PCR – amplifying DNA by thermal cycling
BiologyBiotechnologyAges 17–18
Use it with your class: students open it from a link or a QR code, with no account needed.
Next in Biotechnology
Each PCR cycle consists of denaturation at 95 °C, primer annealing, and extension at 72 °C, doubling the number of copies of the target DNA segment. Adjust the number of cycles, the annealing temperature relative to the primer's melting temperature, and the initial copy number to see the exponential curve plateau, the gel band brighten, and a nonspecific by-product band appear when annealing temperature is too low — the basis of PCR testing and DNA forensics.
Lesson: Molecular biology
What it shows
The polymerase chain reaction copies a short target stretch of DNA. In each thermal cycle the strands separate, two primers bind at the ends of the target, and a heat-stable polymerase builds new complementary strands, so the copy number ideally doubles: N = N₀·2ⁿ after n cycles. The curve then levels off as primers, nucleotides and enzyme run out. Specificity depends on the annealing temperature relative to the primer's melting temperature (about 60 °C here), and the cycle at which the signal crosses the detection threshold, Ct, reflects the starting amount of target DNA.
How to use
Press “Step through one cycle ▶” to follow denaturation, annealing and extension in the Thermal program panel. Then raise Number of cycles and watch the copy-number curve and the gel band. Set Annealing temperature well above and well below 60 °C and compare the sample lane. Finally, lower Initial DNA copy number (log) one step at a time and record how Ct changes.
Parameters you can change
- Number of cycles 0–40
- Annealing temperature 40–70 °C
- Initial DNA copy number (log) 0–6
Questions to explore
- Why does the number of copies stop doubling after about 30 cycles?
- What happens to the product and the gel band when the annealing temperature is well above the primer's melting temperature?
- How does Ct change when the initial amount of DNA falls tenfold, and why?
