NASBA (molecular biology)

NASBA, or nucleic acid sequence-based amplification, is a method in molecular biology which is used to amplify RNA sequences.

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".[1] Immediately after the invention of NASBA it was used for the rapid diagnosis and quantification of HIV-1 in patient sera.[2] 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 at isothermic conditions – usually at a constant temperature of 41 °C. NASBA can be used in medical diagnostics as an alternative to PCR that is quicker and more sensitive in some circumstances.[3]

Procedure

Explained briefly, NASBA works as follows:

  1. RNA template is given 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[4] 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, the one with the T7 promoter region, 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, produces (-)RNA, and the cycle is complete.

The NASBA technique has been used to develop rapid diagnostic tests for several pathogenic viruses with single-stranded RNA genomes, e.g. influenza A,[5] foot-and-mouth disease virus,[6] severe acute respiratory syndrome (SARS)-associated coronavirus,[7] human bocavirus (HBoV)[8] and also parasites like Trypanosoma brucei.[9]

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References
  1. Compton, J (1991). "Nucleic acid sequence-based amplification". Nature. 350 (6313): 91–2. Bibcode:1991Natur.350...91C. doi:10.1038/350091a0. PMID 1706072.
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. 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.
  7. 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.
  8. 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.
  9. 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.
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