Recombinase Polymerase Amplification

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The Recombinase Polymerase Amplification (RPA, engl. For, recombinase polymerase amplification ') is a method of amplification (amplification) of DNA . It is a variant of isothermal DNA amplification .

properties

The recombinase polymerase amplification uses a recombinase , a single-stranded binding protein and a strangversetzende DNA polymerase . The recombinase amplifies the binding of a primer with a length of 30 to 38 nucleotides . By using a strand-dislocating DNA polymerase, the reaction can take place at a constant temperature of 37 to 42 ° C, at room temperature the reaction proceeds somewhat more slowly. The reaction runs even without equipment by keeping the reaction vessel in the armpit . Analogous to RT-PCR can be obtained by addition of a reverse transcriptase , a reverse transcription are performed. The resulting DNA can be quantified analogously to qPCR . Analogous to multiplex PCR , several DNA sequences can be amplified in parallel.

Alternative methods of amplifying DNA are e.g. B. the polymerase chain reaction , the multidisplacement amplification , the isothermal assembly , the loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification ( NASBA ), the helicase-dependent amplification (HDA), the nicking enzyme amplification reaction (NEAR) who have favourited rolling circle replication (RCA). Further detection methods are e.g. B. Nicking endonuclease signal amplification ( NESA ) and nicking endonuclease assisted nanoparticle activation ( NENNA ), exonuclease-aided target recycling , junction or Y-probes , split DNAZyme and deoxyribozyme amplification , non-covalent DNA catalysis and the hybridization chain reaction ( HCR).

Individual evidence

  1. ^ O. Piepenburg, CH Williams, DL Stemple, NA Armes: DNA detection using recombination proteins. In: PLoS biology. Volume 4, number 7, July 2006, p. E204, doi: 10.1371 / journal.pbio.0040204 , PMID 16756388 , PMC 1475771 (free full text).
  2. ↑ J. Kim, CJ Easley: Isothermal DNA amplification in bioanalysis: strategies and applications. In: Bioanalysis. Volume 3, number 2, January 2011, pp. 227-239, doi: 10.4155 / bio.10.172 , PMID 21250850 .
  3. ↑ L. Lillis, D. Lehman, MC Singhal, J. Cantera, J. Singleton, P. Labarre, A. Toyama, O. Piepenburg, M. Parker, R. Wood, J. Overbaugh, DS Boyle: Non-instrumented incubation of a recombinase polymerase amplification assay for the rapid and sensitive detection of proviral HIV-1 DNA. In: PloS one. Volume 9, number 9, 2014, p. E108189, doi: 10.1371 / journal.pone.0108189 , PMID 25264766 , PMC 4180440 (free full text).
  4. ↑ ZA Crannell, B. Rohrman, R. Richards-Kortum: Equipment-free incubation of recombinase polymerase amplification reactions using body heat. In: PloS one. Volume 9, number 11, 2014, p. E112146, doi: 10.1371 / journal.pone.0112146 , PMID 25372030 , PMC 4221156 (free full text).
  5. ^ HM Amer, A. Abd El Wahed, MA Shalaby, FN Almajhdi, FT Hufert, M. Weidmann: A new approach for diagnosis of bovine coronavirus using a reverse transcription recombinase polymerase amplification assay. In: Journal of virological methods. Volume 193, number 2, November 2013, pp. 337-340, doi: 10.1016 / j.jviromet.2013.06.027 , PMID 23811231 .
  6. ↑ A. Abd El Wahed, P. Patel, D. Heidenreich, FT Hufert, M. Weidmann: Reverse transcription recombinase polymerase amplification assay for the detection of middle East respiratory syndrome coronavirus. In: PLoS currents. Volume 5, 2013, S., doi: 10.1371 / currents.outbreaks.62df1c7c75ffc96cd59034531e2e8364 , PMID 24459611 , PMC 3871419 (free full text).
  7. ↑ X. Xia, Y. Yu, M. Weidmann, Y. Pan, S. Yan, Y. Wang: Rapid detection of shrimp white spot syndrome virus by real time, isothermal recombinase polymerase amplification assay. In: PloS one. Volume 9, number 8, 2014, p. E104667, doi: 10.1371 / journal.pone.0104667 , PMID 25121957 , PMC 4133268 (free full text).
  8. ^ S. Kersting, V. Rausch, FF Bier, M. von Nickisch-Rosenegk: Multiplex isothermal solid-phase recombinase polymerase amplification for the specific and fast DNA-based detection of three bacterial pathogens. In: Mikrochimica acta. Volume 181, number 13-14, 2014, pp. 1715-1723, doi: 10.1007 / s00604-014-1198-5 , PMID 25253912 , PMC 4167443 (free full text).
  9. ↑ EC Oriero, J. Jacobs, JP Van Geertruyden, D. Nwakanma, U. D'Alessandro: Molecular-based isothermal tests for field diagnosis of malaria and their potential contribution to malaria elimination. In: The Journal of antimicrobial chemotherapy. [electronic publication before printing] September 2014, ISSN  1460-2091 , doi: 10.1093 / jac / dku343 , PMID 25223973 .
  10. ↑ L. Yan, J. Zhou, Y. Zheng, AS Gamson, BT Roembke, S. Nakayama, HO Sintim: Isothermal amplified detection of DNA and RNA. In: Molecular bioSystems. Volume 10, Number 5, May 2014, pp. 970-1003, doi: 10.1039 / c3mb70304e , PMID 24643211 .