After Nobel laureate Kary Mullis developed the polymerase chain reaction in 1983, many scientists wanted automation for this laborious process. Just a few years later, the first commercial thermocycler provided that automation. Although usually known as PCR machines that amplify the nucleic acids in a sample, the controlled heating and cooling in a thermocycler can be used in many other applications, including DNA sequencing, cloning, gene-expression studies, and more. To pick the best instrument for a specific lab, it helps to review some device details and a variety of applications.
First, let’s take a look at what a thermocycler does. It’s designed “to thermally cycle your samples for certain periods of time,” says Jeffrey Lai, product supervisor at Blue-Ray Biotech. “A user can program the instrument to change between several incubation temperatures, each for a certain amount of time, and then repeat the temperature change again and again for certain cycles.” The temperatures and timing can be about as complex as a scientist desires.
As scientists develop new methods, thermocyclers appear in even more workflows. For example, thermocyclers can be used in CRISPR-Cas 9 gene-editing technology. Lai also points out that thermocyclers “can be used to perform reactions that need to keep precise incubation temperature, including some enzymatic kinetic reactions.”
New challenges in science and world health are also driving scientists to expand the use of thermocyclers. Plus, new scientific methods and more advanced thermocyclers combine to offer new ways to make use of these devices.
The evolution of accuracy
With the first commercial thermocycler now 33 years old, this is far from new technology, but advances still improve these devices. “The first major improvement was the way they change the incubation temperature,” as Lai tells it. The earliest instruments included several sections at different temperatures. Moving a sample from one section to another cycled its temperature. In the 1980s, Lai says, using a compressor and heating element created thermocyclers that can “heat and cool the samples in the same location.” After that, he says, “The Peltier thermoelectric device replaced the compressor and the heating element for sample incubation, thus reducing the size and the power consumption of the instrument.” Today’s thermocyclers continue to use the Peltier approach.
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Still, thermocyclers keep getting better. Lai points out several improvements, and one is a higher heating and cooling capacity. “The temperature ramp rate can go up to around 10°C per second, and some models can go up to 20°C per second using a specialized incubation block and reaction vessels.” This reduces the time to complete PCR experiments.
Modern thermocyclers also provide more precise and consistent temperatures. “The temperature control accuracy can be less than +/- 0.1°C, precision can be less than +/- 0.2°C, and the temperature variation across the incubation block can be less than +/- 0.2°C,” Lai says. “This ensures that the experiments can be performed in a well-controlled condition that reduces the possibility for error.”
Getting the most out of a thermocycler, though, is not all about increasing speed. In some applications, slow temperature changes matter the most. With CRISPR-Cas9, for example, results must be confirmed after gene editing. “Some of the detection methods require the incubation temperature to decrease very slowly, from 85°C to 25°C in a –0.1°C per second cooling rate,” Lai says.
Scientists can even run a new thermocycler remotely. That alone could increase the options of where or how these devices can benefit scientists. Likewise, remote operation makes it easier to run devices around the clock.
Image: An app on a smart phone provides remote control for some thermocyclers. Image courtesy of Blue-Ray Biotech.
An array of new applications
The breadth of a thermocycler’s utility keeps increasing. One team of scientists used a real-time thermocycler to cultivate bacteria and measure their growth. As the scientists reported, gathering the data in real time “enables a significant simplification and increase in cost efficiency in the generation of bacterial growth curves” and it “was also possible to monitor the susceptibility of bacteria to antibiotic resistance, which opened another important application of this technique.”
Changing scientific challenges also expand the use of thermocyclers. Detecting SARS-CoV-2 is one challenge where thermocyclers can help. For example, a team of scientists from the University of California San Francisco (UCSF) and the UCSF-Abbott Viral Diagnostics and Discovery Center pointed out that the kind of thermocycler used in assays for detecting this virus impacts the detection range.
Other scientists are also exploring the use of thermocyclers against this virus. One interesting example involves miniPCR thermocyclers. Scientists in Mexico used a miniPCR thermocycler and a microplate reader in a system that detects SARS-CoV-2. “This straightforward method allows the detection and amplification of SARSCoV-2 nucleic acids in the range of ~625 to 2×105 DNA copies,” the scientists reported. “The accuracy and simplicity of this diagnostics strategy may provide a cost-efficient and reliable alternative for COVID-19 pandemic testing, particularly in underdeveloped regions where RT-QPCR instrument availability may be limited.” They added that the “portability, ease of use, and reproducibility of the miniPCR makes it a reliable alternative for deployment in point-of-care SARS-CoV-2 detection efforts during pandemics.”
Reasonable expectations
When purchasing a new thermocycler, a scientist should get a robust device. “The thermocycler should be the workhorse in the modern day molecular biology lab, not a fancy toy,” Lai explains. “It needs to be reliable.” A manufacturer should be able to validate and guarantee a thermocycler’s reliability.
A manufacturer should also provide data on a thermocycler’s precision and consistency. The required specifications will depend on a device’s likely use.
After more than 30 years in labs, thermocyclers continue to play a fundamental role, from academics to industry. As scientists extend the applications from PCR to pandemics, it becomes increasingly clear that thermocyclers play foundational roles in projects that improve human health and could even save lives.