Lecture Notes on RT-PCR: Definition, Principle, Enzymes, Types, Steps, Uses

Introduction to PCR

• Polymerase Chain Reaction (PCR): Technique to amplify DNA or RNA
  • Developed by Kary Mullis in mid-1980s
  • Combines nucleic acid hybridization and replication
  • Produces billions of copies of a DNA/RNA segment
• Types of PCR:
  • Reverse Transcriptase PCR (RT-PCR)
  • Quantitative PCR (qPCR)

Introduction to RT-PCR

• RT-PCR: Amplifies RNA using reverse transcriptase
• Converts RNA into complementary DNA (cDNA)
• Analyzes mRNA or micro RNA, studies gene expression

Objectives of RT-PCR

• Amplification of RNA segments
• Diagnose infections, study gene expression

Principle of RT-PCR

• Combines reverse transcription and conventional PCR
• RNA converted to cDNA by reverse transcriptase
• Repeated denaturation, annealing, and elongation cycles

Requirements (Enzymes) of RT-PCR

1. Sample RNA: Used instead of DNA
2. Reverse Transcriptase Enzyme: Converts RNA to cDNA
3. DNA Polymerase Enzyme: Synthesizes DNA strands
   • Taq DNA polymerase is commonly used
4. Primers:
   • Random Primers
   • Oligo (dT) Primers
   • Sequence-specific Primers
5. Deoxynucleotide Triphosphates (dNTPs): Building blocks for cDNA
6. PCR Buffers and Chemicals
7. Thermocycler: Automates temperature cycles

Types of RT-PCR

1. One-Step RT-PCR:
   • Single tube for both reverse transcription and amplification
   • Advantages: Simple, accurate, less contamination
   • Disadvantages: Limited detection, higher chance of reaction failure
2. Two-Step RT-PCR:
   • Separate tubes for transcription and amplification
   • Advantages: cDNA storage, higher efficiency
   • Disadvantages: More complex, higher contamination risk

Steps/Procedure of RT-PCR

1. Preparatory Stage:
   • RNA extraction, preparation of reaction mixture
2. Reverse Transcription:
   • RNA to cDNA conversion
3. Amplification:
   • Cycles of denaturation, annealing, and elongation
4. Product Analysis Stage:
   • Confirmation of desired amplification

Applications of RT-PCR

• Study of gene expression
• Identification of unknown species
• Diagnosis of infectious diseases
• Gene insertion and therapy studies
• Study of mutations and cancer cells
• Tools in genetic engineering

Advantages of RT-PCR

• Rapid and efficient amplification
• High specificity and sensitivity
• Accurate identification of RNA viruses
• Quantitative and qualitative analysis of RNA

Limitations of RT-PCR

• Only amplifies RNA
• Requires prior RNA sequence knowledge
• Sensitive to temperature and contamination

References

• Various academic and scientific sources on RT-PCR and its applications