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European Biopharmaceutical Review

Technique Transition

Recombinase polymerase amplification (RPA) is a wellestablished, versatile isothermal alternative to traditional polymerase chain reactions (PCR). The development of RPA began with the principles behind the recombination systems of Escherichia coli and T4-like bacteriophages in the early 2000s, and the technique is now frequently used for testing outside the laboratory environment, where the use of complex and bulky instrumentation – such as thermal cyclers – is impractical. This flexible technology has recently been turned on its head with the launch of liquid kits that can be applied across a much broader range of applications, while still taking advantage of the speed and sensitivity of the original RPA methodology. In contrast to the lyophilised formats, that are ideal for low throughput point-of-care testing, liquid is a good choice for higher throughput lab-based applications where using very small reaction volumes is more costeffective. Olaf Piepenburg talks about the progression of RPA and discusses how the development of liquid technology is opening up new application opportunities.

The rise of molecular techniques – such as NGS – has caused a genomics explosion across a wide range of applications, from clinical and veterinary diagnostics to food and environmental testing. The mechanisms of these molecular technologies, combined with the demand for ever-greater sensitivity, mean that relatively large amounts of genetic material are required to generate high-quality data, making nucleic acid amplification an essential part of many laboratory workflows. PCR is considered the gold standard for DNA/RNA amplification and, as a result, PCR and reverse transcription polymerase chain reaction (RT-PCR) are now virtually ubiquitous techniques across the life sciences sector. However, is PCR really the best solution in every situation?

A major advantage of PCR is its simplicity, requiring just a single enzyme – two for RT-PCR – plus the application-specific primers. However, this biochemical simplicity comes at the cost of greater physical complexity. PCR relies on repeatedly heating and cooling samples to melt the DNA into single strands that can be recognised and bound by the polymerase enzymes and primers. This means that PCR has relatively high associated equipment costs and is difficult to perform outside of the laboratory environment. The need for rapid and efficient heating and cooling also makes PCR techniques difficult to scale – larger volume reactions require a significant amount of energy to rapidly heat and cool, and very low volume reactions require complex microfluidic systems to avoid evaporation. Finally, the need to heat the sample to in excess of 90°C to separate the DNA strands will, obviously, have a significant detrimental impact on any other enzymes or proteins present in the sample, making PCR incompatible with many other enzymatic processes. This prevents other enzyme-driven reactions being run in parallel to the PCR process, significantly limiting the design of PCR-based assays and workflows.

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Dr Olaf Piepenburg is Chief Scientific Officer at TwistDx (now part of Abbott). He was awarded his PhD for work on transcriptional regulation during the embryogenesis of Drosophila at the Max Planck Institute for Biophysical Chemistry in Goettingen, Germany, in 2000. He was a postdoctoral researcher at the Wellcome Institute for Cancer Research and Developmental Biology, Cambridge, UK, before joining TwistDx in 2003, where he co-developed the company’s core RPA technology.
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Dr Olaf Piepenburg
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