by Caitlin Smith
The thermal cycler, or thermocycler, has become a central piece of equipment for many laboratories. Also known as PCR machines, thermal cyclers are devices that use a sample block that can heat and cool rapidly, often using Peltier technology. The blocks, which commonly are available in 96- and 384-well formats, hold sample tubes containing reagents used in the amplification of target DNA. A recent development in this technology is “fast PCR” cyclers and reagents, which enable researchers to accomplish PCR reactions in less time than ever. “About 90% of molecular biology laboratories use PCR on a routine basis, and thermal cyclers are the workhorse of a PCR laboratory,” says Kerry Lowrie, director of market development in PCR and nucleic acid synthesis at Life Technologies. “They are used in applications ranging from drug discovery and cancer research to forensic science and public health.” This article highlights some recent developments in thermal cyclers and suggests points to consider when looking for a thermal cycler.
Flexibility
As PCR has become a central technique in many research areas, it isn’t just molecular-biology labs that have thermal cyclers anymore. Indeed, PCR machines have become so user-friendly that even non-molecular biologists can use them without significant specialized technical training.
Today labs across many disciplines host thermal cyclers and use them routinely in their work. With such a diverse set of customers, thermal-cycler manufacturers are making their instruments more flexible to accommodate different uses and users. “PCR is one of the most commonly used applications in the lab, and many labs run multiple samples a day,” says Jaimie McLaughlin, product manager for PCR, detection and cell technology at Eppendorf. “They need to be able to obtain accurate results in rapid time with absolute reliability and flexibility. With new questions developing and new experiments forming, it is important that only an open and flexible system fits into your lab. With the Mastercycler® pro you can choose from three block formats, combine up to 30 units for ultimate throughput and use any plate, tube or strip you want.”
Bio-Rad’s C1000™ thermal cycler also offers interchangeable blocks for flexibility in sample configuration. The C1000 also offers a programmable temperature gradient, which allows the user to easily optimize annealing temperature in a single run. Additional features to streamline your PCR set up include automated protocol generation and Bio-Rad’s reduced-mass sample block, which shortens ramp and settling times. The C1000 can also be upgraded to a real-time system.
Agilent’s new SureCycler 8800 also addresses today’s need for flexibility in PCR instrumentation. It is capable of fast PCR, large- or small-volume PCR, gradient PCR (that does not sacrifice temperature uniformity). It also lets users switch between a 96- or 384-well format at any time. “[Some systems] make you choose between a fast version which has limited volumes, less uniformity with gradient or only one plate format,” says David Moore, senior product manager for qPCR and PCR instrumentation at Agilent Technologies. “The SureCycler doesn't require these choices and delivers the reliability researchers need to have faith in their results.”
Another system that flexes to accommodate the needs of different labs (and lab members) is the Applied Biosystems Veriti® Thermal Cycler from Life Technologies. Its Veriflex Block technology gives users flexibility in temperature zones within a run and in how many experiments can run simultaneously. “The Veriflex feature allows the user to set six individually controlled temperature zones to precise temperatures,” says Lowrie. “Unlike traditional gradient blocks, the Veriti thermal cycler is not bound to a computer algorithm to generate a ‘gradient’ of temperatures.” This helps save time by making optimization processes faster and more precise. In addition, the Veriti lets users run concurrent protocols. “The Veriflex feature allows the user to set up to six unique PCR conditions, making it possible to run multiple experiments at the same time,” Lowrie says.
Biometra also offers a thermal cycler that can run multiple independent experiments simultaneously, saving time and trouble. “We offer a triple-block instrument that can perform three independent PCR reactions at the same time,” says Holger Densow, product manager in life science at Biometra, referring to the T3000 Thermocycler. Biometra’s latest thermal cycler is the TProfessional model, which features five different exchangeable blocks. Two of the blocks (the 60- and 96-well modules) are made of silver and come with a gradient option. Because silver conducts heat well, the blocks can equilibrate quickly for better speed and temperature uniformity.
Speed and temperature changes
Fast changes in temperature are ideal for optimizing PCR protocols and for accurate runs. Densow notes important factors to consider are a thermal cycler’s average heating and cooling rates. “In most cases, only the maximum ramping rate is specified,” he says. “But there is no directive of how to measure the maximum rate. Companies can take a single peak value and specify this as the maximum rate. But much more interesting [are] the effective, or average, heating and cooling rates.”
