Thermal Cyclers and the Woes of Non-Linear Optimization

The polymerase chain reaction (PCR) is a powerful molecular technique used to rapidly duplicate short segments of DNA. The success of PCR is highly dependent on achieving and maintaining optimal temperatures during the three main phases of the reaction: denaturation, annealing, and extension. The key instrument used to accomplish this is the thermal cycler, which is equipped with programmable heating and cooling devices (known as Peltier blocks) that automate temperature cycling conditions.

The success of PCR is highly dependent on rapidly achieving and holding optimal temperatures, which is the job of the thermal cycler. Optimal temperatures vary between PCR experiments, and researchers must determine the best temperature and exposure duration for each phase and for each primer pair.

Optimization typically involves instructing the thermal cycler to generate a heat gradient across a plate of samples, such that each row experiences an incrementally warmer temperature. Based on a sample’s location within this gradient, researchers can determine the temperature it was exposed to and how that influenced the reaction’s efficiency.

To establish this gradient, a typical thermocycler will use two Peltier blocks, enabling researchers to set the high temperature at one end, and the low temperature at the other. Diffusion of heat from one side to the other will form a heat gradient.

Traditional thermal cyclers have several drawbacks:

• With only two blocks, practitioners can only control the temperatures on the polar ends of the heated surface. For a standard 8x12 plate, this means only truly knowing the temperature in the first and last columns.
• Data suggest that the temperature gradient produced by two Peltier blocks is not actually linear, instead displaying more of a sigmoidal curve.

This reality means users risk miscalculating the temperatures that samples experienced and thus picking suboptimal temperatures.

To address these drawbacks, Thermo Fisher Scientific has developed VeriFlex technology, a new approach to thermal cycling that uses 3 to 6 Peltier blocks. By increasing the number of discrete heating and cooling units, this design gives more control over reaction temperatures and a true linear heat gradient during optimization.

Each zone of the VeriFlex block is segregated and has its own microcontroller, such that it acts as a ‘mini thermal cycler’. Data on the specific plastics and reaction volumes used is integrated into a Thermo Fisher Scientific–developed algorithm, which allows for sample temperature to be precisely controlled, as opposed to heating block temperatures. While the technology is broadly compatible, the optimization used Applied Biosystems plastics. Find your ideal plastic, tested on the Applied Biosystems family of PCR and qPCR instruments.

Ultimately, VeriFlex technology offers several benefits over traditional thermal cyclers, including:

• Increased control over reaction temperatures
• Ability to achieve a true linear heat gradient during optimization
• Greater peace of mind during PCR experiments

For practical tips about thermal cyclers aimed at delivering more reproducible and consistent PCR results, download the complete article.