NASBA (molecular biology)

Nucleic Acid Sequence Based Amplification (NASBA) is a method in molecular biology which is used to amplify RNA sequences.

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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 (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. T7 RNA polymerase produces a complementary RNA strand which can be used again in step 1, so this reaction is cyclic.

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]

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 (RNAse H only destroys RNA in RNA-DNA hybrids, but not single-stranded RNA)
  4. the second primer attaches to the 3' end of the DNA strand, and Reverse transcriptase synthesizes the second strand of DNA. 
  5. T7 RNA polymerase binds double-stranded DNA and produces a complementary RNA strand which can be used again in step 1, so this reaction is cyclic.

References

1. ^ Compton J. Nucleic acid sequence-based amplification. Nature. 1991;350(6313):91-2.
2. ^ Kievits et al. NASBA isothermal enzymatic in vitro nucleic acid amplification optimized for the diagnosis of HIV-1 infection. J Virol Methods. 1991;35(3):273-86.
3. ^ Schneider et al. Real-time nucleic acid sequence-based amplification is more convenient than real-time PCR for quantification of Plasmodium falciparum. J Clin Microbiol. 2005;43(1):402-5
4. ^ Collins et al. Detection of highly pathogenic and low pathogenic avian influenza subtype H5 (Eurasian lineage) using NASBA. J Virol Methods. 2002;103(2):213-25.
5. ^ Collins et al. A method to detect major serotypes of foot-and-mouth disease virus. Biochem Biophys Res Commun. 2002;297(2):267-74.
6. ^ Keightley et al. Real-time NASBA detection of SARS-associated coronavirus and comparison with real-time reverse transcription-PCR. J Med Virol. 2005;77(4):602-8.