Quantitative PCR (qPCR), also known as real-time PCR, is a sensitive technique for quantifying nucleic acids by monitoring DNA amplification in real time using fluorescent dyes or probes. It differs from conventional PCR by providing real-time detection and higher accuracy in quantifying nucleic acids. qPCR has various applications in gene expression analysis, pathogen detection, and cancer research, while also having advantages and limitations regarding sensitivity, specificity, and cost.

1. Quantitative PCR (qPCR)
Principles, Techniques, and
Applications
Presented by: Dr. S. Aravindh
Institution: Rathinam College of Arts
and Science

2. Introduction to qPCR
• - Also known as Real-Time PCR
• - Used for quantifying nucleic acids
• - Monitors DNA amplification in real-time
• - Highly sensitive and specific technique

3. Principles of qPCR
• - Based on polymerase chain reaction (PCR)
• - Uses fluorescent dyes or probes
• - Monitors amplification after each cycle
• - Quantification is done using Ct (cycle
threshold) values

4. qPCR vs. Conventional PCR
• - **Conventional PCR:**
• - End-point detection
• - Gel electrophoresis for analysis
• - **qPCR:**
• - Real-time detection
• - More sensitive and accurate
• - Quantification of nucleic acids

5. Types of qPCR
• - **SYBR Green-Based qPCR:**
• - Fluorescent dye binds to double-stranded
DNA
• - Cost-effective but less specific
• - **Probe-Based qPCR (TaqMan, Molecular
Beacons, etc.):**
• - Uses fluorescent-labeled probes
• - Higher specificity and multiplexing

6. Components of qPCR Reaction
• - Template DNA or cDNA
• - Forward and reverse primers
• - DNA polymerase enzyme
• - dNTPs (deoxynucleotide triphosphates)
• - Fluorescent dye or probes
• - Buffer system

7. qPCR Workflow
• 1. Sample Preparation (RNA/DNA extraction,
cDNA synthesis)
• 2. Reaction Setup (Master mix preparation)
• 3. qPCR Amplification (Thermal cycling)
• 4. Data Analysis (Ct value determination,
relative/absolute quantification)

8. Data Analysis in qPCR
• - **Ct Value (Cycle Threshold):** Number of
cycles at which fluorescence exceeds
background
• - **Relative Quantification:** ΔΔCt method
comparing gene expression levels
• - **Absolute Quantification:** Standard curve
method for precise measurement

9. Applications of qPCR
• - Gene expression analysis
• - Detection of pathogens and viruses
• - Genotyping and mutation detection
• - Cancer research and biomarker discovery
• - Agricultural and environmental monitoring

10. Advantages and Limitations of
qPCR
• - **Advantages:**
• - High sensitivity and specificity
• - Real-time monitoring of amplification
• - Quantitative analysis
• - **Limitations:**
• - Expensive equipment and reagents
• - Requires precise primer design
• - Potential for contamination leading to false

11. Conclusion
• - qPCR is a powerful tool for nucleic acid
quantification
• - Used in research, diagnostics, and
biotechnology
• - Proper experimental setup and analysis are
key for reliable results

12. References & Q&A
• - Cite relevant sources
• - Open the floor for questions