96-well block of the Automated Thermal Cycler

Thermal cyclers of different models and/or manufacturers may exhibit variations in performance and reproducibility. Such discrepancies can impact not only efficiency of PCR but also accuracy and consistency of the data obtained [1-2]. The reliability of thermal cyclers after extensive use, as well as support provided when issues arise, are also important considerations in choosing thermal cyclers. Therefore, understanding features of thermal cyclers that can impact PCR results can help you maximize your experimental success.

Enable precise, consistent PCR results with a thermal cycler that fits your challenge, application, and budget.

1. Accurate and uniform temperatures across the thermal block

The accuracy of a thermal cycler’s temperature may determine the success or failure of PCR, because of the temperature dependence of the three main steps of PCR [3]. Similarly, well-to-well consistency of the temperature across a thermal block is critical to obtaining reliable and reproducible PCR results [2]. Therefore, it is crucial that the thermal cycler being used achieve the set temperature as accurately and consistently as possible across the block.

One approach to enable thermal accuracy is regular testing with a temperature verification kit (Figure 1) and recalibrating by a trained professional, as needed. Temperature verification tests are usually performed for:

  • Well-to-well accuracy against set-point temperatures in an isothermal mode
  • Well-to-well accuracy against set-point temperatures soon after temperature transition
  • Accuracy of the heated lid’s temperature (which can affect block and sample temperatures)
Testing the block and lid of a thermal cycler with temperature verification probes
Figure 1. Measurement of block and lid temperatures using a temperature verification kit.

Watch top 5 benefits of an accurate thermal cycler and how Thermo Fisher Scientific can help you achieve them.

2. Precise temperature control for optimization of primer annealing

Gradient temperature control is one feature of a thermal cycler to facilitate optimization of primer annealing in PCR. The goal of the gradient setting is to achieve varying temperatures across the block, typically at a ≥2°C increment/decrement per lane, so that a number of temperatures can be assessed simultaneously for optimal primer annealing (Figure 2A).

In theory, a true gradient would exhibit linear temperatures across the block (Figure 2B). However, gradient thermal cyclers are usually constructed with one thermal block whose temperature is controlled by only two heating and cooling elements, one located at each end. This design often results in the following limitations [4]:

  • Only two temperatures can be set: for the low and high limits of primer annealing at each end of the thermal block (Figure 2A). Therefore, precise setting of other temperatures across the block cannot be achieved.
  • Due to heat interaction between lanes, temperatures across the block follow more of a sigmoidal curve instead of a true linear gradient (Figure 2B).
(A) Setting temperatures of a gradient block (B) graph of variations between theoretical and actual temperatures of a gradient block

Figure 2. Gradient temperature setting. (A) Due to the design of the thermal block only two temperatures, representing the lower and upper limits of primer annealing, can be set. The two set-point temperatures are based on the calculated annealing temperature (Tm) and the desired temperature differences between each lane. (B) Theoretical (a true gradient) vs. actual measured temperatures of a gradient thermal block.

Thermal cyclers equipped with “better-than-gradient” technology are alternatives that help improve temperature control for primer annealing [4]. One type of such technology involves designing a thermal cycler with three or more segmented metal blocks, each with a separate heating and cooling element. Compared to a gradient block, this block design offers the following:

  • The ability to set three or more different temperatures independently, so that each unique zone can be defined to allow for better control of temperatures, especially during optimization (Figure 3A).
  • Insulation of the segmented metal blocks so that heat interaction between them is prevented. This results in more precise control of block temperatures, enabling a true linear series of temperatures across the blocks (Figure 3B).
(A) VeriFlex thermal cycler block with independent temperature zones (B) Linear temperature profile of VeriFlex blocks

Figure 3. (A) A thermal cycler block with Applied Biosystems VeriFlex “better-than-gradient” technology for more precise temperature control. (B) Temperature measurements across six independent zones of the VeriFlex blocks when they are set 4°C apart.

3. Achieving accurate sample temperatures: Ramp rate, hold time, and algorithms

The ability of a thermal cycler to control sample temperatures precisely is critical to the accuracy and performance of PCR assays. Instrument-specific parameters such as ramp rate, hold time, and algorithms to predict sample temperatures (in addition to block temperatures) are all keys to helping accurately control sample temperatures [5].

The ramp rate of a thermal cycler indicates the change in temperature from one PCR step to another over time and is usually expressed in degrees Celsius per second (°C/sec). The terms “up ramp” and “down ramp” refer to the heating and cooling of thermal blocks, respectively.

Since it takes time to transfer thermal energy from the block to the samples, a slower ramp rate (than that of the block) is experienced by the samples. Hence, it is crucial to differentiate and understand the following definitions of ramp rates (Figure 4).

  • Maximum or peak block ramp rate corresponds to the fastest temperature changes achievable by the block during a very brief period during the ramp.
  • Average block ramp rate represents the rate of temperature change over a longer period, providing a more representative measurement of a thermal cycler’s speed.
  • Maximum sample ramp rate and average sample ramp rate reflect actual temperatures achieved by the samples. Therefore, sample ramp rates offer more accurate comparisons of a thermal cycler’s performance and its potential impact on PCR results.

