Write principle, working mechanism and uses of PCR.

Principle of PCR

The principle behind PCR is based on the natural process of DNA replication that occurs within cells. However, PCR replicates DNA *in vitro* (outside of a living organism) using a thermostable DNA polymerase and specifically designed DNA primers. The process exploits the complementary nature of DNA strands – adenine (A) pairs with thymine (T), and guanine (G) pairs with cytosine (C). By repeatedly cycling through different temperatures, the target DNA sequence is exponentially amplified.

Working Mechanism of PCR: The Three Steps

PCR involves a cyclical process consisting of three main steps, each performed at a specific temperature:

1. Denaturation (94-98°C)

In this initial step, the double-stranded DNA template is heated to a high temperature (typically 94-98°C) to break the hydrogen bonds between the complementary base pairs, effectively separating the DNA into two single strands. This creates the template for subsequent amplification.

2. Annealing (50-65°C)

The temperature is then lowered (typically to 50-65°C) to allow the primers – short, single-stranded DNA sequences complementary to the flanking regions of the target DNA sequence – to bind (anneal) to their respective complementary sequences on the single-stranded DNA templates. The annealing temperature is crucial; too high and the primers won't bind, too low and they may bind non-specifically.

3. Extension (72°C)

Finally, the temperature is raised to the optimal temperature for the DNA polymerase (usually 72°C). The DNA polymerase, typically a thermostable enzyme like Taq polymerase (isolated from the thermophilic bacterium Thermus aquaticus), extends the primers by adding nucleotides complementary to the template strand, synthesizing new DNA strands. This results in two new double-stranded DNA molecules identical to the original target sequence.

These three steps – denaturation, annealing, and extension – constitute one PCR cycle. The cycle is repeated typically 25-35 times, resulting in exponential amplification of the target DNA sequence. After 'n' cycles, the number of DNA copies is theoretically 2ⁿ.

Key Components of PCR

• DNA Template: The DNA sample containing the target sequence to be amplified.
• Primers: Short, single-stranded DNA sequences (typically 18-25 nucleotides long) that define the region to be amplified.
• DNA Polymerase: A thermostable enzyme (e.g., Taq polymerase) that synthesizes new DNA strands.
• Deoxynucleotide Triphosphates (dNTPs): The building blocks of DNA (dATP, dCTP, dGTP, dTTP).
• Buffer Solution: Provides the optimal chemical environment for the polymerase to function.
• Magnesium Ions (Mg²⁺): A cofactor for the DNA polymerase.

Uses of PCR

• Medical Diagnostics: Detecting infectious diseases (e.g., HIV, COVID-19), genetic disorders (e.g., cystic fibrosis), and cancer.
• Forensic Science: DNA fingerprinting for identifying suspects in criminal investigations.
• Genetic Research: Cloning genes, studying gene expression, and creating genetically modified organisms.
• Paternity Testing: Determining biological relationships.
• Archaeology and Paleontology: Analyzing ancient DNA to study evolutionary relationships.
• Environmental Monitoring: Detecting the presence of specific microorganisms in environmental samples.

Variations of PCR, such as Real-Time PCR (qPCR), allow for quantification of the amplified DNA, providing more detailed information about the initial amount of target DNA. Reverse Transcriptase PCR (RT-PCR) is used to amplify RNA by first converting it into complementary DNA (cDNA) using reverse transcriptase.