by Gina Shaw

Nick Cirino remembers clearly when the urgency of investing in real-time PCR hit home in his laboratory. "Right after the 2001 anthrax events, it got integrated here very quickly," says Dr. Cirino, who directs the Biodefense Laboratory at the New York State Department of Health's Wadsworth Center in Albany. The majority of his research is focused on identifying potential targets for diagnostics, therapeutics, and/or vaccines for infectious diseases and potential biological weapons.

Lakshmi Gopinathan examines the alteration of gene expression using reverse-transcriptase polymerase chain reaction (RT-PCR).
Specificy and speed
"In the public health field, we're all starting to migrate over to real-time PCR to enhance throughput for diagnostics," Dr. Cirino says. Culturing, which has long been the gold standard for diagnosing infectious disease, has one very obvious limitation: you can't force bacteria to grow faster. "It takes 24 to 96 hours to grow some of these organisms and identify them, while with PCR-based approaches, you can have a pretty definitive rule-in diagnostic within about six hours. Not only that, but it's very specific - you can identify not only the type, but actually the species, right down to the strain."

Within the last five years or so, real-time PCR has gone from a tool largely limited to major research institutions and pharmaceutical companies to a must-have resource for even the smallest of labs. Gene amplification using traditional PCR has been a staple of many of these labs for years, but the revolutionary gene-amplification tool had clear limitations. Traditional PCR detects amplification at the end point of the reaction, and is only semi-quantitative at best, while real-time PCR measures the kinetics of the reaction in - of course - real time.

"In the last three or four years, the biggest advance that's really changed what my lab does is real-time PCR. The price of these units has basically dropped in half in that time, and it's made quantification of gene expression really easy for us," says Jack Vanden-Heuvel, PhD, Associate Professor of Molecular Toxicology at Penn State University Institutes of the Environment at University Park. When we used to measure gene expression using PCR, we had to create a new internal standard, which took about three days, and in a day's time we could analyze about 100 reactions. Now, with real-time PCR, we can probably get three times that number of genes done in one day, running three 96-well plates."

Helicase-dependent amplification developer Huimin Kong, shown here in his lab with colleague Jamie Wytiaz, hopes to partner with diagnostic companies to bring HDA technology to the in vitro diagnostic field.
Multiplexing multiplied
That's particularly critical for a public-health focused lab like Dr. Cirino's, where PCR priorities include the ability to handle surge testing during an outbreak, and the ability to multiplex, or run more than one specific amplification reaction in a single tube. "There are now probably about eight different platforms you can use to run real time PCR. All differ slightly in their sensitivities or their level of multiplexing," Dr. Cirino says.

Different-colored dyes are used to detect different products in the multiplexed system. Currently, five is about the limit for existing real-time PCR systems, like the Mx3005P from Stratagene (La Jolla, Calif.), but most vendors aren't supporting them all, says Dr. Cirino. "The more dyes that can be spectrally resolved on the system, the more useful it's going to be. We're working with several dye developers to look at bleed-through, light intensity, and other factors, and then provide feedback to the system manufacturers to make them more amenable to multiplexing. Within two or three years, we'd like to get the capacity up to ten dyes."

Amplification without thermal cycling
For many virologists, toxicologists and molecular biologists in fields like public health and environmental science, a crucial limitation of existing PCR technology is this: you can't take it anywhere. "In the field, you need miniaturization and portability, which means features like nanotechnology and microfluidics," says Pamela "Scottie" Adams, Manager of the Molecular Biology Core Facility at the Trudeau Institute (Saranac Lake, NY).

But standard methods of denaturing the targeted DNA - using a thermal cycler to achieve temperatures above 90 degrees Celsius - mean that current PCR approaches can't be used in the field, whether at the scene of a disease outbreak, potential biological weapons attack, or an environmental disaster, or simply at many point-of-care locations.

"That's why there's now a big push for isothermal PCR," says Dr. Cirino. "Instead of using heating and cooling to amplify, you add an enzyme which will amplify the nucleic acid at one temperature. There are a couple of promising systems out there now; they're not as efficient and robust as real-time PCR, but they have important advantages for certain purposes."

One way to skip the thermal cycler is to uncoil DNA using the same tool as in nature: the enzyme helicase. "We wanted, as closely as possible, to mimic nature," says BioHelix Corp. (Beverly, MA) founder and CEO Huimin Kong, the developer of helicase-dependent amplification, or HDA. "We can do our reaction in a simple water bath. If you have a stable reagent, you don't need a fancy, expensive instrument. As a proof of principle, we have developed products that are being marketed by a leading educational supply company, such as a sickle-cell anemia test that is a teaching tool for high school students."

If your tenth-grader can amplify DNA in the classroom, that should tell you something about how simple and cost-effective HDA must be. Of course, it's also far from ideal for every situation. It can only amplify about 100 to 150 base pairs, which is Lilliputian in scale compared to what's required for the amplification of entire genes. Kong is under no illusions that DNA amplification using HDA will replace other methods in most settings. But he knows his market. "Point of care diagnostics is one of the things we can do better than standard PCR, because in most cases all you need to know is a small fragment, and HDA requires less amplification," he says.

Further miniaturization
In larger commercial labs, the 96-well plate systems that seem Autobahn-fast to many noncommercial institutions are starting to look like yesterday's VCR, as researchers are already looking beyond 384-well plate systems (to instruments such as the Applied Biosystems' 7900HT, which can run a full set of amplification cycles in under 40 minutes) to an exponentially faster horizon.

"We do everything now in 384-well format, and our next jump up will be 1536-well plates, but we're really looking further downstream to use nanotechnology to create arrays in the 10,000-well capacity," says Eric Fedyk, Senior Scientist II, Discovery Technologies with Millennium Pharmaceuticals (Cambridge, MA). "There are a lot of hurdles to making something that small, but it's critical from a cost perspective. There are a number of prototypes out there for a massive throughput approach that have just really emerged in beta-testing in the last year or two." Each has its own obstacles, Fedyk says, though he declined to identify the players: one group has been having problems with reproducibility, and another company has been running short of cash for what Fedyk calls a "very interesting" system.

Such nanotechnology approaches, if successful, will also address another challenge for commercial labs: continuing to boost sample throughput while at the same time increasing gene throughput. "Real-time PCR and microarrays are like yin and yang: with an array, you may only be able to afford to look at ten samples, but you see the whole genome in those ten samples," says Dr. Fedyk. "With real-time, for the same money, you can study thousands and thousands of samples, but only a couple of genes."

Multiplexing is one option - but unlike in Dr. Cirino's lab, pharmaceutical scientists aren't looking to go from four or five genes in a sample to ten - but rather, to 20, 30, and beyond. "That's very difficult to develop in multiplex, and you sometimes lose the sensitivity of the overall assay and some of the dynamic range," says Dr. Fedyk.

So instead, Millennium is investing in a "mega-parallel approach," using many more single or duplex assays. "The goal is to harness PCR's sensitivity, reproducibility, and dynamic range, but to be able to use it in a wider format," Dr. Fedyk says. "The road's been paved. If we can reduce the scale and increase the number of different genes being looked at, that will really usher in the era of pharmacogenomics."