Guide to Selecting a Thermal Cycler

Thermal cyclers (a.k.a. thermocyclers, a.k.a. PCR machines) are workhorses of the lab. Of course, they’re great for performing the polymerase chain reaction (PCR), but they’re likely the perfect tool for a host of other applications, from cloning and molecular biology to enzymology and thermal shift assays, that benefit from a steady, or controllably changing, temperature as well.

Thermal cyclers can, at this point, be considered a mature technology, with not many disruptive improvements over the past few years. But that doesn’t mean that they’re all the same as one another, or even the same as they were last time you paid attention. There are some extended features, as well as other differentiating factors, to be aware of when purchasing for the stable for the first time, or putting one out to pasture and replacing it with a new thoroughbred.

Priority list

There are a few things on a customers’ list of what’s most important, with the ability of the instrument to generate reproducible results generally being among the key points. Accuracy and sensitivity of the device—generating good results—is also on the list. “And then we talk about how compatible is the device throughout the whole workflow of the lab,” says Hanaë-Agathe König, Global Marketing Manager for PCR at Eppendorf.

She advocates an “open system,” which should be able to fit all kinds of consumables—high and low profile, skirted and non-skirted plates, for example. It should also be able to interface with other, upstream and downstream, systems.

Lab managers have their own lists, which might include pricing, supply, maintenance issues, and the like, she notes.

Just on paper—looking at the manufacturers’ published specifications—you probably won’t find too many black and white differences. But there is always “soft knowledge behind those numbers,” König says. She recommends evaluating instruments during a sales demonstration, using the customer’s own temperature verification system. Test the homogeneity across the block, whether the accuracy is what it says on paper.

A thermal cycler’s performance relies heavily on temperature control, and stability, and so periodic calibration and maintenance are crucial to assure their performance, says Jeffrey Lai, Product Supervisor of Blue-Ray Biotech.

An added benefit to having their own verification system is that customers can also check from time to time if the cycler still reaches the temperature that’s set in the display, König points out, and avoid the downtime of sending the instrument out.

Mike Mortillaro thinks that “probably the number one [purchasing consideration] is reliability. Back in the ‘90s and even the early 2000s … it was not uncommon for certain brands to have failure rates of (on average) within two years,” says the owner of Bulldog Bio. “Nowadays, I think you can get four or five plus years out of a thermal cycler without any chance for failure, and we certainly have systems that have been out there for 10-plus years … A lot of this is driven by use.”

You can ask a sales representative or product specialist what the failure rates are, and if they even have any data on those, he says. Another indicator of reliability is to look at the warranty coverage. Two years is pretty standard, but “there are companies that will add extended years because they have confidence in their systems.”

Look at me

Nearly every new thermal cycler is controlled by touchscreen. This is already a mature technology, with more or less comparable sensitivity between cyclers, König says. Where these may differ is in the logic and software behind the way the user interacts with and operates the instrument. If that operation “is too difficult, or if it’s not user-friendly, then it … could get quite frustrating to work with.”

Mortillaro echoes the importance of an interface “that’s easy and intuitive, … that everyone is comfortable with.”

In his experience, most labs are not interested in remotely accessing their thermal cyclers: “I’m not sure what that gets you because almost all systems, whether they have remote access or not, will tell you how long it takes for the system to run. And everything is loaded manually, so you need to be in front of the system to load it.”

Yet operation and monitoring a single instrument during an experiment is just one function of an interface, whether local or remote.

Documentation of the runs—especially important for regulated labs, as well as those planning to publish their results—is controlled and accessed by the interface.

It determines who gets access. “Most of the cyclers nowadays have user management—it’s just a matter of how comprehensive the user management function is,” points out König. Some cyclers will allow two levels of access (administrator, and guest not requiring a password) while others allow for at least three levels (administrator, normal user, and restricted user). “Especially for regulated labs, where they have technicians who are meant to only run a program and cannot do anything else, restricted user is a very good function to control … the security of the experimental process.”

It can facilitate control of multiple cyclers connected to a “master” cycler. Supplementary units may be ordinary thermal cyclers, as is most often the case; or they may be pared down units lacking their own touchscreen, reducing the cost of the supplementary devices.

Multiple blocks

An interface can also allow for independent control of multiple, independent, blocks in a single instrument.

Many instruments offer modular heating blocks. For example, Constantine Garagounis “can easily swap from 96-well to a 3x21-well format,” the PCR Biosystems Marketing Specialist relates. “The former is good for running plates or gradients, while the latter allows running of up to three completely different thermocycling programs independently … without affecting the two other parallel runs. This comes in handy when running parallel small experiments yourself, or when multiple people need access to the thermocycler at once.”

Multi-block instruments are a very common feature for labs to consider, says Mortillaro. They’re less expensive than purchasing multiple single-block units, and take up less space. Though that needs to be balanced with the reality that “any time the system is down, all of the blocks are down.”

Gradients

A gradient function is an option on many newer thermal cyclers. The goal, as the name implies, is to achieve a gradient across the block to be able to simultaneously assess several temperatures for optimum primer annealing. But, as Mortillaro points out, a lot of labs don’t realize that the denaturing temperature can make a difference in PCR performance as well: one polymerase may be a little more heat sensitive than another, for example, or the complexity of the template may require additional heat. “The gradient function allows you to optimize around those as well.”

He points out that many enzymatic assays—both cellular (for example the Cellular Thermal Shift Assay (CETSA))¹ and biochemical—require temperature control, and gradients can help find the optimal temperature for those assays.

Ramp rate and fast PCR

Sometimes it’s important to control the transition time between temperatures. Take the T7 endonuclease assay for validation of CRISPR/Cas editing, for example. For some steps “the sample should be cooled down from 85°C to 25°C in a -0.1°C/second cooling rate,” notes Lai. To cope with these new applications, newer thermal cyclers must be more flexible in protocol programming. In addition, he points out, the temperature of the heated lid used to be fixed at 105°C; now it is often designed to be adjustable.

And sometimes users just want those transitions to be as fast as possible, and “there are some super-fast systems out there,” says Mortillaro. But the time savings in transition time versus total run time is subject to the law of diminishing returns.

König reminds us that “you cannot just rely on the specification,” since a manufacturer generally reports a maximum heating and cooling ramp rate between two specific temperatures—say, from 70°C to 75°C—“and most of the time it is running at a very much lower ramp rate.” Instead of comparing maximum ramp rates, she recommends comparing total run times of a standard protocol. Fast PCR also depends on the consumables’ heat transfer properties as well as the speed at which reagents react to the temperature change, and “each represents a bottleneck in the entire PCR workflow.”

The race is on. Once the basics—such as reliability, accuracy, and precision, perhaps cost and compatibility—are taken care of, look at the bells and whistles you want or can’t afford to be without, and place your bets. May the best thermocycler win.

Reference

1. Martinez, N.J., Asawa, R.R., Cyr, M.G. et al. A widely-applicable high-throughput cellular thermal shift assay (CETSA) using split Nano Luciferase. Sci Rep 8, 9472 (2018). https://doi.org/10.1038/s41598-018-27834-y