This year marks the 25th anniversary of commercial thermal cyclers for the polymerase chain reaction (PCR). In brief, this technology amplifies segments of DNA. In such a device, PCR tubes go in holes in a thermal block. With everything loaded, the instrument runs the sample through cycles of temperature increases and decreases. Each cycle makes more nucleic acid.
As explained by Trisha Dowling, director of product management for PCR at Life Technologies (Carlsbad, CA), “Thermal cyclers use Peltier-based heating and cooling, and there’s not much variation across instruments outside that core technology for thermal cycling.” She adds, “Thermal cyclers are workhorse instruments and every molecular biology lab has at least one.”
Although the basic technology seems fairly stable among thermal cyclers, some trends keep pushing this technology to new capabilities. In large part, those trends push for faster speed in a variety of ways.
“One challenge for researchers using thermal cyclers today,” says Dowling, “is that they can be a bottleneck in their research.” This happens because these instruments make up an integral step of much of modern molecular biology’s workflow. “A thermal cycler is used for PCR, which is required in numerous genomic techniques including cloning, genotyping, and sequencing,” she says. “Given such a broad range of uses, scientists may have to sign up to use the thermal cycler in the lab.” So a waiting line of sorts can form at the thermal cycler.
Dowling notes that the throughput issue does not lie in individual thermal cyclers. Instead, the problem comes from so many users sharing them. She points out that users don’t always fill the block. “Someone might only run 10 or 20 samples, leaving significant capacity underutilized,” she says. “It’s like putting half a load of laundry in a washing machine. It still runs just as long to get done.” Depending on the manufacturer, thermal-cycler blocks come in a wide range of sizes, including 24-, 96-, 384-, and 1,536-well designs. Some blocks even include less common formats, such as 60 wells. At any block size, though, other scientists wait in line while a partial PCR load processes
To make better use of the space in blocks, some thermal cyclers can run more than one protocol in the same block. So some samples can be treated differently from others in the same run. This helps a lab make more efficient use of even a single thermal cycler.
Even when putting thermal cyclers through grueling schedules, including frequent use over many years, researchers demand that the instrument remains accurate. “Researchers see thermal cyclers as part of a very routine step, and the instrument needs to be very reliable,” Dowling says. “Scientists expect it to work the same every time.”
Perhaps because of the reliability of thermal cyclers, some researchers like to stick with an old favorite. “Some scientists like to use one specific block in one particular thermal cycler, or use the same thermal cycler every time,” Dowling says.
The need for speed
At the real-time PCR research and diagnostic core facility of the School of Veterinary Medicine at the University of California, Davis, director Emir Hodzic, D.V.M., Ph.D., often gets requests for fast turnarounds on samples. “Sometimes our clients ask for results within a couple of hours,” he says. “To go that fast, we need to change the thermal cycler’s block. That’s an obstacle to speed, and then you need to do some optimization.”
Dowling points out that “there are differences in thermal cyclers that are optimized for faster PCR reactions.” For example, she says, “Our Veriti thermal cycler is optimized to run both standard and fast PCR protocols at a range of volumes. Our GeneAmp PCR System 9700 offers multiple block choices of different alloys for faster sample ramp rates.” To run the process faster, the thermal-cycler block needs to accommodate faster temperature changes and the enzyme must also work at the new rate. By using a smaller volume, the sample’s heat can be changed faster and the enzymatic reaction works faster across the sample, too. “All those components must work together,” she says.
Another key factor to faster reactions is ease of setup and programming. As Dowling says, “Our Veriti thermal cycler has an intuitive touchscreen that makes it very easy to input and quickly start your PCR protocol, or to easily access one of the many preprogrammed methods.”
Today’s thermal cyclers offer many user benefits. In terms of actually running the device, Hodzic says that it’s so simple a child can do it. “It’s very easy,” he says, “like using a Mac.” The complexity arises in designing the experiments and determining what samples to test and when. “With Lyme disease, for example, you cannot get blood and look for the presence of infection at all time points, because it’s a very short time window.”
Standardization would also improve thermal cyclers, according to Hodzic. “There are so many different thermal cyclers from different companies,” he says, “and they all have different software.” He adds that some PCR master mixes work for one thermal cycler but not another.
Continuing to make thermal cyclers faster and more efficient will improve the vast range of genomic technologies that rely on this platform. As genomics itself expands into an increasing range of basic research and clinical applications, the need for thermal cyclers will grow even more intense. Beyond improving the data produced with thermal cyclers, Hodzic sees an even bigger goal. “We are here to help human health, animal health, and the environment,” he says. To do that, thermal cyclers must produce consistent results that can be compared from one experiment to another and between labs around the world.