What are the principles of PCR?

The core principle of Polymerase Chain Reaction (PCR) is the enzymatic amplification of a specific DNA sequence through repeated cycles of denaturation, annealing, and extension.

PCR allows for the rapid and exponential amplification of a targeted DNA region. It relies on several key components and principles:

Key Principles of PCR

• DNA Template: PCR requires a DNA template containing the region to be amplified. This serves as the starting point for DNA synthesis.
• Primers: Short, synthetic DNA oligonucleotides (typically 18-30 base pairs) that are complementary to the sequences flanking the target region. Primers define the start and end points of the DNA fragment to be amplified, and they provide a free 3'-OH group, essential for DNA polymerase to initiate DNA synthesis. They are designed to bind to opposite strands of the template DNA.
• DNA Polymerase: A thermostable enzyme, most commonly Taq polymerase, which synthesizes new DNA strands complementary to the template. The enzyme extends the primers by adding nucleotides to the 3' end, using the template strand as a guide. Its thermostability is crucial, as it must withstand the high temperatures required for denaturation.
• Deoxynucleotide Triphosphates (dNTPs): The building blocks of DNA (dATP, dCTP, dGTP, and dTTP). DNA polymerase uses these dNTPs to build the new DNA strands.
• Buffer Solution: Provides a suitable chemical environment for the DNA polymerase to function optimally, including the correct pH and salt concentration.
• Thermal Cycling: PCR involves repeated cycles of three temperature-dependent steps:
  1. Denaturation: Heating the reaction to a high temperature (e.g., 94-98°C) to separate the double-stranded DNA template into single strands.
  2. Annealing: Lowering the temperature (e.g., 50-65°C) to allow the primers to bind (anneal) to their complementary sequences on the single-stranded DNA template. The annealing temperature is crucial for primer binding specificity.
  3. Extension/Elongation: Raising the temperature (e.g., 72°C) to the optimal temperature for the DNA polymerase to extend the primers and synthesize new DNA strands complementary to the template.

The PCR Cycle

Each PCR cycle doubles the amount of the target DNA sequence. A typical PCR reaction involves 25-35 cycles, leading to exponential amplification of the target DNA. The newly synthesized strands then serve as templates in subsequent cycles.

Summary

In essence, PCR harnesses the specificity of primers and the enzymatic activity of a thermostable DNA polymerase to selectively amplify a target DNA region through repeated cycles of denaturation, annealing, and extension. This results in the exponential increase in the amount of the desired DNA fragment.