PCR, from the past to the future

/PCR, from the past to the future

PCR, from the past to the future

The polymerase chain reaction (PCR), sometimes described as ‘molecular photocopying’, is a laboratory technique that allows researchers and technicians to replicate and amplify DNA or RNA from small or damaged samples, even down to a single strand. The technology was first created in 1983 by biochemist Kary Mullis, who was working for the biotechnology company Cetus (now part of Novartis) (1).

A brief introduction to PCR

PCR is now a standard tool in research, analytical and diagnostics laboratories, creating millions or even billions of copies of the target DNA or RNA in just a few hours or less. Before PCR, this process involved cloning DNA or RNA into vectors for transfer and expression in bacteria, a laborious and time-consuming method that could take weeks (2).

The PCR process, to create multiple copies of DNA, involves three key steps, all carried out in a thermal cycler at different temperatures, for different lengths of time:

  • Denaturing at 94-98 °C for 20-30 seconds, which ‘melts’ the hydrogen bonds between the two strands of DNA, separating them
  • Annealing at 54-65 °C for 20-40 seconds, which adds primers (short strands of DNA) to either end of the DNA to be copied – these match a stretch of DNA on the section to be copied, allowing the addition of new nucleotides, and also mark out the beginning and end of the piece of DNA to be copied
  • Extension (72 °C) where an enzyme (generally Taq DNA polymerase) reads the two mirror-image strands of DNA and uses them as templates to build two new double strands of DNA from a buffer solution containing nucleotides

Each cycle doubles the number of strands of DNA, as the strand from the previous cycle are used as templates, and the cycles are repeated up to 40 times – for example, 40 cycles can create as many as a billion copies in anywhere between 20 minutes and two hours. Once sufficient copies have been created, a final elongation step (70-74 °C for 5-15 minutes) extends any partially-completed strands.

When Mullis originally developed PCR, it required polymerase adding in every cycle – in 1986 he refined it to use a heat-stable polymerase so that it only needed to be added once every run (1).

To ensure accuracy and consistency, the temperatures and timings are critical, meaning that it’s vital that PCR thermal cyclers must be able to reach and maintain temperature quickly, evenly and accurately (3; 4; 5; 6; 7).

Facing the challenges

Much of the development of PCR has focused on three key issues – getting the DNA and RNA samples to the right temperature, keeping them at the right temperature for the right length of time, and maintaining this across all of the samples.

The earliest forms of PCR heated the samples using lamps, and cooled them in water (8). Most modern forms of PCR use Peltier effect devices, which rely on the temperature changes created when an electric current is run across the joint between two metals – one direction raises the temperature and the other direction lowers it.

The DNA or RNA samples and the required reagents are placed in plastic tubes or microtitre plates in wells in heat exchange plates or blocks. The blocks transmit the heat from the Peltier effect devices to the samples, and heating or cooling is passive, along temperature gradients. While the Peltier effect can be controlled precisely, the temperature change can be slow to transmit as the temperature in the block equilibrates, and this leads to under- and overshoots of temperature in the sample, increasing the time taken to get to the right temperature. Overshoots can damage the sample and undershoots lead to incomplete reactions, and both add further delays to the process.

Because of the nature of passive heat exchange, the wells around the edges of the block will take longer to get to the optimum temperature, and are likely to cool more quickly because of heat dissipation. This makes it harder to replicate exactly the same conditions in all wells, which is important in experimental research, and in diagnostics.

The structure of the sample container can also have an impact. If, as in many PCR devices, the samples sit in tubes inside wells in the heat transfer block, the plastic wall of the tube or microtitre plate and the air gap both insulate the sample from the heat exchange block, which can introduce a time lag of up to 10 seconds.

All of these factors work together to increase variability and reduce the reliability of the final results. The time taken for different types of Peltier effect-driven thermal cyclers to run 40 cycles can vary from 30 minutes to 2 hours (4; 3; 6).

