Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR)

• A technique used to create numerous copies of a specific DNA segment from a small initial amount.

Applications of PCR

• Creating recombinant DNA.
• Increasing DNA quantity for forensic testing.
• Amplifying mitochondrial DNA for evolutionary studies.

Components of PCR Mixture

• DNA Template: The DNA segment to be amplified.
• Deoxyribonucleotides (dNTPs): Building blocks for synthesizing new DNA strands.
• Taq Polymerase: A heat-stable DNA polymerase.
  • Essential due to the high temperatures involved in PCR.
• DNA Primers: Short DNA sequences complementary to the ends of the target DNA segment.
  • Provide a starting point for Taq polymerase to begin synthesis.

PCR Process - Step-by-Step

1. Denaturation:
   • The mixture is heated to a high temperature (e.g., 95°C). This high temperature breaks the hydrogen bonds between DNA strands.
   • DNA{\ double-stranded} \rightarrow 2 \times DNA{\ single-stranded}
2. Annealing:
   • The mixture is cooled to allow DNA primers to anneal (bind) to each end of the single-stranded DNA template.
   • The temperature varies (typically 50-65°C) depending on the primers' sequences.
3. Extension/Elongation:
   • Taq polymerase synthesizes a new DNA strand complementary to the template strand.
   • It starts at the primer and extends along the template.
   • The temperature is usually around 72°C, the optimal temperature for Taq polymerase activity.

PCR Cycling

• The denaturation, annealing, and extension steps are repeated multiple times (cycles).
• Each cycle doubles the amount of the target DNA segment.
• DNA{copies} = DNA{initial} \times 2^{cycles}

Thermal Cycler

• An instrument that automates the PCR process by precisely controlling temperature changes and cycle durations.

Amplification Example

• After 21 cycles, one molecule of DNA can be amplified to over a million copies.
• 1 \xrightarrow{21 \ cycles} > 1,000,000 \ copies