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.
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 prime 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 oligonucleotide probe may also be blocked at the 3’ end preventing equivalent extension of the probe, but this is not essential. 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. This generation of target specific primer occurs in parallel with standard PCR amplification under standard PCR conditions.
Representation of a typical multiplex RC-PCR for generating amplicon libraries for downstream analysis by Illumina next generation sequencing. Multiple targets are amplified and dual indexed in a single closed tube reaction. 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.
Advantages
Diagrammatic representation of relative reaction components of a RC-PCR reaction compared to a standard PCR reaction.
RC-PCR provides significant advantages over other methods of amplicon library preparation methods. 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.
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.
The generation of the target specific primer in the reaction as it progresses also leads to more balanced reaction components. Concentrations of target specific primer are more aligned with target molecule concentration thereby reducing the potential of both off target priming and primer dimerisation.
Variations
Multiplex RC-PCR – where two or more universal primer probe sets are present in the reaction mixture to amplify two or more targets simultaneously.
RT-RC-PCR – This modification is used when the template material supplied in the reaction is RNA rather than DNA. In this modification the reaction mixture also contains reverse transcriptase enzymes and reverse transcriptionprimers as well as the universal primers and Reverse complement probes of the method. This approach permits reverse transcription of the provided RNA template, the formation of tailed target specific primers and the amplification of the desired targets in a single closed tube reaction.
Single ended RC-PCR – This variation of the method is used when only one complementary universal primer probe pair is provided in the reaction to generate one target specific primer. The other target specific primer is provided as a traditional primer as per standard PCR.
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.[1]
The patent application was filed in the UK in 2015 and awarded in 2020. Patent applications have been filed in other jurisdictions worldwide and are currently pending.
In May 2019 the Intellectual property was licensed to Nimagen B.V.[2] to develop, manufacture and market kits exploiting the technology. Currently commercially available kits employing the technology include those for Human identification[3][4] and more recently for the whole genome sequencing of the SARS-CoV-2 virus for variant identification, tracking and treatment response.[5][6] In August 2022 Nimagen officially launched a range of products employing the RC-PCR technology for human forensics applications under the trademark IDseek®.
References
^Mattocks, Christopher; Ward, Daniel; Mackay, Deborah (2021-03-05). "RT-RC-PCR: a novel and highly scalable next-generation sequencing method for simultaneous detection of SARS-COV-2 and typing variants of concern". medRxiv: 2021.03.02.21252704. doi:10.1101/2021.03.02.21252704. S2CID 232119608.
^"NimaGen Licenses PCR Tech From Salisbury NHS Foundation Trust". Genomeweb. 2019-01-14. Retrieved 2021-04-22.
^Kieser, Rachel E.; Buś, Magdalena M.; King, Jonathan L.; van der Vliet, Walter; Theelen, Joop; Budowle, Bruce (January 2020). "Reverse Complement PCR: A novel one-step PCR system for typing highly degraded DNA for human identification". Forensic Science International. Genetics. 44: 102201. doi:10.1016/j.fsigen.2019.102201. ISSN 1878-0326. PMID 31786458. S2CID 208535138.
^Bus, Magdalena M; de Jong, Erik AC; King, Jonathan L; der Vliet, Walter van; Theelen, Joop; Budowle, Bruce (2021-08-05). "Reverse complement-PCR, an innovative and effective method for multiplexing forensically relevant single nucleotide polymorphism marker systems". BioTechniques. 71 (3): btn–2021–0031. doi:10.2144/btn-2021-0031. ISSN 0736-6205. PMID 34350776.
^Wolters, Femke; Coolen, Jordy P. M.; Tostmann, Alma; Groningen, Lenneke F. J. van; Bleeker-Rovers, Chantal P.; Tan, Edward C. T. H.; Geest-Blankert, Nannet van der; Hautvast, Jeannine L. A.; Hopman, Joost; Wertheim, Heiman F. L.; Rahamat-Langendoen, Janette C. (2020-10-29). "Novel SARS-CoV-2 Whole-genome sequencing technique using Reverse Complement PCR enables fast and accurate outbreak analysis". bioRxiv: 2020.10.29.360578. doi:10.1101/2020.10.29.360578. S2CID 226228646.
^Donovan-Banfield, I’ah; Penrice-Randal, Rebekah; Goldswain, Hannah; Rzeszutek, Aleksandra M.; Pilgrim, Jack; Bullock, Katie; Saunders, Geoffrey; Northey, Josh; Dong, Xiaofeng; Ryan, Yan; Reynolds, Helen; Tetlow, Michelle; Walker, Lauren E.; FitzGerald, Richard; Hale, Colin (2022-11-26). "Characterisation of SARS-CoV-2 genomic variation in response to molnupiravir treatment in the AGILE Phase IIa clinical trial". Nature Communications. 13 (1): 7284. doi:10.1038/s41467-022-34839-9. ISSN 2041-1723.
