Describe the procedure, requirements and efficiency level of gene amplification through Polymerase Chain Reaction (PCR).

Procedure of Polymerase Chain Reaction (PCR)

PCR is a cyclical process consisting of three main steps, repeated typically 25-35 times, to exponentially amplify the target DNA sequence.

• Denaturation: The reaction mixture is heated to a high temperature (typically 94-98°C) for a short period (20-30 seconds). This breaks the hydrogen bonds between the complementary DNA strands, separating them into single strands.
• Annealing: The temperature is lowered (typically 50-65°C) to allow primers – short, single-stranded DNA sequences complementary to the flanking regions of the target sequence – to bind (anneal) to the single-stranded DNA templates. The annealing temperature is crucial and depends on the primer sequence.
• Extension/Elongation: The temperature is raised to the optimal temperature for the DNA polymerase enzyme (typically 72°C). The polymerase enzyme extends the primers, synthesizing new DNA strands complementary to the template strands, using deoxyribonucleotide triphosphates (dNTPs).

Each cycle doubles the amount of target DNA, leading to exponential amplification.

Requirements for PCR

Successful PCR requires several key components:

• 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. Forward and reverse primers are required.
• DNA Polymerase: A heat-stable DNA polymerase enzyme, such as Taq polymerase (isolated from the thermophilic bacterium Thermus aquaticus), is essential to withstand the high temperatures of the PCR cycle. Other polymerases with higher fidelity (proofreading ability) are also used.
• Deoxyribonucleotide Triphosphates (dNTPs): The building blocks of DNA (dATP, dCTP, dGTP, dTTP).
• Buffer Solution: Provides the optimal chemical environment for the polymerase enzyme to function, including pH and salt concentration.
• Magnesium Chloride (MgCl₂): A cofactor for the DNA polymerase enzyme. Its concentration is critical for optimal enzyme activity.

Efficiency Level of PCR

The efficiency of PCR is typically expressed as the percentage of target DNA amplified per cycle. Ideally, PCR efficiency should be close to 100%, meaning the amount of target DNA doubles with each cycle. However, several factors can affect PCR efficiency:

• Primer Design: Poorly designed primers (e.g., with self-complementarity or incorrect annealing temperature) can reduce amplification efficiency.
• Template Quality: Degraded or contaminated DNA template can inhibit PCR.
• Magnesium Concentration: Suboptimal MgCl₂ concentration can affect polymerase activity.
• Polymerase Fidelity: Polymerases with lower fidelity can introduce errors during amplification.
• Inhibitors: Substances present in the DNA sample (e.g., heme in blood samples) can inhibit the polymerase enzyme.
• GC Content: Extremely high or low GC content in the target sequence can affect primer annealing and amplification.

Real-time PCR (qPCR) allows for monitoring the amplification process in real-time, providing a more accurate assessment of PCR efficiency. qPCR uses fluorescent dyes or probes to quantify the amount of amplified DNA at each cycle. Efficiency is calculated based on the cycle threshold (Ct) values.

Factor	Impact on Efficiency
Primer Design	Poor design leads to reduced annealing and amplification.
Template Quality	Degraded DNA inhibits amplification.
MgCl2 Concentration	Suboptimal levels affect polymerase activity.
Inhibitors	Inhibit polymerase, reducing amplification.