NASBA (molecular biology)

Nucleic acid sequence based amplification (NASBA) is a method in molecular biology which is used to amplify RNA sequences.

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 has been introduced into medical diagnostics, where it has been shown to give quicker results than PCR, and it can also be more sensitive.[3]

Explained briefly, NASBA works as follows:

  1. RNA template is given to the reaction mixture, the first primer attaches to its complementary site at the 3' end of the template.
  2. Reverse transcriptase synthesizes the opposite, complementary DNA strand.
  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 DNA strand.
  5. Reverse transcriptase again synthesizes another DNA strand from the attached primer resulting in double stranded DNA.
  6. T7 RNA polymerase continuously produces complementary RNA strands of this template which results in amplification. The amplicons, however, are antisense to the original RNA template.
  7. Now a cycle can begin starting with the RNA strands from the previous step, the second primer and reverse transcriptase and copying the mechanisms of steps 1–6.

The NASBA technique has been used to develop rapid diagnostic tests for several pathogenic viruses with single-stranded RNA genomes, e.g. influenza A,[4] foot-and-mouth disease virus,[5] severe acute respiratory syndrome (SARS)-associated coronavirus,[6] Human bocavirus(HBoV)[7] and also parasites like Trypanosoma brucei.[8]

References

  1. ↑ Compton, J (1991). "Nucleic acid sequence-based amplification". Nature. 350 (6313): 91–2. 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 540116Freely accessible. PMID 15635001.
  4. ↑ 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.
  5. ↑ 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. doi:10.1016/S0006-291X(02)02178-2. PMID 12237113.
  6. ↑ 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. PMID 16254971.
  7. ↑ B�hmer, A; Schildgen, V; L�sebrink, 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. replacement character in |last3= at position 2 (help); replacement character in |last1= at position 2 (help)
  8. ↑ 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 2650916Freely accessible. PMID 19116352.
This article is issued from Wikipedia - version of the 8/14/2016. The text is available under the Creative Commons Attribution/Share Alike but additional terms may apply for the media files.