External links
RC-PCR animation
WIPO patent filing information page
March 26, 2023
reverse, complement, polymerase, chain, reaction, modification, polymerase, chain, reaction, primarily, used, generate, amplicon, libraries, sequencing, next, generation, sequencing, technique, permits, both, amplification, ability, append, sequences, function. 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 Contents 1 Principles 2 Advantages 3 Variations 4 History 5 References 6 External linksPrinciples Edit 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 prime 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 oligonucleotide probe may also be blocked at the 3 end preventing equivalent extension of the probe but this is not essential 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 This generation of target specific primer occurs in parallel with standard PCR amplification under standard PCR conditions Representation of a typical multiplex RC PCR for generating amplicon libraries for downstream analysis by Illumina next generation sequencing Multiple targets are amplified and dual indexed in a single closed tube reaction 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 Advantages Edit Diagrammatic representation of relative reaction components of a RC PCR reaction compared to a standard PCR reaction RC PCR provides significant advantages over other methods of amplicon library preparation methods 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 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 The generation of the target specific primer in the reaction as it progresses also leads to more balanced reaction components Concentrations of target specific primer are more aligned with target molecule concentration thereby reducing the potential of both off target priming and primer dimerisation Variations EditMultiplex RC PCR where two or more universal primer probe sets are present in the reaction mixture to amplify two or more targets simultaneously RT RC PCR This modification is used when the template material supplied in the reaction is RNA rather than DNA In this modification the reaction mixture also contains reverse transcriptase enzymes and reverse transcription primers as well as the universal primers and Reverse complement probes of the method This approach permits reverse transcription of the provided RNA template the formation of tailed target specific primers and the amplification of the desired targets in a single closed tube reaction Single ended RC PCR This variation of the method is used when only one complementary universal primer probe pair is provided in the reaction to generate one target specific primer The other target specific primer is provided as a traditional primer as per standard PCR History EditFollowing 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 1 The patent application was filed in the UK in 2015 and awarded in 2020 Patent applications have been filed in other jurisdictions worldwide and are currently pending In May 2019 the Intellectual property was licensed to Nimagen B V 2 to develop manufacture and market kits exploiting the technology Currently commercially available kits employing the technology include those for Human identification 3 4 and more recently for the whole genome sequencing of the SARS CoV 2 virus for variant identification tracking and treatment response 5 6 In August 2022 Nimagen officially launched a range of products employing the RC PCR technology for human forensics applications under the trademark IDseek References Edit Mattocks Christopher Ward Daniel Mackay Deborah 2021 03 05 RT RC PCR a novel and highly scalable next generation sequencing method for simultaneous detection of SARS COV 2 and typing variants of concern medRxiv 2021 03 02 21252704 doi 10 1101 2021 03 02 21252704 S2CID 232119608 NimaGen Licenses PCR Tech From Salisbury NHS Foundation Trust Genomeweb 2019 01 14 Retrieved 2021 04 22 Kieser Rachel E Bus Magdalena M King Jonathan L van der Vliet Walter Theelen Joop Budowle Bruce January 2020 Reverse Complement PCR A novel one step PCR system for typing highly degraded DNA for human identification Forensic Science International Genetics 44 102201 doi 10 1016 j fsigen 2019 102201 ISSN 1878 0326 PMID 31786458 S2CID 208535138 Bus Magdalena M de Jong Erik AC King Jonathan L der Vliet Walter van Theelen Joop Budowle Bruce 2021 08 05 Reverse complement PCR an innovative and effective method for multiplexing forensically relevant single nucleotide polymorphism marker systems BioTechniques 71 3 btn 2021 0031 doi 10 2144 btn 2021 0031 ISSN 0736 6205 PMID 34350776 Wolters Femke Coolen Jordy P M Tostmann Alma Groningen Lenneke F J van Bleeker Rovers Chantal P Tan Edward C T H Geest Blankert Nannet van der Hautvast Jeannine L A Hopman Joost Wertheim Heiman F L Rahamat Langendoen Janette C 2020 10 29 Novel SARS CoV 2 Whole genome sequencing technique using Reverse Complement PCR enables fast and accurate outbreak analysis bioRxiv 2020 10 29 360578 doi 10 1101 2020 10 29 360578 S2CID 226228646 Donovan Banfield I ah Penrice Randal Rebekah Goldswain Hannah Rzeszutek Aleksandra M Pilgrim Jack Bullock Katie Saunders Geoffrey Northey Josh Dong Xiaofeng Ryan Yan Reynolds Helen Tetlow Michelle Walker Lauren E FitzGerald Richard Hale Colin 2022 11 26 Characterisation of SARS CoV 2 genomic variation in response to molnupiravir treatment in the AGILE Phase IIa clinical trial Nature Communications 13 1 7284 doi 10 1038 s41467 022 34839 9 ISSN 2041 1723 External links EditRC PCR animation WIPO patent filing information page Retrieved from https en wikipedia org w index php title Reverse complement polymerase chain reaction amp oldid 1141132052, wikipedia, wiki, book, books, library,