Finding solutions

PCR is a widely used technology, and because of this, there have been many different approaches. While this coverage is by no means exhaustive, it gives a feel for the different approaches:

Changing the construction of the Peltier blocks

Using thicker metal blocks, including those made of silver, which is highly heat conductive, improves the conductivity of the heat exchange block. However, the increased thermal mass increases the time taken to raise the entire block to the same even temperature, and can lead to undershoots and overshoots of temperature (3).

In another approach, using a hollow heat exchange block with a circulating conductive fluid improves temperature control and heat uniformity, for example the Illumina ECO Real-Time PCR system and the Roche Lightcycler 480. The Illumina system takes around 40 minutes for 40 cycles of PCR, and the Roche system between 40 minutes and an hour (4; 9).

Moving away from Peltier-based technology

Other PCR thermal cyclers have moved away from Peltier effect-based heating altogether. Passing heated air increases the speed of heating and improves the variability – examples using this approach include Roche’s Lightcycler 1.5 and 2.0, and Corbett’s Rotor-Gene 6000 (also known as QIAGEN’s Rotor-Gene Q). Roche’s thermal cycler takes about 30 minutes for 40 cycles, and Corbett’s about 40 minutes for 40 cycles of PCR, but the thermal mass and conductivity of the air still cause some levels of delay (10; 4).

In another step away from Peltier-based technology, Cepheid has used an I-CORE ceramic heating plate in its Smart Cycler, heating samples in disposable tubes and cooling using force-air cooling. This system takes 20-40 minutes for 40 cycles (5; 4)

Despite the fact that improving PCR technology has reduced the time for 40 PCR cycles down to as little as 20 minutes over the years, there is still room for greater speed, for improved consistency and accuracy, and for simpler workflows for the researchers and technicians.

Creating a new approach

By going back to first principles, rather than adapting existing technologies, BJS Biotechnologies has created an ultra-high speed thermal cycler, known as xxpress. This can run 40 cycles of PCR in 15 minutes or less, potentially making it the fastest PCR thermal cycler in the world. Its innovative heating and cooling techniques also mean that it has potentially to be the most thermally accurate technology as well.

Rather than a Peltier effect block and plastic sample tubes, it uses resistive heating technology built into the sample plate itself – xxpress’ flat-bottomed sample wells are lined with 10 microns of polypropylene and incorporated into a highly conductive thin metal disposable plate with a low thermal mass. The plate has six electrical contact fingers allowing a range of resistive heating paths in multiple heating zones, with the amount of heat being directly proportional to the level of current. Finely-controlled and continuously-variable cooling air jets cool the zones. In contrast with Peltier effect-based devices, which often need changeover of entire heat exchange blocks and recalibration to use different microtitre plates or tube sizes, xxpress’ 24-, 54- and 96-well plates are entirely interchangeable, cutting down the time and staff input needed between runs (10).

The heating paths and cooling jets actively heat and cool nine zones in a three by three grid, each of which has its own highly accurate infrared temperature sensor. This feeds information back, allowing the system to adjust the heating and cooling constantly, to maintain the target temperature. Because the plate has a very low thermal mass, the plate’s temperature can rise and fall very quickly as required, at over 10 °C per second (3).

Because the samples sit within the disposable test plate, samples in the xxpress PCR system are closer to the heating element than those in Peltier effect-based systems. The combination of this and the fast response time for the heating and cooling reduce under- and overshoot and lead to a thermal uniformity between samples of ±0.3 °C, as well as reducing dwell times between cycles (3). These shorten each PCR cycle and contribute to improved accuracy, reliability and reproducibility. The system also includes five-colour fluorescence measurement every cycle.

The smaller sample sizes reduce the amount of costly reagents used, and mean that test samples go further, which can be important in diagnostics and research.

The system workflow

BJS Biotechnologies’ xxpress technology is designed by scientists for scientists to be comprehensive enough to handle the most complex DNA analysis, but simple enough not to require intensive training. The system has a touch-screen user interface that guides users through the process step by step, asking them to make choices or input experimental information. The three key steps are:

  • Identify the type of qPCR and the reagents used.
  • Choose sample size and number of tests (24, 54 or 96 wells).
  • Set up the experiment, include choosing genes of interest and reference genes, and adjusting the thermal profile and measurement information.

