as subsequent genetic analysis, including sequencing."

A RainDance spokesperson told PCR Insider that the company's RainDrop digital PCR system currently does allow for emulsions to be broken following thermal cycling so the amplicons can be rescued and subsequently sequenced. However, RainDance is "just starting to see requests for this kind of thing but it is not a commercial solution on offer at this point," the spokesperson added.

Annette Tumolo, vice president and general manager of Bio-Rad's Digital Biology Center, told PCR Insider recently that the company has also received similar requests for the company's QX100 and QX200 Droplet Digital PCR systems, but that the company does not currently offer such a capability. Richard Kurtz, marketing manager for amplification at Bio-Rad, noted that one workaround that some customers have been performing is splitting a sample before the PCR step, running one portion through the droplet generator and reader, and recovering genetic material from the other for subsequent analysis.

At any rate, Zeng underscored the importance of this feature on his group's technology, and further noted that the system can generate an almost unlimited number of individual PCR reaction volumes, which could be important for increasing the sensitivity of a digital PCR assay.

"The sensitivity or accuracy of any digital-based method is based on the number of events, or the reactions that you can perform," Zeng said.

"You can do millions, or if you let it run long enough, tens of millions of single-molecule reactions," added Joe Shuga, Joe Shuga, another co-first author on the paper and former UC-Berkeley postdoc who is now a senior scientist at Fluidigm. "Basically you can do as many droplets as you want … you just let it run as long as it needs to, so you can get a much higher number of reactions than you can with" chip- or plate-based systems.

"RainDance also has the continuous process ... and is more similar to what we've done than the Bio-Rad or Fluidigm systems," Shuga said.

Further, breaking an emulsion to recover amplicons on individual beads is a relatively easy process, the researchers said.

"We trap those post-PCR beads, and then we can just run them through a flow cytometer, which is available everywhere, so really I think this is more compatible with … equipment that research labs or universities already have," Zeng said.

Shuga agreed, adding that "after we break the emulsion and recover the beads, you can use flow cytometry to count the ones that are fluorescently labeled, and now they're highly stable in buffer, so you've basically got all your colonies stored for downstream analyses."

In their Nucleic Acids Research paper, the researchers used their platform to determine the overall concentration in a healthy study population of the BCL-2/IgH translocation, t(14;18), which is strongly associated with follicular lymphoma.

They were able to quantitatively detect and sequence a single t(14;18) copy in 9 µg (about 3 x 106 copies) of genomic DNA from individuals in the healthy study population. Further, they were able to use the nested PCR assay to develop a quantitative genomic map of t(14;18) by sequencing and quantifying the unique t(14;18) clones found in individual subjects.

"The genomic map that we produced represents a baseline for this healthy population, and further sampling of this population can be used to monitor for expansion of particular clonal forms as part of disease progression," the researchers wrote.

Expanding on this idea, Shuga told PCR Insider that "the natural extension of this work … is to encapsulate single cells … and do a DNA digest on them, and then you have all of the genomic DNA from a single cell encapsulated into these same sort of droplets. From there you can do multiple allele detection from single cells." This, in turn, could lead to new ways to study tumor heterogeneity and co-incidence of mutations, he added.

The researchers would now like to make their technology more widely available to researchers, but first this may require at least a modicum of product development. UC-Berkeley holds the intellectual property surrounding the technology, much of which was developed in the laboratory of UC-Berkeley researcher Richard Mathies, who has had a hand in commercializing several microfluidic-based technologies in the past. Mathies did not respond to an email seeking comment on commercialization plans.

"We are trying to push it that way," Zeng said, though. "I really think we can do something to make it work for other people, not just a well-trained microfluidics person. Eventually [we'd like to] come up with a similar plan to Bio-Rad, but with one machine that can do all the jobs." Bio-Rad's QX100 and QX200 systems consist of separate modules for droplet generation and analysis.

Another limitation of the UC-Berkeley technology is that the throughput is hampered by the use of second-round PCR to amplify bead-bound DNA for standard Sanger sequencing. "Hopefully in the future [our technology] will be better if we can combine it with next-generation sequencing, so we can [increase] throughput for the second part of the technology," Zeng said.

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