PCR – past, present and future

PCR, a development that drastically changed the ability of researchers to study DNA, has become a common laboratory staple – but where next for this ubiquitous method? Suzanne Elvidge thinks speed will be king for the next generation of PCR Biochemist Kary Mullis first discovered the polymerase chain reaction (PCR), a method for replicating small quantities of DNA, in 1983 when he was working at the biotech company Cetus. It was then an exciting and revolutionary breakthrough, but is now fully-integrated into the routine of research laboratories as part of every-day bench work. However, the PCR process itself can be slow, taking up to two hours¹ and much research is going into developing faster technologies.

Analysing RNA or DNA is difficult when there are only small amounts available. PCR, sometimes described as 'molecular photocopying', can be used to amplify these small samples, creating millions of copies in just a few hours. This was much faster than the existing process, which involved cloning pieces of genetic material into vectors and then transferring them into bacteria, which could take weeks.

PCR has three key steps – denaturing, annealing and extension – and each is carried out at different temperatures. The first step (denaturing, at 94-98 °C for 20-30 seconds) 'melts' the DNA, separating the strands and the second step (annealing, at 54-65 °C for 20-40 seconds), joins primers to either end of the DNA to be copied. In the third step (extension, at 72 °C), an enzyme (usually Taq DNA polymerase) uses the two single DNA strands as templates to create two double-stranded pieces of DNA. The three steps are repeated up to 40 times, and in each cycle, each double strand, containing one old and one new single strand, can be used to create new copies. In those 40 cycles, PCR creates up to a billion copies, and once the cycles are complete, a final elongation step, at 70-74 °C for 5-15 minutes, makes sure that all the partial-completed strands are fully extended. The temperatures are critical for complete reactions, so any PCR technology needs to be able to reach the appropriate temperatures both quickly and accurately.

The basic challenges in PCR are getting the samples to the correct temperature, and then maintaining them at that temperature. There have been a number of attempts to increase the speed of heating and cooling in PCR, and to improve the uniformity of the temperature across all the wells.

Initially, PCR used techniques such as heating lamps and water-cooling² but most current PCR technologies rely on one or more Peltier-effect devices for heating and cooling. These use the Peltier-effect, where running an electric current across the joint between two metals generates heat, raising the temperature; running it in the opposite direction lowers the temperature.

The PCR instruments are known as thermal cyclers and use the Peltier-effect devices to cycle the temperatures. Heat exchange blocks connect the Peltier devices to the samples. The DNA samples are heated and cooled with the requisite primers and enzymes in plastic tubes or plastic microtitre plates. These tubes or plates are held in close contact in conforming wells within the metal heat exchange block; it is this heating and cooling triggers the amplification process.

While the temperature in Peltier devices can be controlled accurately using the flow of current, because heating and cooling relies on the passive transfer of heat along temperature gradients, it can take a while for the blocks to react to temperature changes adding time to each cycle. Different Peltier-driven PCR thermal cyclers take between 30 minutes and 2 hours to run 40 cycles^3,1.

Undershooting or overshooting the temperature targets adds delays, and the overheating can result in damage to the sample. Conversely, undershooting can lead to incomplete reactions, as the samples are not at the target temperature for long enough. PCR reactions need dwell time after each temperature change in the cycle to ensure that the all samples have reached the right temperature (the stabilisation time); and then further time to make sure that the reaction has completed (the reaction time).

Once the samples reach the correct temperature, it is important to maintain them at that temperature in order to reduce variability and increase efficiency. However, heat dissipating from the edges of the metal block means that the temperature can vary across different wells. This makes it hard to replicate the same conditions for each sample within a set, vital in both research and medicine.

A further issue is caused by the construction of the sample tubes and microtitre plates. In most Peltier-driven thermal cyclers, the samples sit in a disposable polypropylene microtitre plate in wells in the heat transfer block. This relies on heat transfer from the block to the sample through the sample tube walls, which are usually around 0.2mm thick. The insulation caused by the tube walls, and the lack of close contact with the heating element, can create a time lag of more than 10 seconds between the temperature change in the block and in the sample¹.

There have been a number of attempts to improve PCR technology. Still using passive heating, some systems have used thicker metal blocks – this does improve the conductivity, but increases the thermal mass, which slows the process of heating. It is possible to increase the speed that the thermal cycler drives the heat into and out of the metal block, but the temperature still needs to even out across the block as a whole to avoid temperature variations between individual wells and DNA samples. This is limited by the heat conductivity of the metal – for example, silver is one of the most heat conductive of metals, but the temperature of blocks made from silver still can only change at around 3°C per second¹. Trying to overcome the issues of the thermal mass can mean that the temperature undershoots or overshoots the target, delaying things further as the block is allowed to equilibrate again.

The Illumina ECO RT-PCR system uses a smaller, hollow silver thermal block containing circulating conductive fluid to improve the temperature control and the uniformity of heat across the samples. However, this system still takes around 40 minutes for 40 cycles of PCR⁴. Roche uses its Therma-Base technology as part of its Peltier block in its Lightcycler 480 to even out the temperature across all the wells, and this takes 40 minutes to an hour for 40 cycles⁵.

In an attempt to improve the speed of heating and reduce the variability, Roche’s Lightcycler 1.5 and 2.0 uses heated air, with 40 cycles taking around 30 minutes⁵. Corbett’s Rotor-Gene 6000 (also known as QIAGEN’s Rotor-Gene Q) uses this approach too, and takes about 40 minutes for 40 cycles of PCR^1,5. However, the thermal ramp rates for heated air techniques, whilst faster, are still limited¹, in this case by the thermal mass and conductivity of air.

