OVERVIEW OF THE POLYMERASE CHAIN REACTION METHODOLOGY

The Polymerase Chain Reaction (PCR) is a powerful and widely used technique in molecular biology for amplifying specific DNA sequences. Developed by Kary Mullis in 1983, PCR has become an indispensable tool in various fields, including genomics, diagnostics, and forensics.

PCR Methodology Components:

1. DNA template: The starting material for PCR is a DNA molecule, which can be from any source, such as a bacterial culture, a tissue sample, or a blood sample.
2. Primers: These are short, single-stranded DNA sequences (usually 18-25 base pairs long) that specifically bind to the target DNA region. Primers are designed to anneal to the DNA strand and provide a starting point for DNA synthesis.
3. DNA polymerase: This enzyme is responsible for catalyzing the replication of the target DNA region. In PCR, a heat-stable DNA polymerase (such as Taq polymerase) is commonly used, as it can withstand the high temperatures required for the process.
4. Nucleotides: These are the building blocks of DNA, consisting of deoxyribonucleotide triphosphates (dNTPs) that include adenine, guanine, cytosine, and thymine.
5. Reaction buffer: This is a solution containing salts, enzymes, and other components necessary for the proper functioning of the DNA polymerase enzyme.
6. Thermal cycler: This is an apparatus that maintains the specific temperature conditions required for PCR, including the denaturation, annealing, and extension steps.

Step-by-step Process of PCR:

1. Denaturation: The DNA template is heated to separate the two strands of the double helix, typically at 94-96°C. This process is called denaturation.
2. Annealing: The reaction mixture is cooled to a temperature (usually 50-65°C) that allows the primers to bind to the complementary sequences on the separated DNA strands. This process is called annealing.
3. Extension: The DNA polymerase enzyme is added to the reaction mixture and is activated at a temperature of 72°C. The enzyme synthesizes a new complementary DNA strand, starting from the 3’ end of each primer and extending to the end of the target DNA region. This process is called extension.
4. Cycle repetition: The entire process of denaturation, annealing, and extension is repeated for a specified number of cycles (usually 20-40 cycles), resulting in an exponential amplification of the target DNA region.
5. Final extension: After the last cycle, the reaction mixture is incubated at 72°C for a longer period to ensure complete extension of the target DNA region.