Historically, PCR instrumentation has relied on Peltier technology to power the process of heating and cooling samples, resulting in similar run times between instruments and few significant improvements in run times over the last 30 years. The NEXTGENPCR thermocycler introduces a new technology that enables samples to heat and cool almost instantly. In this blog post, we will briefly discuss the Peltier technology and discover how NEXTGENPCR eliminates ramp times.
The standard PCR cycle
The first step of any PCR is a denaturation step that usually takes place between 94°C and 98°C. This allows the double-stranded DNA to separate into two single strands. The second step is annealing, which lowers the temperature to between 55°C and 72°C so that primers can bind to the targeted region for amplification. The final step is extension, which requires an additional temperature change. Depending on the enzyme's optimal temperature, the extension temperature can range from 68°C to 72°C. During this step, the enzyme extends the primer molecules to complete the copy of the desired fragment. These three steps are repeated for a specific number of cycles in order to amplify the target DNA fragment.
A brief review of Peltier technology
Today’s common thermocyclers have an aluminum or silver block with wells specifically sized for the reaction tubes or plates being used. Using a thermoelectric method, the block is heated and cooled to reach the necessary temperatures for the PCR steps, and the temperature is transferred to the contents of the tube. The time required to change the block temperature is defined as ramp time. A ramp time can take many seconds depending on the temperature change, and ramp rates vary between instruments. The fastest ramp rates on Peltier thermocyclers are on the order of 5°C or 6°C per second.
In addition, some time is required to transfer the heat from the block through the tube to the sample to get the reaction to the desired temperature. Over the years, the use of thinner tubes, with a thickness of about 200 microns, has quickened the transfer of heat to the sample. The heat block must be precisely designed for the tubes being used in order to efficiently transfer the heat from the block to the sample. A drawback is the possibility of variability in the heating of the block from side to side or among the wells for each sample. A block change is also required if switching between different-sized samples as the block and sample must fit exactly.
How NEXTGENPCR is different
With NEXTGENPCR, the temperature change of the PCR is different. The samples are loaded into a microplate with wells that have a wall thickness of 40 microns, five times thinner than the usual thin-walled PCR tubes. The plate is sealed using a heat sealer and aluminum- or polyester-backed polypropylene seal that seals each well individually yet simultaneously. The microplate moves between thermal zones that are maintained at the precise temperature for each PCR step, which eliminates ramp times. The plate moves between each thermal zone in less than a second. In each thermal zone, the microplate gets pressed between two heat blocks at the correct temperature, which compresses the wells slightly and immediately brings the sample temperature to the desired temperature and mixes the sample.
The heat blocks are flat, enabling the use of any sample plate configuration. For example, a 96-well format or 384-well format is possible without requiring any change except the sample plate. The currently available 96- and 384-well plates match standard microplate formats. The microplates have a structural, outer frame of rigid plastic and are capable of being handled by robotics. The plate face is made of thin polypropylene into which the wells are formed. The heat blocks sandwich only the inner polypropylene section of the plate so heat transfer is instantaneous.
By eliminating ramp rates and optimizing the conditions for NEXTGENPCR, the typical PCR protocol can be shortened by 70-80%. A 100 bp fragment using a three-step, 30-cycle protocol was amplified in less than two minutes. By increasing speed, it is possible to add flexibility and throughput in your lab.