LAB-3- REAL TIME PCR12215489652312222.pptx

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1. Real time PCR Quantitative PCR

2. What is Real-Time PCR? In conventional PCR, the amplified product, is detected by an end-point analysis, by running DNA on an agarose gel after the reaction has finished. In contrast, real-time PCR allows continuous measurement of the amplified product to be detected and measured as the reaction progresses, that is, in “real time”.

3. Real-time PCR products is made possible by including a fluorescent molecule that reports an increase in the amount of DNA with a proportional increase in fluorescent signal. The fluorescent chemistries employed for this purpose include (1) DNA-binding dyes (2) fluorescently labeled sequence specific primers (3) fluorescently labeled sequence specific probes.

4. Specialized thermal cyclers equipped with fluorescence detection modules are used to monitor the fluorescence as amplification occurs. The measured fluorescence reflects the amount of amplified product in each cycle

5. What are the steps of Real time PCR? 1- DNA Extraction 2- Preparation of the master mix: a. Taq DNA Polymerase b. PCR Buffer: contains MgCl2. And dNTP Mix: ( dATP , dGTP , dCTP , dTTP ). c. Virus-specific forward and reverse primers and a dual-labeled probe. d. Internal control e. Template DNA (Sample DNA), or positive control, or Quantitation Standards

6. 3- Programming the Real-Time PCR Instrument: Thermal profile programming(*) (Denaturation, Primer annealing, and Extention ) Identifying and labeling of unknown samples, standards, positive and negative controls, and assigning the quantitative values for the standards (plate setup)(**) What are the steps of Real time PCR?

7. (*)

8. Plate Setup (**)

9. Analysis the Results of Real time PCR By the end of the thermal protocol, the Real-Time PCR Instrument software automatically calculates the baseline cycles and the threshold. The standard curve is plotted using the data obtained from the defined standards, with the (Y) axis is the CT-Threshold Cycle, and the (X) axis is the Log Starting Quantity the point at which the signal rises above background level and becomes distinguishable, is called the threshold cycle (CT)

10. Standard Curve

11. Advantages of Real time PCR 1- The main advantage of real-time PCR over conventional PCR is that real-time PCR allows you to determine the starting template copy number with accuracy and high sensitivity Real-time PCR that is quantitative is also known as qPCR. In contrast, conventional PCR is at best semi-quantitative.

12. 2- Real-time PCR data can be evaluated within a short time (one to two days) 3- Because reactions are run and data are evaluated in a closed-tube system, (reduced contamination with no post-amplification manipulation) . 4- for follow up of antiviral therapy, e.g HBV, HIV Advantages of Real time PCR