Understanding PCR, qPCR and RT-PCR

Polymerase chain reaction (PCR) is a laboratory technique used to amplify specific DNA sequences, making millions to billions of copies from a small initial sample.

The PCR cycle 1. **Denaturation** — the double-stranded DNA is heated to around 94–98 °C to separate it into two single strands. 2. **Annealing** — the temperature is lowered to 50–65 °C to allow primers (short DNA sequences complementary to the target region) to bind to the single-stranded DNA. 3. **Extension** — the temperature is raised to 72 °C, the optimum for DNA polymerase, which extends the primers by adding nucleotides to form a new DNA strand.

These steps repeat for 20–40 cycles, exponentially increasing the number of copies. PCR is essential in genetic research, medical diagnostics, forensic science and biotechnology.

qPCR — adding quantification Real-time (quantitative) PCR measures fluorescence every cycle. The cycle at which the signal crosses the threshold (Cq/Ct) reflects the starting amount of target — enabling quantitative interpretation where the assay is calibrated. Cq values are assay-specific and should not be compared across platforms without calibration.

RT-PCR — detecting RNA viruses RNA viruses (influenza, SARS-CoV-2, dengue) first require reverse transcription of their RNA into cDNA, which is then amplified by PCR. Specimen quality and timing are critical because RNA is labile.