The system remembers the user’s preferences and choices, and includes data files on reagents, shortening the setup process. The setup process can also be carried out on a separate computer, freeing up the xxpress machine for other users.

The scientists or technicians pipette the prepared DNA samples into the plates. These are interchangeable and require no additional setup or changes to heat exchanger blocks, unlike the process of changing plate size or format in many Peltier effect-based machines. The xxpress machine automatically recognizes the test plate format and crosschecks with the information inputted by the user.

Once the required number of cycles of PCR is complete, the user is taken through the analysis step by step, and this can also be carried out offline, on another machine.

The need for speed

As PCR technologies have evolved, so have their speed and thermal accuracy. This opens up the possibility of PCR as a ‘while you wait’ test, allowing doctors to diagnose disease and prescribe the most appropriate medication all in one visit, particularly important in remote areas. Rapid testing also has a role in manufacturing, picking up suspected contamination quickly and reducing the time that production lines are halted, or the amount of spoiled product that has to be destroyed.

Rapid, simple and reliable PCR tests can also have an important role in research, as shorter PCR cycle time, lower batch cost and quicker turnaround between batches allows researchers to follow trains of thought much more intuitively.

References:

1. Elvidge, Suzanne. Who invented PCR? xxpress blog. [Online] 8 June 2012. [Cited: 20 September 2013.] http://xxpresspcr.com/who-invented-pcr/.

2. Stöppler, Melissa Conrad. PCR (Polymerase Chain Reaction). MedicineNet. [Online] 30 January 2009. [Cited: 2013 September 2012.] http://medicinenet.com/pcr_polymerase_chain_reaction/article.htm.

3. Burroughs, Nick and Karteris, Emmanouil. Ultra High Speed PCR Instrument Development. [book auth.] Tania Nolan and Stephen A Bustin. PCR Technology: Current Innovations. Third. 2012.

4. Jogan, J M J and Edwards, K J. An Overview of PCR Platforms. [ed.] Julie Logan, Kirstin Edwards and Nick Saunders. Real-Time PCR: Current Technology and Applications. s.l. : Caister Academic Press, 2009, 2.

5. Jain, Tarun. Cepheid’s SmartCycler. Biocompare. [Online] 11 April 2006. [Cited: 2 September 2012.] http://biocompare.com/Product-Reviews/41185-Cepheid-s-Smart-Cycler/.

6. Elvidge, Suzanne. PCR – past, present and future. Laboratory News. 19 February 2013.

7. —. PCR 101: An introduction to PCR. xxpress blog. [Online] 4 July 2012. [Cited: 20 September 2013.] http://xxpresspcr.com/pcr-101-an-introduction-to-pcr/.

8. Ultrafast DNA amplification with a rapid PCR system. Hermann, Hanno, Knippschild, Claus and Berka, Alexander. November 2010, BTi.

9. Illumina. Eco Real-Time PCR System User Guide. 2012.

10. Burroughs, Nick and Karteris, Emmanouil. Chapter 10: Ultra High Speed PCR Instrument Development. [book auth.] Tania Nolan and Stephen A Bustin. PCR Technology: Current Innovations. Third edition. 2012.

 


Suzanne Elvidge is a freelance science, biopharma, business and health writer with more than 20 years of experience. She has written for a range of online and print publications including FierceBiomarkers, FierceDrugDelivery, European Life Science, the Journal of Life Sciences (now the Burrill Report), In Vivo, Life Science Leader, Nature Biotechnology, New Scientist, PR Week and Start-Up. She specialises in writing on pharmaceuticals, biotechnology, healthcare, science, lifestyle and green living, but can write on any topic given enough tea and chocolate biscuits. She lives just beyond the neck end of nowhere in the Peak District with her second-hand bookseller husband and two second-hand cats.

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