Principle of Real-Time PCR

The principle which real time PCR is based on;
(a) Amplifying and simultaneously quantifying a target DNA
(b) Analyzing the PCR product at a predetermined time
(c) Reverse transcription
(d) None of the above

The correct answer is (a) Amplifying and simultaneously quantifying a target DNA.

✅ Correct Answer: (a) Amplifying and simultaneously quantifying a target DNA

Explanation

Real-Time PCR (qPCR) is a technique used to:
✔️ Amplify a specific DNA sequence
✔️ Simultaneously quantify the DNA concentration during the amplification process

Principle of Real-Time PCR

1. Amplification:

   • The target DNA sequence is amplified using a thermostable DNA polymerase, primers, and a fluorescence-based detection system.
   • The process follows the standard PCR cycle:
     • Denaturation (94–98°C) – Separation of DNA strands
     • Annealing (50–65°C) – Binding of primers to target DNA
     • Extension (72°C) – DNA polymerase synthesizes new DNA strand

2. Quantification:

   • Fluorescent dyes (e.g., SYBR Green) or probe-based systems (e.g., TaqMan) bind to the amplified DNA.
   • The fluorescence signal increases proportionally with the amount of amplified product.
   • The intensity of fluorescence is measured at the end of each cycle, allowing real-time quantification of DNA.

3. Threshold Cycle (Ct):

   • The Ct value represents the PCR cycle at which the fluorescence signal crosses a predetermined threshold.
   • Lower Ct values indicate a higher starting concentration of target DNA.

Types of Real-Time PCR

Why Other Options Are Incorrect

Advantages of Real-Time PCR

✅ High Sensitivity: Detects even low levels of DNA.
✅ Quantitative Analysis: Measures DNA concentration in real-time.
✅ Speed: Faster than traditional PCR; no need for post-PCR analysis.
✅ Reproducibility: High precision and accuracy.

Challenges of Real-Time PCR

❌ Primer-Dimer Formation: Can lead to false signals.
❌ Probe Design: Poorly designed probes reduce specificity.
❌ Inhibition by Contaminants: PCR inhibitors in samples can affect accuracy.

Applications of Real-Time PCR

✅ 1. Gene Expression Analysis

• Measurement of mRNA levels using RT-qPCR.

✅ 2. Pathogen Detection

• Rapid diagnosis of viral and bacterial infections (e.g., COVID-19, influenza).

✅ 3. Genotyping

• Identification of single nucleotide polymorphisms (SNPs).

✅ 4. Cancer Diagnostics

• Detection of oncogenes and tumor markers.

Example of qPCR Output

• Ct value = 18 → High initial concentration of target DNA.
• Ct value = 35 → Low initial concentration of target DNA.

Summary

• Real-time PCR works by amplifying and simultaneously quantifying DNA.
• Fluorescence-based detection allows real-time monitoring of amplification.
• Lower Ct values indicate higher initial target DNA concentrations.