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nucleic acid sequence-based amplification (NASBA)

A method for amplification (up to 109-fold) of a single-stranded RNA template. A reaction mixture contains RNA polymerase, ribonuclease H, reverse transcriptase, nucleoside triphosphates and two polydeoxynucleotide primers; primer 1 contains a promoter for the RNA polymerase and a sequence complementary to the 5'-end of the RNA template's coding region, and primer 2 is the DNA analogue of the 3'-end of the template. The amplification begins as primer 1 anneals to the template, the reverse transcriptase forms a cDNA:RNA hybrid, the RNA of which is hydrolysed by ribonuclease H. With formation of the resulting single-stranded (ss)DNA, the reaction enters a cyclic phase: the ssDNA anneals to primer 2, and the reverse transcriptase then synthesizes a complementary copy of the first-synthesized DNA to generate a double-stranded promoter region which the polymerase recognizes and uses to make 10-100 RNA transcripts. As each new RNA transcript is a template for the reverse transcriptase, the cycle continues to amplify the original RNA template. Compton, J. (1991) Nature (London) 350, 91-92

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