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Illumina Reps Provide More Detail on New Benchtop RT-qPCR Platform at Industry Event

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By Ben Butkus

DUBLIN – About a month and a half after Illumina announced its foray into the PCR space with the launch of its Eco real-time benchtop PCR system, marketing specialists from the company shared some additional information about the system and its potential applications at Select Biosciences' qPCR Europe conference, held here this week.

In a technology showcase session at the conference, Judy Macemon, director of PCR marketing at Illumina, highlighted some of the rationale behind Eco's development as well as technical specifications of the system that help it maintain industry-standard performance at a price point of about $13,900.

Meantime, in a separate conference presentation, Sandrine Miller, senior product manager at Illumina, discussed the Eco's amenability to conducting affordable genotyping using high-resolution melt analysis.

Illumina unveiled the Eco system in late July along with the announcement that it had acquired privately held real-time PCR and nucleic acid-analysis firm Helixis in a deal worth as much as $105 million (PCR Insider, 7/29/10).

Helixis, founded in 2007 by the California Institute of Technology's David Baltimore, Alex Dickinson, and Axel Scherer, had developed a qPCR system called Pixo that the company claimed would cost about a quarter the price of other qPCR systems while maintaining the same level of performance.

Illumina took over development of the platform leading up to its acquisition of Helixis, and at this week's qPCR conference, Macemon said that the new platform began shipping in August, "rebranded and looking a little different." She also identified the National Institutes of Health's National Institute of Dental and Craniofacial Research as one of the first paying customers.

Macemon, who joined Illumina from Helixis, said that the company's rationale for developing the platform was market research showing that the biggest barrier to wide adoption of real-time qPCR was pricing.

According to Macemon, Helixis' research revealed that typically 10 or more people in a laboratory share an RT-qPCR instrument. In addition, she said that despite the recent industry trend to increase the throughput of RT-qPCR systems, about 80 percent of researchers use fewer than 50 sample wells per run; hence Helixis and Illumina's decision to develop a 48-well platform.

In essence, she added, scientists were being forced to choose between high performance and low cost. Illumina is banking on the idea that the ubiquity of RT-qPCR has led at least the academic research market to the point where nearly every scientist would want their own affordable benchtop system to run everyday routine experiments.

The company is also claiming that, despite its low price, the Eco can deliver many of the same performance specifications as other, more expensive machines.

Macemon said that the Eco has demonstrated five-fold improved thermal uniformity and detection sensitivity as other platforms, though she didn't identify specific competing machines.

The key to this performance is the core technology developed at CalTech: a hollow silver thermal plate system containing an electromagnetic-driven conductive fluid, which offers very precise and fast temperature control, low power requirements, and well-to-well uniformity.

Most other instruments use a solid block for thermal cycling, and the best of these offer somewhere in the neighborhood of 0.5°C well-to-well temperature variation. Meantime, in instrument specs Illumina claims Eco delivers well-to-well variation of 0.1°C, but Macemon said that researchers at the company have been able to achieve variations as little as 0.05°C.

The upshot of this temperature control is "higher quality and more uniform data," Macemon said.

Other features of the instrument include LED array illumination, which provides four-color multiplexing; and proprietary "adaptive LED control," which regulates the duration of LED exposure to reaction wells during cycles, thereby equalizing fluorescence across the plate and allowing for faster data collection.

Melting Away

Illumina is also marketing Eco's ability to perform high-resolution melting, or HRM, a technique that has been gaining popularity as a way to extract information about DNA based on the dissociation or melting of double strands.

Illumina's Miller, who also joined the company from Helixis, outlined how HRM, which is an enhancement of more traditional melt-curve analysis, can be used to conduct affordable SNP genotyping and other mutation analyses.

Miller said HRM is more cost-effective than sequencing- or probe-based genotyping, and that use of the technique in the scientific literature has spiked in the last few years, much in the way that publications involving RT-qPCR spiked rapidly when it was first introduced in the early 1990s.

According to Miller, HRM has three major requirements: chemistry, instrumentation, and software. By chemistry, Miller was referring to saturating dsDNA-binding dyes that are non-inhibitory to PCR, such as Life Technologies' SYTO 9, Idaho Technologies' LCGreen, or Biotium's EvaGreen.

Meantime, HRM instrumentation requires precise temperature control and fast data acquisition; while software must feature specialized normalization algorithms and difference plots that show the difference in fluorescence from selected reference samples.

Miller noted that Life Tech's ABI 7500 and Roche's LightCycler are both capable of HRM, but that the Eco also offers all the necessary features at a lower price. Further, Miller said that the Eco may in fact offer superior HRM analysis, again due to its five-fold better well-to-well thermal uniformity.

Specifically, Miller highlighted data obtained from in-house research showing the ability of the Eco to distinguish DNA molecules down to a SNP using HRM. Further, she outlined how the Eco is capable of distinguishing Class 4 SNPs, which describe an A-to-T base change that has traditionally been very challenging to detect with HRM because it results in an extremely small melt curve shift – as little as 0.2°C.

Illumina will continue to develop HRM-based applications for the Eco as the instrument begins to gain traction in the market, Miller said.

The company has tested the Eco HRM application using four different HRM master mixes: Roche's LightCycler HRM Master Mix; Life Technologies' ABI MeltDoctor HRM Master Mix and Invitrogen Express SYBR GreenER SuperMix; and Bio-Rad's SsoFast EvaGreen Supermix. Miller said that all mixes work well and none really performed better than others.

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