Overall, RPA positions itself very favourably for widespread exploitation in kits and assays for use at the point-of-care or point-of-need, as well as in affordable, sensitive, specific, user friendly, rapid, robust, equipment-free and delivered (ASSURED) devices, in low-resource settings. RPA is remarkable due to its simplicity, high sensitivity, selectivity, compatibility with multiplexing, extremely rapid amplification, as well as its operation at a low and constant temperature, without the need for an initial denaturation step or the use of multiple primers. The characteristics of these isothermal approaches are summarised in Table 1Īnd the advantages and disadvantages of each technique has been extensively reviewed elsewhere. To address requirements of amplification for use in low-resource settings, or at the point-of-need, isothermal DNA amplification methods have been developed, including nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), rolling circle amplification (RCA), the loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), as well as the recombinase polymerase amplification (RPA). However, these techniques inherently require the use of thermocycler and a reliable power supply, thus restricting their use to laboratories. To date, the vast majority of genotyping techniques require a previous step of amplification, routinely carried out using the robust PCR thermal cycling methodology, and more recently quantitative real-time PCR (qPCR). The last decade has seen an avalanche of information gleaned in the post-HGP era, such as gene assignation, identification of disease related mRNA biomarkers, as well as the discovery of the importance of single nucleotide polymorphisms (SNPs) and methylated DNA. The 1953 discovery of the structure of DNA ushered a revolution in molecular biology, leading to an increased understanding of the central dogma and the subsequent development of invaluable molecular biology techniques, including the polymerase chain reaction (PCR), electrophoresis and automated sequencing, culminating in the completion of the human genome project (HGP) in 2003.
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