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Bio-Rad Study Demonstrates EvaGreen-based Digital Droplet PCR and Multiplex Applications

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In a study published this month, Bio-Rad researchers have demonstrated the capability of the company's new droplet digital PCR system using DNA-binding dyes with comparable results to TaqMan and that it's possible to use this new dye-based approach for the quantification of multiple targets in a single reaction.

The paper, published in Analytical Chemistry, describes a head-to-head comparison of TaqMan probe detection and DNA-binding dye-based ddPCR, with both approaches yielding equivalent results, according to the authors. It also demonstrates several strategies for using EvaGreen to quantify more than one target species.

Geoff McDermott, the paper's first author, and George Karlinn-Neumann, a co-author of the study, both of whom are Bio-Rad senior scientists, told PCR Insider this week that the study hopefully demonstrated to customers some of the dimensions where DNA-binding dye-based ddPCR can be particularly "fruitful."

"It's early for people to get their hands on [the new system] but we are already seeing users taking some of the things Geoff demonstrated in the paper and running with them," Karlinn-Neumann said.

In the study, the Bio-Rad researchers used the company's second-generation QX200 Droplet Digital PCR system in several experiments with EvaGreen dye to show how fluorescence emission — determined by the mass of DNA in a droplet — could be used to account for off-target products and enable equivalent sensitivity to TaqMan-based chemistry, as well as allow the quantification of multiple targets in a single well of droplets.

EvaGreen and its predecessor SYBR Green have become popular among researchers because they don't require the design and use of probes, only primer pairs, and thus can be used more easily and cheaply.

In the study, the Bio-Rad team started with a series of experiments to examine the effect of DNA mass on fluorescent amplitude to establish appropriate operating conditions for EvaGreen-based ddPCR.

According to the paper, the greatest degree of separation between primer and background DNA concentrations (and thus the greatest ability to distinguish target from non-specific products) occurred with primer concentrations of 100-200 nM. Higher concentrations "may affect the ability to draw a threshold between the two population types and obtain an accurate target concentration," the authors wrote.

The group then compared a total of 10 EvaGreen and TaqMan-based ddPCR assays for the quantification of five genes, using the same primer pairs for both versions of each reaction to compare the two approaches.

Overall, the measured concentrations of the targets were analogous and separation between positive and negative populations were comparable when using either EvaGreen or TaqMan forms of each assay, the authors wrote, with the exception of three of the assays — B2M, IL-4, and EEF2 — for which distinguishing between positive and negative populations was easier with EvaGreen.

The group also looked at the influence of non-specific amplification products on EvaGreen-based ddPCR assays.

"Adapting [ddPCR for DNA-binding dyes] was exciting," McDermott said, "because a lot of the disadvantages of using these dyes in real-time PCR, like the fact that EvaGreen associates non-specifically with any double-stranded or single-stranded DNA, actually become a benefit in droplet digital PCR."

"With these dyes, you can't distinguish if fluorescence is due to primer-dimers or to non-specific amplification … However, you can distinguish different products based on the fluorescent amplitude of the droplets," he explained.

"You are getting both analog and digital information with ddPCR," Karlinn-Neumann added. "So you distinguish negative and positive droplets — that’s the digitization. But then you also have the fluorescence amplitude, which gives you a different set of information, and that's what's being leveraged here."

According to Karlinn-Neumann, the paper demonstrates to potential adopters of the system how this difference in fluorescence allows for visualization of the extent to which off-target products might be present in a particular assay.

"Based on what we showed, if researchers see that [the influence of off-target products is] fairly minor and decides not to redesign the assay, they can eliminate these off-target products from their calculations based on their fluorescence amplitude," he said.

"And we showed that the precision and sensitivity you get out of that is indistinguishable from what we get out of TaqMan probes."

The group also took advantage of fluorescence amplitude in using the system to detect and quantify multiple targets, demonstrating several different strategies for this single-color multiplexing.

"By tuning the amount of DNA within each droplet, we are able to get multiple populations and multiplex different targets. We had three approaches to controlling the mass of DNA within a droplet so that when we looked at a 2D cluster plot, we had different clusters belonging to different targets," McDermott said.

One approach relied on the group's finding that fluorescence amplitude was greater for longer amplicons than for shorter lengths of DNA.

In the study, the researchers combined two previously-used RPP30 and ACTB assays into one ddPCR reaction to detect both targets based on the difference in their amplicon lengths. This resulted in four distinct droplet populations when viewing the results in both 1D and 2D plots, and the ability to quantify both genes using a 2D droplet plot.

The droplet cluster with the lowest amplitude represented negative droplets, the group wrote, while the next two bands comprised droplets possessing only one or the other target: first RPP30 at 62 base pairs, then ACTB with 137 bp. Finally, a third population of droplets contained both targets at the highest fluorescent amplitude based on their combined mass.

The group's second approach to multiplexing relied on varying input primer concentrations to create different masses of amplified DNA (and associated fluorescence) for two different target sequences.

Using MRGPRX1 and RPP30 — targets with only an 8bp difference in length — the researchers found that by tripling concentration of RPP30 primers compared to those for MRGPRX1, they could once again use 2D clustering to distinguish four distinct populations and quantify both targets with similar results to those from parallel individual reactions.

In a third, alternate strategy, the team used TaqMan-probe and DNA-binding dye chemistries combined in the same reaction. The group demonstrated this approach to detect BRAF V600E wild-type DNA in combination with a probe-based RPP30 reference assay in one well of droplets, with results directly equivalent to those from single-plex versions of each assay.

According to Karlinn-Neumann, DNA-binding dyes allow researchers to design assays much more quickly, and potentially less expensively.

Moreover, he said, "a lot of people have assays that work well for them that are SYBR-based and they either haven't designed it with the thought in mind to be able to place a specific TaqMan probe in the midst of their primers, or they don't want to."

One specific application of DNA-binding dye-based ddPCR, Karlinn-Neumann said, was demonstrated earlier this year in a Bio-Rad collaboration with Elizabeth Blackburn, a researcher at the University of California, San Francisco (PCR 4/18/2013).

"If you picture a telomere, there is a six-base repeating sequence and that's not something that's amenable to putting a TaqMan probe in the midst of… We demonstrated that we can convert a radioactive assay [that] people use to measure telomerase activity to a ddPCR telomere repeat amplification assay that relies on the EvaGreen dye," he said.

"I don't know if in some repeat diseases like Fragile-X or so forth, if [a similar approach] might benefit," he added. "We haven't explored it, but you could potentially generalize from the case of the telomere sequences to [other similar problems]."

Karlinn-Neumann also said that some QX200 users are doing work that follows on elements of the Bio-Rad group's recent paper, such as multiplexing BRAF mutant alleles and wild-type alleles.

"With this paper we say, 'Look what you can do in principle, now run with it,' and we are seeing users doing that for clinically important determinations … with absolutely superimposable results with TaqMan," he said.

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