Transcription Mediated Amplification

Transcription-mediated amplification (TMA) uses RNA as the template, two primers and two enzymes: reverse tran-scriptase and RNA polymerase. One primer contains a promoter sequence that binds RNA polymerase. As the amplification process begins, the promoter-containing primer hybridizes to the target RNA at a complementary site (Figure 2-5). Reverse transcriptase then synthesizes a cDNA copy of the target RNA template by extension of the 3' end of the promoter-primer. The result is an RNA: DNA duplex. The RNA component is degraded by the activity of the enzyme RNase H inherent in reverse transcriptase. The other primer in the reaction mixture hybridizes to the DNA

Figure 2-5. Transcription-mediated amplification cycle (TMA):

Step 1. Promoter-primer binds to rRNA target.

Step 2. Reverse transcriptase (RT) creates DNA copy of rRNA target.

Step 3. RNA:DNA duplex.

Step 4. RNAse H activities of RT degrades the rRNA.

Step 5. Primer 2 binds to the DNA and RT creates a new DNA copy.

Step 6. Double-stranded DNA template with a promoter sequence.

Step 7. RNA polymerase (RNA Pol) initiates transcription of RNA from DNA template.

Step 8. 100 to 1000 copies of RNA amplicon are produced.

Step 9. Primer 2 binds to each RNA amplicon and RT creates a DNA copy.

Step 10. RNA:DNA duplex.

Step 11. RNAse H activities of RT degrades the rRNA.

Step 12. Promoter-primer binds to the newly synthesized DNA. RT creates a double-stranded DNA and the autocatalytic cycle repeats, resulting in a billion-fold amplification.

(Reprinted with permission from Gen-Probe.)

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copy, and a new DNA strand is synthesized from the end of the primer by RT, generating a dsDNA molecule. The other enzyme in the mixture, RNA polymerase, binds the promoter sequence in the DNA template and initiates transcription. Each of the resulting newly synthesized RNA amplicons reenters the TMA cycle,serving as a template for a new round of replication and exponential expansion of the RNA target. Each DNA template can generate 102 to 103 copies of RNA amplicon, with the potential for 108- to 109fold amplification in less than 1 hour. The process is auto-catalytic and isothermal. Acridinium ester-labeled DNA probes are added on completion of the reaction to initiate detection and quantitation based on chemiluminescence.

Examples of Applications of TMA

1. Chlamydia trachomatis detection55

2. Neisseria gonorrhoeae detection

3. HCV detection (qualitative)56

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