Write in brief about: (i) Real time PCR (ii) Hepcidin (iii) VNTR (iv) Tag Polymerase

(i) Real-Time PCR (Quantitative PCR)

Real-Time PCR (qPCR) is a laboratory technique based on the polymerase chain reaction (PCR) that allows for the amplification and quantification of a specific DNA sequence in real-time. Unlike conventional PCR, qPCR monitors the amplification process as it happens, rather than at the end. This is achieved by incorporating fluorescent dyes or probes into the reaction mixture.

• Principle: qPCR utilizes fluorescent reporters that increase in signal as the amount of amplified DNA increases. The cycle threshold (Ct) value – the number of cycles required for the fluorescent signal to cross a defined threshold – is inversely proportional to the initial amount of target DNA.
• Applications: Diagnosis of infectious diseases (e.g., COVID-19 detection), gene expression analysis, cancer detection (detecting oncogenes), and genetic testing.
• Recent Advancements: Digital PCR, a more recent development, offers absolute quantification of DNA targets with even greater precision.

(ii) Hepcidin

Hepcidin is a peptide hormone produced by the liver that plays a central role in systemic iron homeostasis. It is considered the master regulator of iron metabolism.

• Principle: Hepcidin binds to ferroportin, the only known iron exporter in cells, causing its internalization and degradation. This reduces iron absorption from the gut and iron release from macrophages and hepatocytes.
• Applications: Diagnostic marker for iron deficiency anemia, anemia of chronic disease, and hereditary hemochromatosis. It's also being investigated as a therapeutic target for iron overload disorders.
• Clinical Significance: Low hepcidin levels are observed in iron deficiency anemia, while high levels are seen in inflammation-related anemias.

(iii) VNTR (Variable Number Tandem Repeat)

VNTRs are regions of DNA that contain repetitive sequences of a specific number of base pairs. The number of repeats varies significantly between individuals, making them highly polymorphic.

• Principle: VNTRs are inherited, and the variation in repeat number creates unique genetic fingerprints. These variations are used for DNA fingerprinting and genetic mapping.
• Applications: Forensic science (identifying individuals from biological samples), paternity testing, population genetics studies, and mapping genes associated with diseases.
• Limitations: While highly informative, VNTR analysis can be complex and requires careful standardization.

(iv) Tag Polymerase

Tag polymerase is a thermostable DNA polymerase enzyme originally isolated from the thermophilic bacterium Thermus aquaticus. It is a crucial component of PCR.

• Principle: Tag polymerase can withstand the high temperatures required for DNA denaturation during PCR without being denatured itself. This allows for repeated cycles of amplification.
• Applications: Essential for PCR-based applications, including DNA cloning, sequencing, and diagnostics.
• Advancements: Modified versions of Tag polymerase with enhanced fidelity (reduced error rate) and processivity (ability to synthesize long DNA strands) have been developed. Hot-start polymerases are also commonly used to prevent non-specific amplification.