**2. Principles of PCR**

PCR typically involves a series of 20–40 repeated temperature changes, called cycles, with each cycle usually comprising three discrete temperature shifts (**Figure 3**) [2, 4, 7–10].

1.Denaturation: 94–96°C

2.Primer annealing (depending on the primer): 45–60°C

3.Primer extension: usually 72°C

The cycling steps often start and end with a temperature step called "hold" where product extension is performed at (>90) and (~72°C), respectively. The final product is kept at 4°C before its analysis or storage. The most of PCR methods typically amplify DNA fragments of up to ~10 kilo base pairs (kb). However, some techniques can amplify up to 40 kb.
