Question

Explain the different steps carried out in Polymerase Chain Reaction, and the specific roles of the enzymes used.

Answer 1

Polymerase Chain Reaction (PCR) is a molecular biology process that multiplies a gene or DNA to produce many copies. Each cycle has three steps:

1. Denaturation: It occurs when double-stranded DNA molecules are heated to (94°C), causing the two strands to split and form a single stranded DNA molecule. This process is known as denaturation. Each strand serves as a template for DNA synthesis.
2. Annealing: The two oligonucleotide primers anneal (hybridize) to each single-stranded DNA template, as their sequences are complementary to the template DNA’s 3’ ends. This step is performed at a lower temperature based on the length and sequence of primers. This causes duplication of the original DNA molecule.
3. Extension of primers: DNA polymerase (Taq polymerase) expands primers with nucleotides delivered in the process. The optimal temperature for this polymerization stage is 72°C. Repeating this method numerous times yields multiple copies of the rDNA fragment. The enzyme in PCR is thermostable. Taq polymerase enzyme derived from the bacteria Thermus aquaticus.