Courtesy of Bio-Rad Laboratories
In less than three decades, the polymerase chain reaction has evolved from a slow, labor-intensive practice that was initially performed manually and only by the initiated few, to a fast, powerful, easy-to-use tool found in life science laboratories everywhere. With the introduction of real-time thermocyclers in the late-1990s, the technology has become more versatile as well.
Real-time, or quantitative PCR (qPCR) allows experimenters to monitor PCR quantitatively as it progresses, providing the means to accurately detect and measure starting sample sizes or gene expression levels. Traditional PCR, on the other hand, provides an end product whose quantity is generally unrelated to the amount of starting material.
Most of the industry representatives interviewed for this article agree that one of the key features of recent growth in PCR technology has been the increasing availability and development of real-time instruments. In 2001 the US and European markets for real-time thermocyclers earned combined revenues of $252 million. Moreover, according to data that the consulting firm Frost & Sullivan compiled, that figure is expected to rise to an estimated $776 million by 2006.
Meanwhile, traditional PCR remains a strong contender. Chris Cunanan, PCR instrumentation product manager at Applied Biosystems in Foster City, Calif., estimates that only about 25% of all thermocycler users are switching from traditional to real-time PCR. Most researchers, he says, are not interested in the kinds of quantitative questions that real-time PCR can address. Consequently, they have no need to invest in the more costly real-time instruments and reagents.
Either way, the market continues to expand. In the months since
A year ago, Eppendorf introduced what was then the fastest traditional PCR instrument on the market, the Mastercycler ep, which could run a traditional 30-cycle protocol in just 15 minutes. Analytik Jena's new SpeedCycler beats even that. Described in company literature as "ultrafast," the German company's SpeedCycler runs a typical PCR protocol of 30 to 40 cycles in 13 minutes. Designed for either high-throughput needs or as a powerful standard cycler, the SpeedCycler can be purchased with either a 96-well or 36-well sample block, and can operate either as a stand-alone unit or be (optionally) PC-controlled. In the United States, the basic unit sells for about $8,000, or $9,500 with a computer.
Like the SpeedCycler, Applied Biosystem's new 9800 Fast Thermal Cycler is, well, fast. Taking typical PCR reactions from two hours down to less than 25 minutes, it may not be quite as fast as the SpeedCycler or Mastercycler ep, but according to Cunanan, that doesn't matter: Sample temperature, not block temperature, is what sets this instrument apart. Its competitors may have faster ramp times – that is, the time it takes for the whole sample block to reach a particular temperature – but, he says, the 9800 "reaches uniform sample temperature faster than any thermocycler on the market." Cunanan calls it a "hot-rod thermocycler." He says that the 9800 was originally designed for high-throughput labs, but all sorts of customers, including researchers from smaller academic labs, are buying it.
The system currently lists for $8,995, but Cunanan says the company is running a special promotional bundle at least through the end of 2005. Customers can purchase the instrument, a 96-well aluminum sample block module, and enough reagents to run reactions for about three months, all for $7,200.
Applied Biosystems also recently launched its 7500 Fast Real-Time PCR System. Selling for just under $50,000 and based on the same hardware as last year's 7500, the 7500 Fast includes an upgraded 96-well sample block that decreases run time from about two hours to 35 minutes.
In August 2004 Bio-Rad Laboratories of Hercules, Calif., purchased MJ GeneWorks, parent company for PCR industry stalwart MJ Research. As a result, Bio-Rad now sells MJ's thermocyclers, which have long been favorites among smaller research labs. Two new additions, the MJ Mini Gradient Thermal Cycler and the MiniOpticon Real-Time PCR Detection System, both released in May, might attract the attention of investigators who have been curious about real-time PCR but who were put off by the price of the instruments.
The 48-well Mini, says product manager Alex Vira, stands apart thanks to its high-performance capabilities. As Vira explains, thermocycler performance can be measured in three ways: well-to-well (or sample) uniformity, instrument-to-instrument uniformity (or accuracy), and speed. Most traditional instruments in the sub-96-well market are not subject to an especially high performance standard, Vira says. But the Mini performs as well as any high-end 96-well instrument, he says, particularly with respect to sample uniformity and accuracy. And its speed is comparable to Bio-Rad's other personal cyclers.
