1️⃣ Real-Time PCR (qPCR)
📌 Mechanism:
Amplifies DNA and monitors fluorescence in real time.
Fluorescent dyes/probes emit signals proportional to DNA quantity.
📌 Advantages:
Used for quantification.
High sensitivity & specificity.
Standard method for viral load detection (e.g., SARS-CoV-2).
2️⃣ Conventional PCR
📌 Mechanism:
Three classical steps: Denaturation → Annealing → Extension.
Amplified products visualized using gel electrophoresis.
📌 Advantages:
Qualitative only (presence/absence).
Simple, cost-effective.
Widely used in routine labs and teaching.
3️⃣ Digital PCR (dPCR)
📌 Mechanism:
Sample is partitioned into thousands of tiny reactions.
Each partition acts as an individual PCR.
Counts positive vs negative partitions → absolute quantification.
📌 Advantages:
Ultra-sensitive for rare mutation detection.
No need for standards.
Ideal for oncology, GMO quantification, and low viral loads.
4️⃣ Reverse Transcription PCR (RT-PCR)
📌 Mechanism:
RNA → (Reverse Transcriptase) → cDNA → PCR amplification.
📌 Advantages:
Used for RNA viruses (Rabies, Influenza, SARS-CoV-2).
Foundation for qRT-PCR.
Detects gene expression levels.
5️⃣ Hot-Start PCR
📌 Mechanism:
DNA polymerase is inactive at room temperature due to antibody/chemical blocking.
Activation happens only after initial heating.
📌 Advantages:
Prevents non-specific amplification.
Reduces primer-dimer formation.
Best for high-sensitivity applications.
6️⃣ Multiplex PCR
📌 Mechanism:
Multiple primers targeting different gene regions in a single reaction.
Amplifies multiple targets simultaneously.
📌 Advantages:
Saves time & reagents.
Used in pathogen panels, genetic testing, STR profiling, etc.
Requires precise primer design to avoid cross-reactivity.
🧬 Why PCR Keeps Evolving?
Because scientific questions are becoming more complex, and we need faster, more sensitive, and more accurate technologies to answer them. PCR continues to be the backbone of modern diagnostics and molecular biology.
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