PCR (Polymerase Chain Reaction)

May 17, 2024

PCR (Polymerase Chain Reaction) Lecture Notes 🧬

Introduction

  • Appreciation for technology: Mention of technology we appreciate, like phones, laptops, etc.
  • Copy machine anecdote: The speaker believes the copy machine always malfunctions at the worst times.
  • Transition to biotechnology: Compares copy machine to PCR, which is for DNA replication.

What is PCR?

  • Full form: Polymerase Chain Reaction.
  • Purpose: Makes numerous copies of a specific portion of DNA.
  • Doesn’t require a cell: Can happen in a test tube.

How PCR Works

  1. Preparation

    • Materials needed:
      • DNA segment to be copied
      • Buffer
      • Primers
      • DNA polymerase (e.g., Taq polymerase, which is heat-resistant)
      • DNA nucleotides

  2. Steps Involved

    • Step #1: Denaturation
      • Use heat to separate DNA strands.
    • Step #2: Annealing
      • Cool down so primers can bind to specific DNA segments.
    • Step #3: DNA Synthesis
      • DNA polymerase builds new DNA strands using nucleotides.
      • Temperature adjusted for optimal polymerase activity.

  3. Repetition of Steps

    • Doubling of DNA molecules with each cycle.
    • Automated machines can speed up the process.

Why Use PCR?

  • Common Applications:
    1. DNA Fingerprinting
      • Crime scene investigations.
      • Creates enough DNA for gel electrophoresis.
    2. Disease Diagnosis
      • Example: COVID-19 testing.
      • Specifically, rRT-PCR for SARS-CoV-2, which uses RNA.
      • Reverse transcription step needed to convert RNA to DNA.
      • Fluorescent probes for detection.

Detailed Example: COVID-19 rRT-PCR Test

  • RNA to DNA conversion: Necessary due to RNA being the viral genetic material.
  • Primers Binding: Helps identify viral RNA.
  • Role of Reverse Transcriptase: Converts RNA to cDNA.
  • Amplification: Regular PCR steps to make multiple cDNA copies.
  • Detection: Use of fluorescent probes.

Conclusion

  • Multiple uses of PCR: Beyond the examples given, more uses available in further readings.
  • Future of PCR: Will remain an indispensable technology.
  • Closing remark: Stay curious!