BOGObiology: Polymerase Chain Reaction (PCR)

Introduction

• Topic: Polymerase Chain Reaction (PCR)
• Focus: What PCR is, its uses, reagents, steps in PCR, and PCR in COVID-19 testing.

Overview of PCR

• PCR is a genetic copying process used in biotechnology.
• Nicknamed "molecular photocopying."
• Amplifies small genetic material samples to millions or billions of copies.
• Used in forensics, agriculture, and medicine.

Uses of PCR

• Forensics: Amplify genetic material for comparison and solving crimes.
• Agriculture: Plant genotyping for breeding and cloning.
• Medicine: Diagnostic tool for genetic testing, cancer tracking, and COVID-19 testing.

Reagents in PCR

• DNA Sample: Contains the sequence to be copied (template strand).
• Polymerase Enzyme: Taq Polymerase, heat-resistant due to origin from Thermus aquaticus.
• DNTPs: Building blocks for new DNA copies.
• Primers: Single-stranded DNA that marks the start of the target region.
• Buffer Solution: Maintains optimal reaction conditions.
• Magnesium Cofactor (MgCl2): Assists primer adhesion and Taq polymerase function.

PCR Process

1. Denaturation
   • Heat DNA to ~95°C to break hydrogen bonds and create template strands.
2. Annealing
   • Lower temperature to ~55°C for primers to attach to the template.
   • Primers need to be specific to the target sequence.
   • Magnesium ions aid primer orientation and buffer stabilizes primer-template bonding.
3. Extension
   • Heat to ~72°C where Taq polymerase synthesizes new DNA strand from DNTPs.
   • Magnesium helps bond formation between primer and first DNTP.

• PCR cycle: Denaturation, annealing, and extension repeat for exponential DNA amplification.
• Example: 25 cycles result in ~33 million copies.

PCR in COVID-19 Testing

• RT-qPCR: Modified PCR for detecting SARS-CoV-2.
  • RT (Reverse Transcription): Uses reverse transcriptase to convert RNA to complementary DNA (cDNA).
  • q (Quantitative): Measures DNA quantity with fluorescence.

Fluorescent Reagents in RT-qPCR

• SYBR Green: Binds to double-stranded DNA, increases fluorescence with DNA amplification.

• Taqman Probe: Probes with reporter and quencher, glow when probe is broken during DNA synthesis.

• Detection: Positive result if fluorescence exceeds threshold.

  • False Positives: Rare due to specificity.
  • False Negatives: Possible if testing too early with low viral amount.

• Conclusion: PCR is a crucial tool in biotechnology for various applications including diagnostics.
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