Nucleic acid sequence-based amplification, commonly referred to as NASBA, is a method in molecular biology which is used to produce multiple copies of single stranded RNA.[1] NASBA is a two-step process that takes RNA and anneals specially designed primers, then utilizes an enzyme cocktail to amplify it.[2]

Background

Nucleic acid amplification is a technique used to produce several copies of a specific segment of RNA/DNA.[3] Amplified RNA and DNA can be used for a variety of applications, such as genotyping, sequencing, and detection of bacteria or viruses.[4] There are two different types of amplification, non-isothermal and isothermal.[5] Non-isothermal amplification produces multiple copies of RNA/DNA through reiterative cycling between different temperatures.[6] Isothermal amplification produces multiple copies of RNA/DNA at a constant reaction temperature.[7] NASBA takes single stranded RNA, anneals primers to it at 65°C, and then amplifies it at 41°C to produce multiple copies of single stranded RNA.[8] In order for successful amplification to occur, an enzyme cocktail containing, Avian Myeloblastosis Reverse Transcriptase (AMV-RT), RNase H, and RNA polymerase is used.[9] AMV-RT synthesizes a complementary DNA strand (cDNA) from the RNA template once the primer is annealed.[10] RNase H then degrades the RNA template and the other primer binds to the cDNA to form double stranded DNA, which RNA polymerase uses to synthesize copies of RNA.[11] One key aspect of NASBA is that the starting material and end product is always single stranded RNA. That being said, it can be used to amplify DNA, but the DNA must be translated into RNA in order for successful amplification to occur.

Loop-mediated isothermal amplification (LAMP) is another isothermal amplification technique.

History

NASBA was developed by J Compton in 1991, who defined it as "a primer-dependent technology that can be used for the continuous amplification of nucleic acids in a single mixture at one temperature".[12] Immediately after the invention of NASBA it was used for the rapid diagnosis and quantification of HIV-1 in patient sera.[13] Although RNA can also be amplified by PCR using a reverse transcriptase (in order to synthesize a complementary DNA strand as a template), NASBA's main advantage is that it works under isothermal conditions – usually at a constant temperature of 41 °C or two different temperatures, depending on the primers and enzymes used. Even when two different temperatures are applied, it is still considered isothermal, because it does not cycle back and forth between those temperatures. NASBA can be used in medical diagnostics as an alternative to PCR that is quicker and more sensitive in some circumstances.[14]

Procedure

Explained briefly, NASBA works as follows:

  1. RNA template added to the reaction mixture, the first primer with the T7 promoter region on its 5' end attaches to its complementary site at the 3' end of the template.
  2. Reverse transcriptase synthesizes the opposite complementary DNA strand extending the 3' end of the primer, moving upstream along the RNA template.
  3. RNAse H destroys the RNA template from the DNA-RNA compound (RNAse H only destroys RNA in RNA-DNA hybrids, but not single-stranded RNA).
  4. The second primer attaches to the 5' end of the (antisense) DNA strand.
  5. Reverse transcriptase again synthesizes another DNA strand from the attached primer resulting in double stranded DNA.
  6. T7 RNA polymerase binds to the promoter region on the double strand. Since T7 RNA polymerase can only transcribe in the 3' to 5' direction[15] the sense DNA is transcribed and an anti-sense RNA is produced. This is repeated, and the polymerase continuously produces complementary RNA strands of this template which results in amplification.
  7. Now a cyclic phase can begin similar to the previous steps. Here, however, the second primer first binds to the (-)RNA
  8. The reverse transcriptase now produces a (+)cDNA/(-)RNA duplex.
  9. RNAse H again degrades the RNA and the first primer binds to the now single stranded +(cDNA)
  10. The reverse transcriptase now produces the complementary (-)DNA, creating a dsDNA duplex
  11. Exactly like step 6, the T7 polymerase binds to the promoter region to produce (-)RNA, and the cycle is complete.

