by Caitlin Smith
Who wouldn’t want to cut their 90-minute PCR protocol down to a 30-minute run? It can be done with fast PCR, but scientists are reluctant to embrace this new take on their tried-and-true method. After all, why rock the boat with such an important technique?
Taking a second look could save you time and money. And many labs, from big to small, are incorporating fast PCR steps to save time. There are three ways to make PCR faster: quicker temperature ramping, protocol changes that shave time off of most steps (and sometimes even combine steps), and enhanced enzyme functions. (An illustrated tutorial is available on Biocompare.) With this range of ways to speed PCR comes a myriad of new products aimed at those interested in fast PCR, with some systems promising a standard PCR run in less than 30 minutes.
Is this too good to be true? In some cases no, but there are points of caution that should be navigated. For example, the improvements in PCR instrumentation’s ability to change temperatures quickly must be matched simultaneously with improved enzymatic abilities. “As instrumentation ramp rates continue to improve, the move to protocol modifications using wild-type enzymes can increase the chances of reaction failure and poor performance,” says John Foskett, technical director at Kapa Biosystems. “As the ramp rates of thermocyclers increase, the extra time provided for each step in the cycle is reduced.” Kapa Biosystems focuses on polymerase engineering to keep pace with advances in instrumentation speed. As fast PCR evolves, all the components must improve together for the technique to continue as a true advance.
Overcoming fears and misconceptions
Even though fast PCR offers obvious advantages, many scientists are wary of replacing reliable techniques that work, even if they do take three or more times as long to run. “The main challenge facing customers who want to run fast PCR is data quality,” says Applied Biosystems’ Edwin Hauw, product manager for real-time PCR reagents. “Many researchers fear that by cutting down the reaction times, they may lose the high quality data they require.”
There are other reasons for their reluctance: “Even with fast PCR reagents such as ours that do not require a specialized thermal cycler, many researchers are reluctant to try a new method, despite the considerable time savings,” says Ken Doyle, Epicentre Biotechnologies’ director of technical and marketing communications. “[Some] fast PCR kits also do not perform well with long or difficult amplifications.”
Companies are trying to make it as easy as possible for researchers to take the plunge and get started in fast PCR, so convinced are they that people won’t look back once they have tried it. Applied Biosystems, for example, offers four new instruments that differ in levels of throughput and are designed to convert people to fast PCR. “The Applied Biosystems instruments have built-in fast protocols to make transitioning to fast easy for the customer,” says Hauw. Besides selling the complete range of fast PCR reagents individually, Applied Biosystems also sells them together as validated “off-the-shelf assays.”
Epicentre is even offering free sample sizes of their fast PCR kits to encourage scientists to try them. “We are confident that once customers try the new kits, they'll never go back to ‘slow PCR’ again!” says Doyle.
Instrumentation complications
One of the key components to shaving time from PCR protocols is ramping the temperature changes more quickly; consequently, most major cycler manufacturers now offer thermocyclers that are capable of fast ramping. But simply speeding the ramp may not be enough to obtain the results you need.
“A key component of overall protocol run time is the time to reach target temperature, which is determined by the average ramp rate and the time for the sample block to reach thermal uniformity,” says Hilary Srere, marketing manager for amplification in the gene expression division at Bio-Rad Laboratories. “Maximum ramp rate is less important, because it can fluctuate significantly during the ramp.” To address this potential problem, Bio-Rad offers their C1000 and S1000 thermal cyclers, whose “tight temperature control produces high average ramp rates and uniformity during ramping, to yield fast time-to-target-temperature and faster protocol run times,” according to Srere. These cyclers also have six independently controlled heating elements, which maintain a tighter temperature uniformity across all wells at each step – allowing a precision that is especially important in real-time PCR.
