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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