PCR [Polymerase Chain Reaction]

Polymerase Chain Reaction (PCR)

Source

PCR was developed by Kary Mullis in 1983. It utilizes Taq DNA polymerase, an enzyme derived from the thermophilic bacterium Thermus aquaticus, which remains active at high temperatures required for DNA denaturation.

Principle

PCR is a technique used to amplify specific DNA sequences. It involves repeated cycles of denaturation, annealing, and extension.

  1. Denaturation: Heat (94–98°C) separates the double-stranded DNA into single strands.
  2. Annealing: Cooling (50–65°C) allows primers to bind to complementary sequences on the target DNA.
  3. Extension: DNA polymerase synthesizes a new DNA strand by adding nucleotides to the primers at ~72°C.

Instrumentation

  1. Thermal Cycler:
    • Programmable machine to perform temperature cycling for denaturation, annealing, and extension.
  2. Reagents:
    • Template DNA: The target DNA to be amplified.
    • Primers: Short oligonucleotides complementary to the target sequence.
    • dNTPs: Building blocks for DNA synthesis.
    • Taq Polymerase: Heat-stable enzyme for DNA replication.
    • Buffer: Maintains optimal conditions for the polymerase.
  3. Detection Systems (for quantitative PCR):
    • Fluorescent dyes like SYBR Green.
    • Probes like TaqMan for sequence-specific detection.

Types of PCR

  1. Conventional PCR: Standard method for DNA amplification.
  2. Real-Time PCR (qPCR): Quantifies DNA in real-time using fluorescent markers.
  3. Reverse Transcription PCR (RT-PCR): Converts RNA into cDNA before amplification.
  4. Multiplex PCR: Amplifies multiple targets simultaneously with different primer sets.
  5. Nested PCR: Uses two sets of primers to increase specificity.
  6. Digital PCR: Provides absolute quantification of DNA molecules.
  7. Hot-Start PCR: Reduces non-specific amplification by activating Taq polymerase only at high temperatures.
  8. Touchdown PCR: Gradually decreases annealing temperature to enhance specificity.
  9. Asymmetric PCR: Amplifies one DNA strand more than the other, used for sequencing or probe generation.
  10. Colony PCR: Amplifies DNA directly from bacterial colonies without prior extraction.

Applications of PCR

  1. Molecular Diagnostics:
    • Detection of genetic diseases (e.g., cystic fibrosis, sickle cell anemia).
    • Identification of pathogens (e.g., Mycobacterium tuberculosis, HIV).
  2. Forensics:
    • DNA fingerprinting for crime scene investigations.
    • Paternity testing.
  3. Genomics and Research:
    • Gene expression studies.
    • Sequencing and cloning of genes.
    • Analysis of mutations (e.g., SNP genotyping).
  4. Evolutionary Biology:
    • Amplifying ancient DNA for phylogenetic studies.
    • Identification of extinct species' DNA.
  5. Food Safety and Agriculture:
    • Detection of genetically modified organisms (GMOs).
    • Identifying foodborne pathogens.
  6. Cancer Research:
    • Detection of oncogenes or tumor suppressor gene mutations.
    • Monitoring minimal residual disease.
  7. Environmental Studies:
    • Detection of microbial communities in soil, water, or air samples.
    • Identifying bioengineered organisms.
  8. Therapeutics:
    • CRISPR-mediated genome editing validation.
    • Monitoring viral load in antiviral treatments.






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