PCR Thermal Cycler Selection – Which instrument fits your needs?

Be careful with your purchase: 6 critical factors to help you choose the right thermal cycler.

Not all thermal cyclers are created the same. When selecting a cycler, be sure to consider these six main parameters to avoid experimental frustration.

When the polymerase chain reaction (PCR) was first introduced, the process was challenging and time consuming. Did you know that denaturation, annealing, and extension was performed by manually moving the samples to different water baths and adding new enzyme after each cycle? This took forever! Luckily, thermal cyclers were created to automate the PCR process and the technology continues to evolve to make our experiments easier and faster. Because of this technological evolution, however, the process of deciding which thermal cycler to use can be difficult and confusing. In addition, your instrument choice can vastly impact your PCR performance and reproducibility. Here are six helpful tips to consider when selecting a thermal cycler.

Consideration #1: Accurate and uniform temperatures
The three main steps of PCR (denaturation, annealing, and extension) are highly dependent on temperature. If you do not have a thermal block that has both accurate and uniform temperature from well to well, the reproducibility of your experiment may be compromised. If you can’t reproduce your experiment, it also brings into question how accurate and reliable your results are. When selecting a thermal cycler, be sure that the well-to-well accuracy has been tested in isothermal and transition modes, as well as testing of the heated lid which can greatly affect the temperatures of your samples. This is typically done by trained personnel and should be a feature included with the service of your thermal cycler.

Consideration #2: Precise optimization control
To optimize primer annealing, you will need to find the ideal temperature for your PCR primers to bind to your target DNA. This typically involves setting varying temperatures across the block so that several settings can be tested simultaneously. Gradient enabled cyclers typically allow for two temperatures to be set (low and high) so that a gradient occurs across the block. The limitation of this approach is that the user cannot set any temperatures between the high and low calculated settings of the instrument, which makes precise optimization difficult. In addition, a true gradient cannot be accomplished as the heat interactions between the lanes interfere with each other. To overcome these limitations, a “better-than-gradient” technology was developed using three or more blocks, each with its own heating and cooling element. This allows for three or more temperatures to be set independently, and also prevents the heat interaction between wells as each block is insulated from the others. As such, more precise temperature control is achieved, and primer annealing temperature is more accurately optimized.

Consideration #3: Sample vs. block temperatures
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 to consider for the precise temperature control that is required. The ramp rate is the time it takes a thermal cycler to change temperature from one step to another during the PCR process. While many thermal cyclers report block ramp rates, the more critical factor is the sample ramp rate which will differ due to the time it takes to transfer the heat from the block to the samples. Sample ramp rates provide a more accurate comparison of a thermal cycler’s potential impact
When running a PCR experiment, steps in the protocol should not occur until the samples have reached the desired temperatures for each step. This capability is called the hold time of the thermal cycler. If your cycler does not have this capability, you run the risk of not having accurate protocols completed, thus affecting your overall PCR results and reproducibility. Predictive algorithms allow accuracy of sample temperatures and times based on the volume of sample and the thickness of the PCR plastic being used. Without this feature, samples do not reach the programmed temperature as fast as possible, and the accurate temperature of the samples cannot be guaranteed.