When the polymerase chain reaction (PCR) was first introduced, the process was challenging and time consuming. Denaturation, annealing, and extension was performed by manually moving the samples to different water baths and adding new enzyme after each cycle. Thermal cyclers—or PCR machines—were created to automate the PCR process, and PCR technology continues to evolve to make our experiments easier and faster. Now there is a plethora of options available for thermal cyclers that range from compact sizes for smaller labs and automation capabilities for higher-throughput labs. Here are six useful tips to consider for how to select a PCR machine.
The three main steps of PCR (denaturation, annealing, and extension) are highly dependent on temperature, so you need a thermal block that has both accurate and uniform temperature from well to well. Otherwise, the reproducibility of your experiment may be compromised.
When selecting a thermal cycler, determine if they have been quality tested for temperature accuracy and what options you have for regular testing. There should be regular testing with a temperature verification kit (Figure 1) and recalibrating by a trained professional to enable the accuracy of a thermal cycler’s temperature. Temperature verification tests are performed to measure:
A thermocycler should maintain temperature uniformity across all sample wells, ideally within 0.5°C of the set temperature. The better the temperature uniformity, the better the opportunity for amplification uniformity. A temperature non-uniformity test can be performed using a temperature verification probe as shown in Figure 1.
To optimize primer annealing of PCR primers to target DNA, you will need to find the ideal temperature, called the annealing temperature. Optimization typically involves setting varying temperatures across the block so that several settings can be tested simultaneously. When selecting a thermal cycler, determine what type of blocks are being used (gradient or alternative) and if it is compatible with the nature of your experiments.
The annealing temperature in PCR is dependent on the DNA fragment you are targeting to amplify. This is the temperature that is ideal for your PCR primers to bind to your target DNA.
Gradient temperature control is one feature of a thermal cycler designed 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. Limitations of this design include:
Thermal cyclers equipped with “better-than-gradient” technology are alternatives that help improve temperature control for primer annealing. 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 VeriFlex Block design offers the following:
The ability to control the temperature of your sample is critical to the accuracy of your PCR reactions. Ramp rates, hold times, and predictive algorithms are all important factors affecting PCR experimental results. When selecting a thermal cycler, determine what type of precise temperature control is required for the nature of your experiments.
PCR ramp rate is the time it takes a thermal cycler to change temperature from one step to another 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. Sample ramp rates provide a more accurate comparison of a thermal cycler’s potential impact on PCR results and reproducibility (Figure 4).
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).
Hold time and predictive algorithms are two key factors affecting PCR accuracy.
Hold time refers to the time between each step in the PCR protocol. It's important to make sure that the thermal cycler maintains the desired temperature for each step before moving to the next one. Without this, the PCR results may not be accurate, which could affect the reproducibility of the experiment.
Predictive algorithms help to control sample temperatures and times based on the volume of the sample and the thickness of the PCR plastic. This feature ensures that the sample reaches the set temperature as quickly as possible, without overshooting or undershooting. This helps to minimize errors and ensure reliable results.
When selecting a thermal cycler, identify if the nature of your experiments requires a specific design or calls for flexibility. Features of thermal cyclers that can improve throughput include ramp rate, thermal block construction, and integration with automation platforms. In addition, it is important to consider the number of reactions that can be performed in a single PCR run. The design of your thermal cycler block can determine the number of reactions, and therefore greatly affect your results and how soon you can get results.
Options like interchangeable blocks (that can be swapped out when higher throughput is needed) and independent blocks (instruments with independently operating blocks that allow separate PCR runs simultaneously) can greatly decrease the time spent on optimization and allow for multiple users to utilize the same instrument at the same time.
3 x 32-well block for running up to three independent experiments simultaneously.
96-well block for standard PCR applications.
Dual 96-well block for high-throughput sequencing.
Dual 384-well block for higher-throughput sequencing.
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).
For automated high-throughput PCR, thermal cyclers should be programmable and compatible with software for controlling a 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.
Another option for thermal cyclers, especially when using a fleet of thermal cyclers, is the ability to remotely control the instruments through the cloud or an on-premise server, which are more convenient and desirable over wire connections.
Purchasing a thermal cycler is an investment. Therefore, the thermal cycler you select should be able to withstand your lab’s levels of use, environmental stress, and shipping conditions (Figure 7). Some thermal cycler manufacturers report how the instruments have been tested for reliability and durability. When selecting a thermal cycler, identify if what reliability, durability, and quality testing is done on the machine.
Table 1. Durability and environmental testing results for Applied Biosystems thermal cyclers.
|Test performed||Test methods||Requirements||Results|
|Component reliability||Temperature cycles||1 cycle = 95°C (15 sec), 60°C (60 sec)||>350,000 cycles||Pass|
|Heated cover opening and closing||Lid actuation robot: 1 cycle = close, open, close||>29,000 cycles||Pass|
|Touchscreen touches (not performed on Applied Biosystems Automated Thermal Cycler)||Touchscreen actuation robot: 1 cycle = touch, release||>2.900,000 cycles||Pass|
|Docking mechanism (ProFlex PCR System only)||Docking actuation robot: 1 cycle = dock, release, dock||>5,000 cycles||Pass|
|Environmental testing||Temperature||Thermal cycler performance in environmental chamber||15–30°C||Pass|
|Humidity||Thermal cycler performance in environmental chamber||15–80%||Pass|
|Elevation||Thermal cycler performance in environmental chamber||6,000 ft (812 mbar)||Pass|
|Shipping testing||ISTA*-recommended shock and vibration testing||Pass||Pass|
* International Safe Transit Association, www.ista.org
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. When you are in a bind with a non-functional instrument, these types of offerings can make a huge difference. Ask about:
For Research Use Only. Not for use in diagnostic procedures.