Cepheid has gone down an alternate heating technology route – its Smart Cycler has disposable reaction tubes that sit in wells in the company’s I-CORE ceramic heater plates, designed to improve heat uniformity and heat transfer. The system cools the samples using forced-air cooling, and 40 cycles take around 20-40 minutes^1,3. However, even the fastest of these, at 20 minutes, still limits the use of the technology in emergency or even routine clinic situations, as well as slowing the progress of research.

BJS Biotechnologies, based in the UK, has developed an ultra-high speed thermal cycling technology, known as xxpress, which uses a direct resistive heating technology to improve the speed and reliability of qPCR (quantitative PCR) and can run 40 cycles of PCR in less than 10 minutes.

The xxpress technology is based around a highly-conductive thin metal plate divided into a three by three grid, creating nine heating zones, each of which has a temperature sensor. Electrical current can be directed through each of these nine zones allowing direct local heating of the plate itself. The plate heats using the Joule effect from within and is proportional to its electrical resistance. The developers at BJS chose this technique as it allowed very precise heating control, because the amount of heat is directly proportional to the current flowing through multiple pathways in the plate.

The plate is made from a thin sheet of aluminium, which has a very low thermal mass, which means that it can heat and cool very quickly, at rates of more than 10°C per second¹. Its six electrical contact fingers allow a range of simultaneous heating paths throughout the plate, each one heating a number of zones. The xxpress includes air jets driven by individual, continuously-variable pumps that can cool the sample at more than 15°C per second, with the resistive heating adding in an element of fine control.

The system constantly adjusts the heating at specific zones across the plate to achieve the target temperature. After each temperature adjustment, the control system uses a complex algorithm to calculate the ratio between the energy needed to make the change and the actual temperature change, and uses this information to work out what energy input will be needed for the next heating cycle. This technique of direct temperature control maintains a more uniform temperature across the plate, with a thermal uniformity between samples of ±0.3°C¹. This solution required highly accurate sensors, and BJS developed these in collaboration with the UK’s National Physical Laboratory (NPL), creating an infrared system of sensors that was repeatable, accurate and had a very fast response time.

To reduce the time lag in sample heating, the BJS xxpress technology incorporates the sample tubes into the aluminium resistive heating plate as flat-bottomed wells lined with only 10 microns of polypropylene. This almost eliminates any time lag as the sample is in close contact with the heating source and gives much greater consistency from well to well.

The combination of the active control system and low thermal mass means that the xxpress thermal cycler can reduce under- and overshoot to less than 0.5°C for less than 0.5 seconds, compared with around 4°C for more than 10 seconds for a Peltier-based system. This reduction in under- and overshoots and the faster ramping times reduce the stabilisation component of the dwell time, shortening each cycle and improving the consistency of the results. Overall, the xxpress PCR technology can reduce a 40-cycle PCR test down to less than ten minutes.

BJS's resistive heating technology has potential to make PCR faster, more consistent and more accurate. But what difference will cutting it to ten minutes (or even less) actually make, as some technologies have already brought 40 cycles down to 20 minutes?

A 'while you wait' test really needs to be complete in less than 20 minutes, and the PCR thermal cycle is only part of the process – the sample still needs to be prepared and the results processed. Therefore, a 10-minute PCR cycle is key to making disease testing at the point of care possible, allowing rapid diagnosis and immediate treatment of infectious, genetic and other diseases while the patient is still on site. It also allows doctors to tailor treatment to individual patients by selecting the treatment most likely to be effective (companion diagnostics; CDx). Patients don’t need to visit the clinic twice, once to give a sample and again to get the results, which is particularly significant in the developing world, where transport infrastructures are not necessarily in place.

The benefits are not just in patient testing; tests that are this rapid could be used in real time in food processing and drug manufacturing, particularly to spot contamination. This way, manufacturers could stop the production lines more quickly, reducing the risk of contaminated products reaching the outside world, whilst cutting waste and cost.

Even in research, a ten-minute PCR run will allow researchers to think differently – rather than planning their entire day around a set number of 2 hour cycles, they can follow the evidence, basing the next experiment around the results of the previous one, with barely time for a cup of tea in between cycles.

Author: Suzanne Elvidge is a freelance science, biopharma, business and health writer with more than 20 years of experience.

References:

1. Burroughs, N. & Karteris, E., 2012. Chapter 10: Ultra High Speed PCR Instrument Development. In: PCR Technology: Current Innovations. Third edition ed. s.l.:s.n.
2. Hermann, H., Knippschild, C. & Berka, A., 2010. Ultrafast DNA amplification with a rapid PCR system. BTi, November.
3. Jain, T., 2006. Cepheid's SmartCycler. [Online] Available at: http://www.biocompare.com/Product-Reviews/41185-Cepheid-s-Smart-Cycler/ [Accessed 2 September 2012].
4. 4. Illumina, 2012. Eco Real-Time PCR System User Guide, s.l.: s.n.
5. 5. Jogan, J. M. J. & Edwards, K. J., 2009. Chapter 2: An Overview of PCR Platforms. In: J. Logan, K. Edwards & N. Saunders, eds. Real-Time PCR: Current Technology and Applications. s.l.:Caister Academic Press.