**3.1 DNA template**

*Genetic Engineering - A Glimpse of Techniques and Applications*

PCR typically involves a series of 20–40 repeated temperature changes, called

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

Setting up a basic PCR requires many ingredients, reagents, and conditions

cycles, with each cycle usually comprising three discrete temperature shifts

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

**2. Principles of PCR**

(**Figure 3**) [2, 4, 7–10].

1.Denaturation: 94–96°C

3.Primer extension: usually 72°C

techniques can amplify up to 40 kb.

which are described below (**Figure 1**) [2, 4, 7–10].

**3. The components of PCR**

**14**

**Figure 1.**

*The component of PCR.*

The double-stranded DNA molecule amplified by PCR is called the target or template DNA. The template defines the sequence in which new nucleotides are added during the PCR process [11]. This process is carried out in vitro by cyclically varying temperature, enabling separation of DNA strands, hybridization of primers, and polymerization.

DNA isolated from any source can be used as a template for PCR provided that it contains the target sequence. The DNA used in PCR can be isolated from blood, tissue, forensics specimens, paleontological samples, or microbial/tissue cells grown in the lab. Whatever the source, we need to have some information of the target DNA sequence, so that primers for PCR can be designed [2, 12]. The PCR primers can be designed very easily nowadays owing to the plethora of software tools that only requires target sequence information. If the sequence information is not known, the designing of primers becomes very challenging. This problem can be circumvented by using degenerate primers [2].
