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PCR, or Polymerase Chain Reaction, is a revolutionary biotechnology method to amplify DNA. This tool helps you calculate the optimal annealing temperature for your experiment.

DNA Structure​

DNA is a double-helix molecule composed of nucleotides that pair in specific ways: A-T and G-C. The strands run in opposite directions (5′→3′ and 3′→5′).

DNA Replication​

Replication starts by separating the double helix. Primers attach to the exposed bases, and DNA polymerase creates new strands, replicating the original DNA.

What is PCR?​

PCR amplifies DNA fragments using temperature cycles. Invented by Kary Mullis, it uses a heat-stable polymerase, primers, nucleotides, and a buffer solution to replicate DNA rapidly.

PCR amplifies DNA fragments using temperature cycles. Invented by Kary Mullis, it uses a heat-stable polymerase, primers, nucleotides, and a buffer solution to replicate DNA rapidly.

Ingredients of PCR​

• Target DNA fragment
• Forward and reverse primers
• Taq DNA polymerase
• Free nucleotides (dNTPs)
• Buffer solution with Mg²⁺

Steps of a PCR Cycle​

• Denaturation: 94–98°C – separates DNA strands
• Annealing: 50–65°C – primers bind to strands
• Extension: 72–80°C – polymerase synthesizes new strands

PCR Annealing Temperature​

The annealing temperature depends on the melting temperatures of the primers and target DNA. Use the formula:

Tₐ = 0.3 × Tₘₚ + 0.7 × Tₘₜ − 14.9