Several companies offer thermal cyclers with fast changes in temperature. For example, consider “the extremely high heating and cooling rates of Eppendorf’s Mastercycler pro,” says McLaughlin. “Unsurpassed speed can be achieved with Eppendorf’s Impulse PCR, a device-driven, hot-start function that increases heating rates to 8°C/s. The gradient option of the Mastercycler pro features the SteadySlope® technology to ensure that the heating and cooling ramp rates are identical in both gradient and normal operation.”
The Mastercycler pro also has a newly designed lid with vapo.protect™ technology. “While [some] PCR instruments press a metal plate onto the PCR consumable, the vapo.protect lid utilizes a malleable material that molds to the consumables being used, thus forming a tight seal that drastically reduces evaporation,” says McLaughlin. “The vapo.protect technology leads to improved reproducibility and specificity at the rim and in the corners of the block, making it ideal for use with small sample volumes.”
For a small but powerful thermal cycler, consider the high-performance, small-footprint Piko from Thermo Fisher Scientific. Based on the company’s Piko PCR plates, the Piko thermal cycler is available in 24- and 96-sample formats. “It offers an exceptional thermal performance with extremely fast cycling times—PCR protocol times as short as 15 minutes are possible,” says Hanna Granö-Fabritius, product line marketing manager at Thermo Fisher Scientific. “It also features reduced energy and waste consumption, as the plates are only one-fourth of standard PCR plates. The plates nevertheless are compatible with standard liquid-handling instrumentation and multichannel pipettes.” Granö-Fabritius notes that customers who need a lower-cost “personal” thermal cycler, for one or just a few users or for occasional use, often have to make compromises when it comes to performance. “This is not the case with the Piko thermal cycler, which offers top-notch performance in a tiny box at a very competitive price,” she says.
Another high-performance, yet small, instrument designed for personal benchtop use is Bio-Rad’s MJ Mini 48-well gradient thermal cycler. The MJ Mini includes thermal gradient technology, which allows quicker temperature optimization by incubating at 8 different temperatures simultaneously. The MJ Mini’s 6 x 8 sample array accommodates a range of different formats, such as 0.2 or 0.5 ml tubes, tube strips, or 48-well plates.
User-friendly features for all user levels
Not only does your thermal cycler need to be convenient to use when you are flying through protocol after protocol—it also needs to be user-friendly for all levels of experience. “Ease of use and reliability are key points when selecting a thermal cycler,” says Lowrie. “PCR equipment is often shared within laboratories by all levels of scientists, from expert to novice. The cycler is expected to provide consistent DNA amplification every time. This means that it should be easy to program, intuitive to use and versatile enough to handle all the different PCR applications performed in the laboratory.”
Features that make thermal cyclers easier to program and connect to other equipment also can contribute greatly to ease of use. “In a lab with multiple users, Eppendorf’s intuitive graphic programming and networking capabilities [are] extremely important,” says McLaughlin. “Users are often excited about the intuitive and easy-to-use touch-screen interface of our new cycler,” says Agilent’s Moore. “It was designed from scratch with a touch screen in mind so users could jump right in, create custom protocols from scratch or with our PCR wizard and use productive networking features without buying extra software.”
Central to easy operation is the accessibility of the thermal cycler’s programming software. “Customers should look for a machine with easy-to-operate, intuitive software,” says Densow. The quality and responsiveness of service is important, too—as an everyday piece of equipment that will be used for years to come, it’s important to know that repairs will occur promptly. “Ask for service times and if a loaner unit can be provided while the instrument is repaired,” adds Densow. Also, be aware of the lifetime of the Peltier elements your thermal cycler may use and how the cycler’s lid functions. “The lid should provide a constant [contact] pressure regardless of the height of the plasticware used,” Densow advises.
Future directions
Densow notes that the two things all thermal cyclers can still improve on are speed and temperature uniformity: “Today it is impossible to build a block thermal cycler with a temperature uniformity ±0.0°C.” However, besides getting faster and more uniform, thermal cyclers likely will be used in new places in the coming years. “The next step for thermal cyclers,” says Lowrie, “will be to move out of the research laboratory and into more specific diagnostic-application and clinical-trial environments."
The image at the top of this article is Agilent's SureCycler 8800 thermal cycler.