PCR & gene sequencing · Cells & Genetics

PCR & SEQUENCING SIMULATION

Thirty heating-and-cooling cycles turn one DNA molecule into roughly a billion copies, because each round doubles the count along a 2ⁿ curve. The hard part of the technique sits at the annealing temperature around 55°C: set it too low and the primers stick to near-matches, set it too high and they bind nothing at all. That same amplifying power is what makes contamination the classic false positive, where a handful of stray molecules become a confident signal. Once amplified, the product can be read out base by base in A, T, G and C.

▸ Loading template DNA & forward/reverse primer pair…

▸ Mixing thermostable Taq polymerase + dNTPs

▸ Calibrating thermal cycle 95° / 55° / 72°C

▸ Mounting the 4-colour Sanger sequencing reader

▸ Ready — Online. ✅

Block temp & amplification

🔬 PCR thermal cycle

white needle = target temperature

Current step—

Block temperature—

Cycle—

Target copies (~2ⁿ)—

Annealing temp—

Base read—

Notes

PCR repeats 3 steps: denaturation 95°C strand separation → annealing ~55°C → extension 72°C (Taq). Each cycle the target copy number ×2 → after n cycles ≈ 2ⁿ copies. Only the stretch between the 2 primers is selectively amplified.

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Adjust ANNEALING TEMP & CYCLE COUNT on the right · pick a scenario (Run 2ⁿ · Sequencing · Gel · qPCR) · click “Cycle diagram” to see the 3 steps

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Target DNA copies per cycle (log) 2ⁿ amplification