Figure 1.

Nucleic acid sequenced based amplification (NASBA). An example of an RNA target is shown. Included in the amplification reaction are reverse transcriptase (RT), RNase H, T7 RNA polymerase, nucleotides, primers, and molecular beacons if performing real-time detection.

Phase I. Template amplification

1. Single-stranded sense RNA.

2. Oligonucleotide P1, containing the T7 promoter recognition sequence, binds to the complementary sequence on the target.

3. RT makes a DNA copy of the RNA template.

4. RNase H removes the RNA from the duplex, and P2 binds the antisense DNA strand.

5. RT copies the antisense DNA to form a double-stranded DNA complex.

6. T7 RNA polymerase recognizes the double-stranded T7 promoter sequence and initiates transcription, making hundreds of antisense RNA copies.

Phase II. Exponential amplification

7. P2 binds the complementary sequence on the antisense RNA strands.

8. RT makes a DNA copy of the RNA template.

9. RNase H removes the RNA from the duplex.

10. P2 binds the antisense DNA strand.

11. RT creates a double-stranded T7 RNA polymerase promoter.

12. Additional rounds of transcription occur, resulting in 10⁸ to 10¹⁰ copies of antisense RNA that are templates both for more rounds of amplification and for detection by using probes and electrochemiluminescence or in real time with molecular beacons incorporated into the amplification reaction, as shown. (Figure reprinted with permission of bioMérieux, Inc., Durham, NC.)