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Understanding Thermal Cyclers: An Introduction and Overview

Harnessing PCR is fundamental to modern molecular biology. This process hinges on thermal cycler technology

by Donna Kridelbaugh
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The discovery of polymerase chain reaction (or PCR) in the 1980s sparked a molecular biology revolution. The reaction enables copious amounts of a target DNA sequence to be synthesized from a small amount of starting template for use in downstream applications. PCR also is a sensitive technique to aid in the detection and quantification of nucleic acids from various samples. It is now a ubiquitous laboratory method used for biotechnology applications, forensic testing, and clinical diagnostics—from agricultural crops to COVID-19 testing.

The PCR process is dependent upon heating and cooling cycles, which is facilitated using a thermal cycler (also known as a thermocycler or PCR machine). Thermal cyclers further can be used for other temperature-sensitive applications (e.g., restriction enzyme digestion, thermal shift assay). While PCR enabled genetic technology development, the thermal cycler has been the powerhouse that drove the advent of the modern molecular biology laboratory.

Brief history of the thermal cycler

A PCR reaction involves short oligonucleotides called primers that target a specific sequence of DNA and a thermostable DNA polymerase that amplifies the segment. Millions of copies of the DNA sequence can be fabricated in a short period of time through multiple reaction cycles.

Each cycle requires a series of specific temperature changes that enables denaturation of the DNA template into single strands, annealing of the primers at complementary sequences, and extension by the polymerase to fill in the gaps. A thermal cycler functions by automating the temperature changes required for PCR.

Prior to its invention, PCR was a laborious and time-intensive process requiring scientists to manually manipulate sample tubes among various water baths. The first thermal cycler prototype, named “Mr. Cycle,” used hoses attached to water baths to automate the heating and cooling cycles.

Subsequently, the first commercial thermal cycler model, called “TC1,” advanced it towards a fully programmable and compact instrument that could fit on the lab bench. This design used a metal sample block and internal heating and cooling elements similar to today’s models.

How a thermal cycler works

Thermal cyclers are designed to regulate heat transfer and deliver precise temperature control. The instrument can be fully programmed for each stage of a PCR protocol and multiple protocols stored for repeated use. Additional software options may include user controls to restrict editing of protocols, automatic report generation, and remote monitoring capability.

Key design elements of a standard thermal cycler model include a sample block composed of a thermal-conducting metal (e.g., aluminum, silver), Peltier devices (or thermoelectric modules) for rapid heating and cooling function, and a heated, sealing lid to prevent evaporation of samples. Notably, the use of thermoelectric modules is significant in achieving a self-contained instrument with dual heating and refrigeration requirements.

A thermoelectric module is a solid-state device that can function as a heat pump. The core of a module is a thin wafer of ceramic plates fixed to blocks of a semiconductor (e.g., bismuth telluride) in the middle. When an electric current is applied, heat is transferred to one side of the device or the other, depending on the direction of current. This enables both heating and cooling of samples. Excess heat is removed with the use of a heat sink and proper ventilation.

Design considerations when purchasing

The price of a thermal cycler can vary widely at an estimate of $1,500 to $50,000 for a new model. Ultimately, the selection of an instrument depends on the design features required for the intended lab applications.

One consideration is the availability of interchangeable and multiple sample blocks to accommodate differing sample sizes and lab user needs. Additionally, thermal cyclers are available in various capacities from automated systems with multiwell plates for high-throughput capability to more portable models for point-of-care settings.

Related temperature-control features to research include the ramp rate (i.e., how fast the block and/or sample heats and cools), which can impact total run times, and the use of gradient technology that enables multiple temperature ranges to be applied across a sample block.

A thermal cycler also is a main hardware component required for other nucleic acid applications and quantitative monitoring protocols (i.e., qPCR, RT-PCR, RT-qPCR). Thus, it is important to consider any additional modular requirements like fluorescence detection. Further purchasing considerations are outlined in Lab Manager’s PCR and qPCR Resource Guide.

Basic PCR troubleshooting

Numerous factors can impact the success of a PCR reaction. In terms of the thermal cycler, the troubleshooting process may involve both operation checks and using the instrument to determine the correct PCR conditions.

Thermal cyclers should be periodically checked to ensure the sample block temperature is operating within normal parameters. This process can be completed with use of a temperature verification kit or thermal validation services by a vendor. Regular cleaning of the instrument, including exterior surfaces like heat vents, is important to ensure effective operations as well.

Other basic PCR troubleshooting includes adjusting protocols for the length or temperature of each stage of a reaction and the total number of cycles. One important parameter is optimizing for the primer annealing temperature: too high of a temperature can yield little DNA results, while too low of a temperature can result in nonspecific amplification. If the thermal cycler is equipped with gradient technology, the optimal temperature can be tested within the same run.

Future of thermal cyclers

Thermal cycler technology has continued to advance over the nearly four decades since the original prototype was developed. One area of development is the design of more compact and portable instruments that has allowed the thermal cycler to go where no thermal cycler has gone before—from the International Space Station to backpack kits for use in the field.

Additional advancements have applied the use of microfluidics to develop digital PCR systems that further reduce sample size and improve both detection and quantification. In other cases, the instrument has gotten a retrofit. Some models of high-throughput thermal cyclers rotate microwell plates through multiple water baths resulting in more stable and rapid temperature changes, while extending the life of the instrument and reducing energy costs.