by Caitlin Smith
Can you complete a PCR run in less than 30 minutes? If not, check out “fast PCR,” a term coined to refer to a standard PCR protocol that underwent a make-over for speed—namely, faster temperature changes, faster enzymes, and more reducing or combining of steps in the protocol. (See Biocompare for an illustrated tutorial.)
But speed is not free. “The biggest challenge in fast PCR is speeding up the PCR reaction without sacrificing reliability,” says Chas André, applications manager at Finnzymes Instruments. “In order to obtain robust and consistent results in fast PCR experiments, it is crucial that each of the different components of the system have been optimized to avoid variability in PCR reaction rates from well to well and run to run.” André says that different components of a PCR system such as “block speed and uniformity, thermal transfer through the reaction vessel, and speed of the polymerase” must be optimized simultaneously. “Therefore, in the future, the trend will be for PCR systems in which all aspects have been co-engineered for maximum compatibility,” says André. Such co-engineering is already happening with enzymes, instruments, and buffers.
Faster enzymes
At Kapa Biosystems, according to technical director John Foskett, their DNA polymerases are engineered using a process of molecular evolution. “Our KAPA SYBR® FAST qPCR Kits contain a polymerase that is significantly less inhibited to SYBR® Green dye while exhibiting a very high affinity to DNA,” says Foskett. “As a result, the KAPA SYBR® FAST qPCR Kits are able to achieve the fastest reaction times while maintaining high reaction efficiency and sensitivity. Our competitors typically increase the concentration of wild-type Taq polymerase to achieve faster cycling conditions, which can come at a cost to reaction efficiency.”
Kapa Biosystems recently released fast PCR kits based on their engineered enzyme technology, including KAPA2G Fast HotStart, KAPA2G Robust HotStart, KAPA SYBR® FAST qPCR Kits, and KAPAHiFi HotStart DNA Polymerase. “All these polymerases are highly processive and are capable of extension times of 1 sec/kb without a loss in performance,” says Foskett. Kapa also plans to release their Fast qPCR Kit for other probe chemistries in April 2009. “We are excited about our KAPA SYBR® FAST qPCR Kits because this is the first time a researcher will be able to have their cake and eat it too, with respect to performing ultra-fast qPCR while maintaining high reaction efficiencies, linearity, and sensitivity,” says Foskett. “This is achieved with the KAPA SYBR® DNA Polymerase evolved specifically for qPCR.”
Engineering enzymes is also the work of Finnzymes Instruments. “The speed and reliability of our Phusion and Phire DNA Polymerases is based on several features,” says André. “One, they are highly processive. Two, they utilize higher denaturation and annealing temperatures allowing shorter hold-times and enabling simpler two-step protocol design. And three, hot start versions of the polymerases are based on a unique technology that allows zero-time reactivation.” André believes that newly designed polymerases that better mimic biological replication complexes (and thus have greater processivity and inhibitor resistance) are an exciting development for fast PCR. “This in turn has enabled ultra-fast PCR, direct PCR, and other highly demanding PCR applications,” says André. “Time is saved not only in the PCR itself, but also in sample preparation, since PCR can be performed directly from a variety of tissues without DNA purification or extraction.” For example, Finnzymes’ Direct PCR lets you amplify directly from the sample source with no DNA purification step. “Direct PCR is possible with Phusion and Phire DNA Polymerases because these robust enzymes are exceptionally tolerant of many PCR inhibitors present in unpurified samples,” says André, “and are also able to withstand the higher temperatures employed in these direct protocols.”
Instruments of speed
Ideally enzymes evolve at a pace with PCR instruments and reaction vessels. Finnzymes also offers the Piko® Thermal Cyclers, whose heating blocks give fast ramp rates (about 5°C/s) and short (less than a second) settling times, and reaction tubes. “The final rate-limiting step in the PCR protocol is the heat transfer between the block and the reaction mixture,” explains André. “The thinner the tube walls, the faster the heat transfer, and thus, the protocol. Finnzymes ultra-thin walled UTW® reaction vessels are manufactured with a novel method of injection molding polymers. UTW vessels have walls that are less than 150 microns thick—half the thickness of conventional thin-wall PCR tubes and plates.” The Piko system also gives you a green option. “‘Green PCR’ means minimizing environmental impact while achieving the best possible results,” according to André, who says the 96-well Piko Thermal Cycler cuts energy and plastics consumption by 95% and 75%, respectively.
Idaho Technology adds another dimension to their new fast PCR system with high-resolution melting analysis, a method that compares melting temperatures to study sequence differences. “The LightScanner 32 is our newest high-speed real-time PCR machine with the highest resolution melting,” says Cameron Gundry, scientist at Idaho Technology; it also offers multiplexing capabilities. “We offer three-channel detection enabling for any chemistry,” says Gundry. “In addition, the one-color hi-res melting block that is hidden nicely within the machine, offers a new type of multiplexing suggested by others. This is the ability to use real-time PCR and hi-res melting information.” Gundry notes that a challenge for fast PCR is maintaining homogeneity among samples throughout the heating block. “Some other manufacturers may offer faster cyclers, but homogeneity is sacrificed. In our LightScanner 32 system, the high-speed air chamber instantly reacts to the heating element, creating extreme homogeneity at the fastest rates of temperature ramping.”
Buffers and kits
New buffers or master mixes may also accelerate your PCR work. For example, QIAGEN’s Fast Cycling PCR Kit is a master mix that includes their new Q-bond molecule that facilitates fast cycling (such as 15 minutes for 200 bp templates), and their HotStarTaq Plus polymerase. “The combination of the polymerase master mix and PCR buffer system provide researchers with a protocol requiring virtually no optimization,” says Quan Dinh, North America marketing manager for application DNA business at QIAGEN. Their Type-it Fast SNP Probe PCR for SNP (single nucleotide polymorphism) genotyping has a unique PCR buffer system that lends itself to “narrower melting peaks leading to higher allelic discrimination, higher call rates, and less error,” according to Dinh. “The combination of the polymerase, the buffering system, and Q-solution provides researchers with a pre-optimized protocol compatible with standard ramping PCR, standard fast-ramping PCR, and real-time PCR thermal cyclers. The complete procedure takes 40% less time than other probe-based SNP genotyping systems.”
Whether you augment your current PCR protocol with engineered enzymes, new instruments, or new buffers, there are likely easy ways to speed your PCR to 30 minutes or less. This will soon have important consequences, according to Foskett: “Coupling high-performance reagents with high-speed instrumentation is a growing trend that will accelerate the pace of discovery in genomics, genetic testing, and molecular diagnostics.”