REAL TIME DATA:
Courtesy of Bio-Rad Laboratories
Fluorescence intensity vs. cycle number as measured by the MiniOpticon real-time PCR detection system.
Currently listed at $4,195, the Mini Gradient Thermal Cycler started shipping to customers last month. With advanced features such as gradient capability and calculated sample mode, the Mini replaces Bio-Rad's older, 25-well Minicycler.
But the Mini's real selling point may be the fact that it sits, or can sit, beneath a real-time system: the MiniOpticon. At about $16,500 (including the cost of the Mini but minus the computer), weighing only about 6.8 kg, and with a footprint smaller than that of a laptop computer, Bio-Rad's MiniOpticon is the smallest and most affordable real-time system on the market.
Customers can upgrade from the Mini or buy the system outright. According to product manager David Unger, the system will appeal to labs with budgetary and/or space constraints. Up until now, most researchers who have wanted to explore or rely on real-time PCR have had to access it through core facilities or share their equipment with colleagues. Unger says the MiniOpticon promises to "open up a whole new market. Anyone can now afford a real-time system. It will be a big seller."
Unger expects the system also will appeal to investigators who are interested in gel-free PCR, since real-time PCR obviates post-PCR electrophoresis, which means they will get answers – not just qualitative, but quantitative information – as the reaction proceeds. Moreover, when optimizing real-time PCR, particularly if probe-specific chemistries such as TaqMan are going to be used (as opposed to the easier-to-optimize but less specific SYBR Green I dye), optimization could be a bit easier than with competing instruments, because Bio-Rad's real-time systems have thermal gradients. They are, according to Unger, the only real-time cyclers on the market with that capability.
This month Bio-Rad plans to launch another new real-time system, the iQ5 Multicolor Real-Time Detection System. Selling for about $38,000 (without a computer), this 96-well premium instrument will have five-color detection and gradient capabilities; the MiniOpticon offers two-color (duplex) detection.
In December 2004, Stratagene of La Jolla, Calif., released its new real-time instrument, the MX3005P. According to instrumentation supervisor Mike Metzler, the new model still has many of the same features as its older sibling the MX3000P. With a list price of $29,000 ($4,000 more than its predecessor), the MX3005P offers a five-color detection system (versus the model 3000's four-color system), and comes bundled with primer- and probe-design software. Metzler describes the system as a good entry-level choice for researchers who are new to quantitative PCR and for smaller labs with small budgets.
According to Metzler, most real-time instruments in the 3005's price range are one-color instruments without multiplexing capability (or, in the case of Bio-Rad's Opticon, with duplexing capabilities only). With five-color detection the MX3005P can save researchers money in the long run, Metzler explains, by decreasing the per-sample reaction cost. He notes, though, that multiplexing poses an initial challenge, in terms of optimizing primers and probes and designing assays.
Arguably one of the most interesting real-time thermocyclers to appear this year could be BioTrove's OpenArray NT Cycler, which boasts a 9,000 plus-assay capacity. Colin Brenan, chief technology officer for the Woburn, Mass., company, calls this nanofluidics-based technology "high-density qPCR." Instead of wells, samples are loaded into tiny through-holes, each of which holds 33 nanoliters. The through-holes are arranged on nanotiter plates, or arrays, each about the size of a microscope slide, with more than 3,072 through-holes per array. The NT Cycler can accommodate three arrays.
Unlike other high-end real-time PCR systems, all 9,216 reactions are single-plex. "That's the beauty of it," says Brenan, who describes the process as "miniaturized PCR." You forego the optimization and other hassles of multiplexing but still get the benefits of cost-effective parallel processing. The cost benefits are even greater than with other high-throughput PCR instruments, Brenan says, because the smaller assay volumes reduce the assay cost five-to 10-fold, compared to current technology. PCR that utilizes 384-well plates costs an estimated 50 to 75 cents per reaction, Brenan estimates. But depending on reagent choice and other variables, the nanoscale volumes required of the NT cycler could bring that expense down to about 10 cents per reaction.
Brenan says both Big Pharma and basic researchers are interested in the new technology. Among other applications, the NT cycler can be used to validate and measure gene expression levels of thousands more different tissue or patient samples than other PCR technologies afford. The excitement, says Brenan, doesn't stem from the fact that reagents will cost less but that scientists can do so much more with the reagents they already have. "Now you can do a lot more analyses with the same amount of money," he says, "and that's really the name of the game."