Genetic techniques: Nucleic acid sequence-based amplification (NASBA)

· Amplify nucleic acid at a constant temperature of 4oC.
· Involve the reaction of three enzymes:
1. Avian Myoblastosis Virus-Reverse Transcriptase (AMV-RT)
2. Ribonuclease H
3. T7 RNA polymerase
· Used to selectively amplify RNA sequences in the presence of DNA strands.
· Two assays:
1. Hybridisation (Northern blotting and southern blotting, depending on whether DNA or RNA is being analyed)
- Joining of single stranded nucleotides
- Correspondence of two sequences: determines their degree if hybridization.
- Procedure:
1) Nucleic acid separate by electrophoresis.
2) Transferred to and fixed on a membrane probed with a labeled probe to detect the sequence of interest.
2. Microarrays (gene chips)
- Provide a miniaturized system for the simultaneous analysis of hybridization to an array of oligonucleotide probes immoblised on a support.
- Enables the analysis of large number of genetic features in a single hybridization experiment.
- Quantitative study of gene expression.

References: K. Sanderson and D. Nichols, University of Tasmania, Australia. 2003. Genetic techniques: PCR, NASBA, Hybridisation and microarrays. In: Thomas A. McMeekin. Detecting pathogens in food. North America: CRC Press LLC.261-262.