As thermocyclers improve with shorter ramp times, other complications arise. Foskett cautions that combining fast PCR reagents with the newer fast thermocyclers can be disastrous for your experiment, though it sounds contradictory. “We've seen that the majority of fast PCR reagents fail completely when the protocols are transferred to fast thermocyclers,” says Foskett. “qPCR is especially prone to reduction in performance (specifically amplification efficiency and sensitivity) when cycling parameters are too fast. This presents a challenge for determining what protocols are required for specific assays on specific instruments.”
Foskett says that a major challenge in the development of fast PCR is determining which applications are best suited for conversion to fast PCR assays. “Often fast PCR protocols tend to compromise one or more of the reaction parameters necessary for efficient amplification,” he says. “For example, insufficient DNA denaturing or primer annealing can have dramatic effects on PCR performance, causing reaction failures for assays already operating on the edge of success.” These effects are exacerbated when fast protocols are transferred to fast thermocyclers, according to Foskett, because the “extra” time provided during the ramping and cooling of a slow block is lost on fast thermocyclers. “The programmed time on a fast cycler is much closer to the actual time of the PCR reaction,” he says.
Reagent and enzyme evolution
While advantages promised by fast PCR are alluring, the price tag of new instrumentation is prohibitive for some researchers. But if you are one of these researchers, take heart—it is still possible to speed your research with new enzymes and/or reagent mixes. For example, Epicentre offers their TAQXpedite™ Fast PCR kits that work with conventional or real-time PCR instrumentation, and can be adapted to high-throughput use. Primers are being optimized for fast PCR, too. Natasha Paul, senior staff scientist at TriLink BioTechnologies, says that their “CleanAmp™ Turbo Primers show significant benefit in fast PCR by improving the specificity of amplicon formation ... as they have a faster rate of deprotection.”
The enzyme
is arguably one of the most important reagents in a PCR reaction. Polymerase engineering improves the speed and performance of fast PCR because the enzyme becomes a limiting factor as you approach the physical and biochemical limits of the reaction. “Addressing fast PCR at the level of the enzyme allows Kapa Biosystems to supply fast PCR kits that achieve significantly reduced reaction times (up to 70%) without compromising performance or the need for specialized consumables or instrumentation,” says Foskett. “This contrasts with other fast PCR kits using standard Taq polymerase that are limited to the extension rate of 30 – 60 seconds per kilobase. To achieve fast PCR these kits must recommend protocol modifications (such as decreased denaturation and annealing times) that can lead to significant reduction in the efficiency of the reaction. Fast PCR becomes slow PCR when you have to repeat your experiments due to reaction failure.”
Two new enzymes offered by Kapa Biosystems are incorporated into their fast PCR kits as well. The DNA polymerase KAPA2G is capable of extension times as low as one second per kilobase. “This speed is possible because the KAPA2G DNA Polymerase catalyzes the synthesis of DNA at a much higher rate than standard wild-type enzymes like Taq and Pfu. The specific activity of KAPA2G DNA Polymerase has been measured to be 2.5x – 3x higher than Taq polymerase,” says Foskett. Another new enzyme, KAPAHiFi DNA Polymerase, was engineered to have both high speed and exceptional fidelity. “Other companies offering fast, high fidelity PCR fuse accessory proteins to wild-type polymerases to achieve improved processivity,” explains Foskett. “The speed of KAPAHiFi DNA Polymerase is the result of increased processivity that is intrinsic to the actual enzyme, without the need for fusion technology.”
Reaching ahead
Doyle believes that the adoption rates of fast PCR will grow significantly, as researchers realize that “fast PCR kits perform as well as conventional PCR kits and save time, thereby increasing their productivity. This is especially true for real-time PCR and/or high-throughput applications, where obtaining results faster or processing more samples per day can offer a competitive advantage to the researcher.” Similarly, Paul forecasts “the development of faster cycling kits with its application to one-step RT-PCR and multiplex PCR.” It won’t be long before PCR, as most of today’s researchers have learned it, becomes a rarity—if not a memory.
