by Caitlin Smith
Thermal cyclers, also known as PCR machines or thermocyclers, have come a long way from the earliest models. Continual demands of researchers for faster, shorter, and more accurate PCR protocols have resulted in beneficial changes to the basic thermocycler model. “You want a cycler that will fulfill all your needs in the lab,” says Beverly Cutson, product manager for PCR and detection at Eppendorf. “It needs to be accurate, reliable, and able to accommodate the changes in the rapidly moving field of molecular biology. These changes include a shift to lower sample volumes, and faster protocols.”
Indeed, the newer protocols, known as “fast PCR,” significantly reduce PCR run time and increase productivity, and they are still gradually replacing old PCR protocols in many instances. Many researchers could still benefit from these newer fast PCR protocols, and the new thermal cyclers that better support them. “PCR continues to be one of the most widely used applications in molecular biology laboratories today,” says Pete Skirpstunas, product manager in the gene expression division at Bio-Rad Laboratories. “Although thermal cyclers have been produced for many years, there is still room for improvement.” This article spotlights some of the technological features of today’s thermal cyclers that are helping to meet the demands of researchers who are pushing their thermal cyclers to give them better data, faster.
Faster temperature changes and shorter settling times
Fast PCR is steadily becoming more accepted. This is significant for a method like PCR, in which researchers get comfortable, almost superstitious, with their normal protocol, and hesitate to change. “The advancement of fast PCR is one development that has an important impact on researchers today,” says Skirpstunas. “By using a combination of protocol adjustment, high performance reaction modules and fast enzymes, researchers are able to generate more data and accelerate their research.”
Methods for rapid changes in temperature are especially important for fast PCR, which uses shorter incubation and ramp segments to save time. Bio-Rad’s 1000-series thermal cycling platform offers innovations to support these changes. Their platform uses a “patented mass-reduced honeycomb reaction block for fast heating and cooling and short settling times,” says Skirpstunas. “Both of these features translate to shorter run times and increased productivity as more runs can be completed in a shorter period of time.”
Skirpstunas believes that “one of the biggest challenges is simply creating and optimizing protocols, particularly for novice users. “With the on-board protocol Autowriter [on the C1000], Ta calculator and thermal gradient, we believe the C1000 offers features that allow researchers to easily overcome these challenges,” he says. “Finally, the 1000-series thermal cyclers also offer a dual 48-well reaction block with gradient capabilities. This allows quick protocol optimization as well as the ability to run two completely different protocols simultaneously. It is like having two cyclers in one.”
An additional exciting area in PCR, according to Skirpstunas, are new methods in sample preparation for next generation sequencing. “In particular, techniques such as emPCR remove the bias of amplifying high abundant sequences in a mixed population,” he says. “Another area of excitement is the development of advanced PCR enzymes. Fusion proteins like the Bio-Rad iProof enzyme with increased processivity offer the advantage of improved speed, sensitivity, and tolerance to inhibitors.” Bio-Rad’s iProof DNA polymerase is a high-fidelity, thermostable enzyme. Engineered using Bio-Rad’s Sso7d fusion technology, iProof gives higher yields, speed, and fidelity. In fact, according to Bio-Rad, iProof is 52 times more accurate than Taq polymerase, and can amplify DNA fragments up to 37 kb in less time with less enzyme.
Reducing edge effects
With PCR being one of the most commonly used applications in the lab, many labs need to run multiple samples a day, yet they also need accurate results in a short time. Cutson thinks that some of the most exciting new developments in conventional thermocyclers today are the “fast ramp rates and innovative technologies, such as [Eppendorf’s] vapo.protect, that address the changing needs of labs,” she says. “With a shift to lower sample volumes, you run an increased risk of evaporation that can negatively affect your results. Evaporation can skew the concentrations of the various components of the PCR reaction, which in turn can adversely affect the amplification of the desired target. A reduction in evaporation can help to ensure a better result.”
To do just that, Eppendorf’s Mastercycler pro offers a newly designed lid using the vapo.protect™ technology. “This new lid utilizes a malleable material that molds to the consumables being used, thus forming a tight seal that drastically reduces evaporation,” says Cutson. Thus, the Vapo.protect™ technology basically eliminates edge effects and is ideal for use with small sample volumes.
Conserving time with other innovations
Another way to speed PCR is to bump up the throughput, as with the Veriti® 384-Well Thermal Cycler by Applied Biosystems. With the Veriti, you can network up to 12 machines together, while controlling them all from one machine. Programming is made easier by large color touch screens, with pre-programmed methods or user-entered thermal cycling steps. Smaller throughput needs can be accomplished by Applied Biosystems’ GeneAmp® PCR System 9700, which is designed to give researchers the flexibility to tailor their work using interchangeable sample block modules. While designed for 384-well plates, the GeneAmp® machines don’t network together as do the Veriti® machines.
At about half the size of most benchtop thermal cyclers, the Piko® Thermal Cycler by Finnzymes can both save you some bench space and speed your PCR work. Indeed, Finnzymes claims that you can complete a PCR protocol in as little as 10 minutes, because the small size helps to facilitate faster ramp rates and quick settling times. The Piko PCR Plate is only one-fourth the size of a conventional microplate, while still using standard sample capacity and well-to-well spacing, making the Piko Plate faster and more economical. The small PikoThermal Cycler also uses less power than other benchtop models, and is portable for use in the field.
It’s likely that we will continue to see a steady increase in throughput and decrease in run times as thermal cycler manufacturers try to meet the needs of researchers. “Thermal cyclers need to be able to address the new applications that are emerging as well as be able to function with various sample types,” says Cutson. “The PCR system needs to be flexible so that it can progress with the times. Scientists are looking for thermocyclers that will address their needs today and in the future.”