Transcription Mediated Amplification | Nucleic Acid Sequence Based Amplification

This section introduces Nucleic Acid Sequence Based Amplification (NASBA) and Transcription Mediated Amplification (TMA), noting their similar mechanisms. NASBA uses three enzymes: reverse transcriptase, RNA polymerase, and RNase H, while TMA uses only reverse transcriptase and RNA polymerase. TMA avoids RNase H because reverse transcriptase itself possesses RNase H activity.

The process begins with isolating mRNA, which is then converted into cDNA (complementary DNA) using a primer and reverse transcriptase. The primer is 45 bases long, with 20 bases specific to the target mRNA and 25 bases containing a T7 promoter sequence. After DNA-RNA hybrid formation, the RNA in the hybrid is hydrolyzed by RNase H or the RNase H activity of reverse transcriptase. It is important to note that RNase H specifically hydrolyzes RNA in an RNA-DNA hybrid, not single-stranded RNA.

Next, a second reverse primer binds to the cDNA. Reverse transcriptase extends this primer to form double-stranded DNA. This double-stranded DNA contains the T7 promoter, which is recognized by RNA polymerase. RNA polymerase then transcribes the DNA to produce multiple copies of RNA.

The newly synthesized RNA copies are further converted back into cDNA by reverse transcriptase, repeating the amplification cycle. This method can achieve billions of times amplification of the target sequence in just two hours. A significant advantage of NASBA and TMA is that all steps occur at a constant temperature, unlike PCR which requires temperature cycling for amplification.