Multiplex RC-PCR – where two or more universal primer probe sets are present in the reaction mixture to amplify two or more targets simultaneously.Concentrations of target specific primer are more aligned with target molecule concentration thereby reducing the potential of both off target priming and primer dimerisation. The generation of the target specific primer in the reaction as it progresses also leads to more balanced reaction components. This significantly reduces the number and length of oligonucleotides required by the laboratory compared to using full length pre-synthesised indexed target specific primers. A Laboratory employing this approach would only require a single set of index primers, which can be used with all target specific probes compatible with that index set. This benefit is used with NGS applications to apply sample specific indexes independently to each end of the amplicon construct. This is currently most advantageous in modern next generation sequencing (NGS) laboratories where a single target specific probe pair can be used with a whole library of universal primers. The technique also provides the significant advantage of the flexibility of appending any desired sequence or functional domain of choice to either end of any amplicon. Most significantly it is a single closed tube reaction, this eliminates cross contamination associated with other two-step PCR approaches as well as utilising less reagent and requiring less labour to perform. RC-PCR provides significant advantages over other methods of amplicon library preparation methods. Target specific primer generation occurs in the first and subsequent rounds with target amplification and amplicon generation occurring in the second and subsequent rounds of thermal cycling.Īdvantages Diagrammatic representation of relative reaction components of a RC-PCR reaction compared to a standard PCR reaction. Multiple targets are amplified and dual indexed in a single closed tube reaction. Representation of a typical multiplex RC-PCR for generating amplicon libraries for downstream analysis by Illumina next generation sequencing. This generation of target specific primer occurs in parallel with standard PCR amplification under standard PCR conditions. The probe is not consumed it is available to act as a template for the universal primer to be ‘converted’ into target specific primer throughout successive PCR cycles. The oligonucleotide probe may also be blocked at the 3’ end preventing equivalent extension of the probe, but this is not essential. Once hybridized, the universal primer can be extended, using the oligonucleotide probe as the template, to yield fully formed, target specific primers, which are then available to amplify the template in subsequent rounds of thermal cycling as per a standard PCR reaction. The oligonucleotides interact with each other in pairs one oligonucleotide probe and one universal primer (containing functional domains of choice), which hybridize with each other at their 3’ ends. A typical reaction employing the approach requires four oligonucleotides. Instead target specific primers are formed as the reaction proceeds. In RC-PCR, no target specific primers are present in the reaction mixture. The 5 prime portion of the RC probe contains the reverse complement sequence of the desired target specific primer sequence. Universal primer hybridises to the RC probe and is extended by the DNA polymerase generating target specific primer. Principles Representation of the interaction of the universal primer and RC probe to generate functional target specific primer. RC-PCR was invented in 2013 by Daniel Ward and Christopher Mattocks at Salisbury NHS Foundation Trust, UK. The technique permits both the amplification and the ability to append sequences or functional domains of choice independently to either end of the generated amplicons in a single closed tube reaction. It is primarily used to generate amplicon libraries for DNA sequencing by next generation sequencing (NGS). Reverse complement polymerase chain reaction (RC-PCR) is a modification of the polymerase chain reaction (PCR). Modification of the polymerase chain reaction
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