Encyclopedia of Experiments: Biological Techniques
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Transcript
Real-time quantitative reverse transcription PCR is useful for identifying viral infections.
Begin with a sample containing the target viral RNA. Add target-specific primers — aiding in both DNA synthesis and amplification — along with reverse transcriptase enzyme and dNTPs.
Add thermostable DNA polymerase — an enzyme for amplifying DNA across PCR cycles — and SYBR green — a dye that intercalates into double-stranded DNA, dsDNA. Place the reaction in a thermal cycler. Set an appropriate temperature for the primers to bind to the target RNA.
The reverse transcriptase adds dNTPs to the primer while simultaneously cleaving the template RNA, synthesizing cDNA.
Increase the temperature, inactivating the reverse transcriptase. Lower the temperature, allowing the primers to anneal to the cDNA for subsequent amplification. Increase the temperature to reach the extension step, where the DNA polymerase extends the primers, synthesizing dsDNA.
Consecutive cycles result in exponential dsDNA amplification. SYBR green binds to the DNA in increasing numbers, causing a proportional increase in fluorescence emission. Plot the fluorescence signal detected across the cycles.
A positive amplification curve indicates the presence of viral RNA in the sample.
Increase the temperature incrementally to reach the melting temperature, denaturing the dsDNA into single strands. The bound dye molecules get released — losing their fluorescence. Plot the decreasing fluorescence against the temperature.
A peak corresponding to the virus-specific melting temperature confirms viral infection.
