Reverse Complement Polymerase Chain Reaction (RC-PCR) is a modification of the Polymerase Chain Reaction (PCR). It is primarily used to generate amplicon libraries for DNA sequencing by Next Generation Sequencing (NGS). 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. RC-PCR was invented in 2013 by Daniel Ward and Christopher Mattocks at Salisbury NHS Foundation Trust, UK. Principles 567x567px|Representation of the interaction of the universal primer and RC probe to generate functional target specific primer. Universal primer hybridises to the RC probe and is extended by the DNA polymerase generating target specific primer. The 5 primer portion of the RC probe contains the reverse complement sequence of the desired target specific primer sequence. In RC-PCR, no target specific primers are present in the reaction mixture. Instead target specific primers are formed as the reaction proceeds. A typical reaction employing the approach requires four oligonucleotides. The oligonucleotides interact with each other in pairs; one oligonucleotide probe and one universal primer (containing functional domains of choice), which hybridise with each other at their 3’ ends. Once hybridised , 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 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. 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. This benefit is used with NGS applications to apply sample specific indexes independently to each end of the amplicon construct. 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 significantly reduces the number and length of oligonucleotides required by the laboratory compared to using full length pre-synthesised indexed target specific primers. History Following the invention of RC-PCR in 2013 the technique was clinically validated and employed diagnostically for a range of both inherited diseases such as Hemochromatosis and Thrombophilia as well as somatically acquired disorders including Myeloproliferative neoplasms and Acute myeloid leukemia in the Wessex Regional Genetics Laboratory (WRGL), Salisbury UK. More recently work has been undertaken to utilise the technology in the fight against the SARS-CoV-2 pandemic.
Reverse complement polymerase chain reaction (RC-PCR) is a modification of the polymerase chain reaction (PCR). It is primarily used to generate amplicon libraries for DNA sequencing by next generation sequencing (NGS). 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. RC-PCR was invented in 2013 by Daniel Ward and Christopher Mattocks at Salisbury NHS Foundation Trust, UK. Principles 567x567px|Representation of the interaction of the universal primer and RC probe to generate functional target specific primer. Universal primer hybridises to the RC probe and is extended by the DNA polymerase generating target specific primer. The 5 primer portion of the RC probe contains the reverse complement sequence of the desired target specific primer sequence. In RC-PCR, no target specific primers are present in the reaction mixture. Instead target specific primers are formed as the reaction proceeds. A typical reaction employing the approach requires four oligonucleotides. 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. 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 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. 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. This benefit is used with NGS applications to apply sample specific indexes independently to each end of the amplicon construct. 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 significantly reduces the number and length of oligonucleotides required by the laboratory compared to using full length pre-synthesised indexed target specific primers. History Following the invention of RC-PCR in 2013 the technique was clinically validated and employed diagnostically for a range of both inherited diseases such as hemochromatosis and thrombophilia as well as somatically acquired disorders including Myeloproliferative neoplasms and Acute myeloid leukemia in the Wessex Regional Genetics Laboratory (WRGL), Salisbury UK. More recently work has been undertaken to utilise the technology in the fight against the SARS-CoV-2 pandemic.
R
Complement Polymerase Chain Reaction
complement polymerase chain reaction
R
Polymerase Chain Reaction
polymerase chain reaction
R
Next Generation Sequencing
next generation sequencing
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hybridise
hybridize
R
hybridised
hybridized
R
Next Generation Sequencing
next generation sequencing
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Hemochromatosis and Thrombophilia
hemochromatosis and thrombophilia