Clinical applications

The NASBA technique has been used to develop rapid diagnostic tests for several pathogenic viruses with single-stranded RNA genomes, e.g. influenza A,[16] zika virus, foot-and-mouth disease virus,[17] severe acute respiratory syndrome (SARS)-associated coronavirus,[18] human bocavirus (HBoV)[19] and also parasites like Trypanosoma brucei.[20]

Recently, NASBA reaction with fluoresce, dipstick and next generation sequencing readout has been developed for COVID-19 diagnosis.[21]

See also

References

  1. ↑ Deiman, Birgit; van Aarle, Pierre; Sillekens, Peter (2002). "Characteristics and Applications of Nucleic Acid Sequence-Based Amplification (NASBA)". Molecular Biotechnology. 20 (2): 163–180. doi:10.1385/mb:20:2:163. ISSN 1073-6085. PMID 11876473. S2CID 28712952.
  2. ↑ Reed, Adam J.; Connelly, Ryan P.; Williams, Allison; Tran, Maithi; Shim, Byoung-Shik; Choe, Hyeryun; Gerasimova, Yulia V. (March 2019). "Label-free pathogen detection by a deoxyribozyme cascade with visual signal readout". Sensors and Actuators B: Chemical. 282: 945–951. doi:10.1016/j.snb.2018.11.147. ISSN 0925-4005. PMC 6713451. PMID 31462856.
  3. ↑ Lamb, Laura E.; Bartolone, Sarah N.; Tree, Maya O.; Conway, Michael J.; Rossignol, Julien; Smith, Christopher P.; Chancellor, Michael B. (December 2018). "Rapid Detection of Zika Virus in Urine Samples and Infected Mosquitos by Reverse Transcription-Loop-Mediated Isothermal Amplification". Scientific Reports. 8 (1): 3803. Bibcode:2018NatSR...8.3803L. doi:10.1038/s41598-018-22102-5. ISSN 2045-2322. PMC 5830622. PMID 29491389.
  4. ↑ Schachter, Julius (1997), "Evaluation of Diagnostic Tests — Special Problems Introduced by DNA Amplification Procedures", Nucleic Acid Amplification Technologies Application to Disease Diagnosis, Boston, MA: Birkhäuser Boston, pp. 165–169, doi:10.1007/978-1-4612-2454-9_12, ISBN 978-1-4612-7543-5, retrieved 2020-11-15
  5. ↑ Biolabs, New England. "Isothermal Amplification | NEB". www.neb.com. Retrieved 2020-11-15.
  6. ↑ Biolabs, New England. "Isothermal Amplification | NEB". www.neb.com. Retrieved 2020-11-15.
  7. ↑ Biolabs, New England. "Isothermal Amplification | NEB". www.neb.com. Retrieved 2020-11-15.
  8. ↑ Malek, L.; Sooknanan, R.; Compton, J. (1994). Nucleic acid sequence-based amplification (NASBA). Methods in Molecular Biology. Vol. 28. pp. 253–260. doi:10.1385/0-89603-254-x:253. ISSN 1064-3745. PMID 7509695. S2CID 30720773.
  9. ↑ Malek, L.; Sooknanan, R.; Compton, J. (1994). Nucleic acid sequence-based amplification (NASBA). Methods in Molecular Biology. Vol. 28. pp. 253–260. doi:10.1385/0-89603-254-x:253. ISSN 1064-3745. PMID 7509695. S2CID 30720773.
  10. ↑ Vasileva Wand, Nadina I.; Bonney, Laura C.; Watson, Robert J.; Graham, Victoria; Hewson, Roger (August 2018). "Point-of-care diagnostic assay for the detection of Zika virus using the recombinase polymerase amplification method". The Journal of General Virology. 99 (8): 1012–1026. doi:10.1099/jgv.0.001083. ISSN 1465-2099. PMC 6171711. PMID 29897329.