Since the ramp rate of a thermal cycler can affect PCR results, when switching from one thermal cycler to another, instruments that are equipped with programs to simulate the ramp rate of previous models are recommended for easy transitioning with minimal impacts on PCR reproducibility.

Changes in temperatures of block vs. samples during ramping and hold time

Figure 4. Ramp rates of block and sample. The block’s thermal overshoot enables the sample to reach the desired temperature quicker. The dotted orange and blue curves depict block and sample temperatures without the block’s overshoot.

Thermal cyclers should be designed to start timing a step only when the sample reaches the set temperature for that step. In this way the hold time, or length of time the sample spends at the set temperature, will be more accurate with respect to the cycling conditions specified in the protocol.

Thermal cyclers often use complex mathematical algorithms to enable samples to reach set temperatures quickly and accurately as programmed [6]. Accounting for the reaction volume entered and thickness of the PCR plastics used, the algorithms predict sample temperatures and time taken to achieve the set points. Relying on the algorithms, thermal cyclers often drive the block temperature past the set point in a process known as the thermal block’s overshoot or undershoot during heating and cooling, respectively. This setting helps ensure the sample reaches the set temperature as fast as possible without itself overshooting or undershooting (Figure 4).

4. Flexibility and design to facilitate experimental throughput

The number of reactions that can be run in a thermal cycler in a given time is important for productivity of your PCR work. Features of thermal cyclers that can improve throughput include ramp rate, thermal block construction, and integration with automation platforms.

The ramp rate of a thermal cycler dictates how fast it takes to reach set temperatures. The higher the ramp rate, the faster the PCR runs, and more experiments can be completed in a given time (Figure 5A) (learn more:sample ramp rate vs. block ramp rate). In addition, use of a DNA polymerase designed for faster synthesis of target DNA can accelerate high-throughput experiments (Figure 5B) [6].

Figure 5. Ramp rate of a thermal cycler and synthesis rate of a DNA polymerase influence PCR run time. (A) The faster the ramp rate, the shorter the ramping time. The impact on PCR run time is more significant at slower ramp rates (e.g., 1–3°C/sec). Hypothetical values are used in this example, and the cycling time refers to the protocol time. (B) Using a DNA polymerase with a faster synthesis rate results in a shorter cycling time and PCR run time.

The design of a thermal cycler block also plays an important role in setup of PCR experiments. For instance, interchangeable blocks provide flexibility in the number of samples that can be included in each run. Moreover, thermal blocks with multiple modules that can be controlled independently are ideal for performing different PCR protocols simultaneously in one thermal cycler (Figure 6).

ProFlext PCR system with 3-in-1 block

Figure 6. A thermal cycler’s block with three independently controllable modules.

Maximize your throughput or run three experiments at once. The ProFlex PCR System fits how you work today and tomorrow with 5 interchangeable block formats.

For automated high-throughput PCR, thermal cyclers should be programmable and compatible with software for controlling the liquid-handling system. Automated systems are ideal for high-throughput PCR since they can run around the clock with little human intervention, thereby minimizing the time needed for manual experimental setup and increasing the number of reactions that can run in a given time. Therefore, easy and flexible integration with the robotic platform being used is desirable, in order to perform hands-free operation with a liquid handler or plate stacker.

Watch customer stories on how the Automated Thermal Cycler has helped them in high-throughput applications with robotic handling.

Lastly, when using a fleet of thermal cyclers, the ability to remotely control the instruments over the cloud or an on-premise server is much more convenient and desirable over “daisy chain” wire connections.

5. Reliability, durability, and quality assurance of thermal cyclers

In addition to performance and throughput capabilities, thermal cyclers should be able to withstand repeated use, environmental stresses, and shipping conditions. Some manufacturers may report how the instruments have been tested for reliability and durability [7]. Tests that thermal cyclers may have undergone include:

  • Component reliability: Robotic assemblies may be used in repeated testing of frequently used instrument components such as the heated lid, control panels/touch screens, and temperature cycling modules (Figure 7A).
  • Environmental stresses: An environmental chamber may be utilized to simulate various conditions in a typical lab, such as temperature, humidity, and elevation (Figure 7B).
  • Shipping testing: Rigorous shock and vibration tests may be conducted according to International Safe Transit Association (ISTA) standards, to help ensure that instruments arrive in uncompromised working condition (Figure 7C).
Testing of a thermal cycler for component reliability, environmental stresses, and shipping conditions

Figure 7. Durability testing of thermal cyclers.

6. Warranty and services to support thermal cyclers

Despite rigorous testing for reliability and durability, technical problems with thermal cyclers are unavoidable in the life of instruments. For peace of mind, the warranty, services, and support offered by the manufacturer should be considered when making purchasing decisions. Ask about:

Watch processes involved in receiving your instrument to shipping it back to you at a Thermo Fisher global repair center.

In conclusion, features of thermal cyclers such as performance, design, and reliability, and available support and services, are important factors to consider when choosing instruments for your PCR work.