Figure 3.

Molecular underpinnings of a nucleic acid sequence-based amplification (NASBA) reaction. [1] Purified template RNA in a reaction well. [2] Forward primer binds to target RNA and reverse transcriptase transcribes a complementary DNA strand to form a cDNA-RNA hybrid. [3] RNase H hydrolyzes the RNA, leaving the cDNA strand free for [4] the reverse primer to bind to it; reverse transcriptase then produces a double-stranded DNA molecule with a complete T7 promoter region. [5] T7 RNA polymerase binds the T7 promoter region and transcribes the DNA to [6] anti-sense RNA strands. [7] A loop-structure hairpin beacon is self-hybridized to quench its fluorophore; however, it is attracted to the anti-sense RNA strands. [8] The beacon binds the anti-sense RNA, releasing a fluorescent signal that can be read on an instrument, either to quantify the concentration of the sample, or as a presence/absence metric.