  11. ↑ "PDB101: Molecule of the Month: RNA Polymerase". RCSB: PDB-101. Retrieved 2020-11-15.
  12. ↑ Compton, J (1991). "Nucleic acid sequence-based amplification". Nature. 350 (6313): 91–2. Bibcode:1991Natur.350...91C. doi:10.1038/350091a0. PMID 1706072. S2CID 4304204.
  13. ↑ Kievits, T; Van Gemen, B; Van Strijp, D; Schukkink, R; Dircks, M; Adriaanse, H; Malek, L; Sooknanan, R; Lens, P (1991). "NASBA isothermal enzymatic in vitro nucleic acid amplification optimized for the diagnosis of HIV-1 infection". Journal of Virological Methods. 35 (3): 273–86. doi:10.1016/0166-0934(91)90069-c. PMID 1726172.
  14. ↑ Schneider, P; Wolters, L; Schoone, G; Schallig, H; Sillekens, P; Hermsen, R; Sauerwein, R (2005). "Real-time nucleic acid sequence-based amplification is more convenient than real-time PCR for quantification of Plasmodium falciparum". Journal of Clinical Microbiology. 43 (1): 402–5. doi:10.1128/JCM.43.1.402-405.2005. PMC 540116. PMID 15635001.
  15. ↑ Arnaud-Barbe, Nadege; Cheynet Sauvion, Valerie; Oriol, Guy; Mandrand, Bernard; Mallet, Francois (1998). "Transcription of RNA templates by T7 RNA polymerase". Nucleic Acids Research. 26 (15): 3550–3554. doi:10.1093/nar/26.15.3550. PMC 147742. PMID 9671817.
  16. ↑ Collins, RA; Ko, LS; So, KL; Ellis, T; Lau, LT; Yu, AC (2002). "Detection of highly pathogenic and low pathogenic avian influenza subtype H5 (Eurasian lineage) using NASBA". Journal of Virological Methods. 103 (2): 213–25. doi:10.1016/S0166-0934(02)00034-4. PMID 12008015.
  17. ↑ Collins, RA; Ko, LS; Fung, KY; Lau, LT; Xing, J; Yu, AC (2002). "A method to detect major serotypes of foot-and-mouth disease virus". Biochemical and Biophysical Research Communications. 297 (2): 267–74. CiteSeerX 10.1.1.328.625. doi:10.1016/S0006-291X(02)02178-2. PMID 12237113.
  18. ↑ Keightley, MC; Sillekens, P; Schippers, W; Rinaldo, C; George, KS (2005). "Real-time NASBA detection of SARS-associated coronavirus and comparison with real-time reverse transcription-PCR". Journal of Medical Virology. 77 (4): 602–8. doi:10.1002/jmv.20498. PMC 7167117. PMID 16254971.
  19. ↑ Böhmer, A; Schildgen, V; Lüsebrink, J; Ziegler, S; Tillmann, RL; Kleines, M; Schildgen, O (2009). "Novel application for isothermal nucleic acid sequence-based amplification (NASBA)". Journal of Virological Methods. 158 (1–2): 199–201. doi:10.1016/j.jviromet.2009.02.010. PMID 19428591.
  20. ↑ Mugasa, CM; Laurent, T; Schoone, GJ; Kager, PA; Lubega, GW; Schallig, HD (2009). "Nucleic acid sequence-based amplification with oligochromatography for detection of Trypanosoma brucei in clinical samples". Journal of Clinical Microbiology. 47 (3): 630–5. doi:10.1128/JCM.01430-08. PMC 2650916. PMID 19116352.
  21. ↑ Wu, Qianxin; Suo, Chenqu; Brown, Tom; Wang, Tengyao; Teichmann, Sarah A.; Bassett, Andrew R. (February 2021). "INSIGHT: A population-scale COVID-19 testing strategy combining point-of-care diagnosis with centralized high-throughput sequencing". Science Advances. 7 (7): eabe5054. doi:10.1126/sciadv.abe5054. ISSN 2375-2548. PMC 7880595. PMID 33579697.
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