Thermal cyclers (a.k.a. thermocyclers) were designed and built to maintain a given temperature for a given time, change to a different temperature and hold it for another given time, and so on. Although their main job remains performing PCR, it may be misleading to call them simply “PCR machines.” With much of what they used to be asked to perform—such as large-scale genotyping—shifting to real-time quantitative PCR (qPCR) platforms, researchers are finding other uses for these laboratory workhorses as well.
These days, “endpoint” (as opposed to real-time) PCR is used mainly for simple yes/no answers and fragment analysis—expression analysis, simple genotyping, and Sanger sequencing, perhaps disease monitoring and forensic research, or laboratory QC—where the researcher doesn’t want a long hassle with assay optimization. It’s used for manufacturing DNA and sub-cloning, with or without mutagenesis. But the instrument itself is also used “more and more for enzyme reactions as well—like restriction digestion and ligation, for example,” says Florian Hilbers, a PCR application specialist at Eppendorf involved with the beta testing program.
Maarit Tiirikainen, director of the University of Hawaii’s Genomics Shared Resource, uses her machines “not only for PCR cycling, but also as a kind of fancy incubator”—for all sorts of sample processes involved with microRNA research, for example. “It’s already there, it’s handy, and you would consider it a little bit more reliable for temperature.”
Here are some things to consider when looking to purchase one of these versatile instruments.
Configuration
Consumers are looking for robust and reliable systems that deliver high-quality, consistent results, and the instruments on the market “tend to work fairly consistently,” says Mike Mortillaro, owner of Bulldog Bio. “There are some differences, but they aren’t as dramatic as they used to be. Everything is within a few 10ths of a degree—within specs. When you’re looking at the majority of basic lab PCR protocols, you’re going to see no difference.” At least in terms of precision and accuracy.
But there are differences in other respects. Perhaps the most obvious of these are the type and number of consumable that can be accommodated. If there is such a thing as a “standard” research lab configuration, it will have a heating block that works with 96-well plates or 96 0.2 ml tubes (or tube strips). But many can handle 48-well or 384-well plates, or perhaps even microscope slides, while others offer the capacity for fewer, or different volume, tubes. Some instruments allow the user to select from interchangeable blocks, while others do not.
It may also be important to pay attention to the type of plate or tube you plan to run. For example, not all instruments will handle deep-well plates. And much to his chagrin, James Shira, research specialist at the University of Arizona Genomics Core, found that the Thermo Fisher Scientific Applied Biosystems Veriti™ thermal cycler that the core was demoing wouldn’t accept skirted plates.
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For those who need even more capacity, or want to run different experiments simultaneously, several instruments come with two, three, or even four blocks in a single instrument. Shira’s core, for example, runs multiple Bio-Rad Tetras (which are no longer manufactured), each with “four separate pods that can be independently programmed. And the footprint is less than the equivalent number of stand-alone thermocyclers,” he notes.
Other manufacturers allow multiple thermocyclers to be connected to and controlled by a single master controller—up to nine additional units, in the case of Eppendorf’s new Mastercycler® X50: “if you link ten 384-well cyclers together, you can run 3,840 samples,” points out Hilbers. These—as are many modern thermocyclers—are vented in the front and back, allowing them to be placed side-by-side without the fear of overheating.
Gradient and beyond
Tiirikainen’s core operates about 20 thermocyclers, and two of these can do gradient PCR—in which each row can be set to run at a different annealing temperature. “That’s good to have—at least on some of your thermocyclers—so that in the same experiment you can optimize the PCR reactions,” she explains.
Veriti’s 96-well VeriFlex™ block is divided into six zones in which the annealing temperatures are independently-controllable. (In contrast, a typical gradient instrument allows setting of only the low and high annealing temperature.) In addition to optimization, some researchers use this feature to simultaneously run separate experiments, as if the instrument had multiple blocks, allowing them to save on consumables as well as instrument usage—which is what attracted Shira to the system.
Eppendorf’s Mastercycler® X50 offers a two-dimensional gradient, “where you can set both a horizontal and a vertical gradient in the same PCR program, so you can basically optimize annealing temperature and also denaturing temperature,” Hilbers says. The company has data showing that optimizing denaturing temperature can produce improved specificity and a higher yield from the PCR reaction.
Wet and dry
Two issues that thermocyclers have notoriously suffered from are evaporation and condensation. Many instruments these days are equipped with a high-pressure lid, which goes a long way toward alleviating the problem of evaporation. Shira—whose Tucson locale may have drier air than most—relies on using the right labware and a good sealing film for his plates, and for good measure adds a squishy silicone mat (what he calls a “cycler implant”) between the metal lid and plastic.
The near ubiquity of heated lids has largely resolved the issue of condensation, notes Mortillaro. Most maintain a temperature of about 100 degrees, to keep the condensate from forming on the top surface—especially during the 4 degree hold step that follows many PCR reactions. (Hilbers recommends holding at 10 degrees instead of 4 degrees, which can also help lengthen the life of the instrument.) Some lids have adjustable temperatures down to about 30 degrees—which is something that appeals to Tiirikainen, who runs in vitro translation reactions overnight at 16 degrees.
Optional, highly thermoconductive blocks made of silver or even gold, for example, allow some manufacturers to boast a faster ramp speed—the rate at which the thermocycler goes between temperature—which in turn allows for a shorter run time of the PCR reaction. This, of course, is not to be confused with the related “fast PCR”, which generally takes advantage of specialized enzymes and protocols.
Some instruments can now be accessed—at least monitored—remotely, and some manufactures are currently developing or testing apps to check up on their thermocyclers.
Most (non-economy level) thermocyclers these days come with a touch screen, allowing users to more easily interface with, program, and run the instrument, and store, organize, and retrieve folders. Not all touch screens—nor user interfaces—are created equal, though. If this matters to you, test out how sensitive the screen is, how fast the response time, and how user-friendly the operation.
In fact, Hilbers recommends bringing in instruments for side-by-side demos—to test out the interfaces, to hear how loud they are, to see the actual run times as opposed to the heating and cooling rates...After all, you’ll probably be living with this workhorse for many years to come.
Image: The new Mastercycler X50. Image courtesy of Eppendorf.