Real -Time PCR.pptx

A real-time polymerase chain reaction (real-time PCR) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR).

1. Real-Time PCR Presented by:- Arvind Prajapati (18/IBT/005 )

2. Contents Introduction Mechanism of Real-time PCR Types of Real-time PCR probes detection Advantages & disadvantages of SYBR Green Advantages & disadvantages of Taqman probe Advantages of Real-time PCR Disadvantages of Real time PCR Applications

3. Introduction A   real-time polymerase chain reaction  ( real-time PCR ) is a  laboratory technique  of  molecular biology  based on the  polymerase chain reaction  (PCR). It monitors the amplification of a targeted  DNA  molecule during the PCR (i.e., in real time), not at its end, as in conventional PCR . It is also known as a quantitative polymerase chain reaction ( qPCR )

4. Mechanism of Real time PCR

5. Types of Real-time PCR probes SYBR Green Detection:

6. Taqman – Probe Detection:

7. Types of rt -PCR probes: SYBR Green Dye:

8. Taqman Probe :

9. Advantages & Disadvantages of SYBR Green: Advantages: Simple operation Lower cost More suitable to relative qPCR Disadvantages Slightly lower accuracy. Specificity lower than Taqman probe method . Do not perform multiplex qPCR .

10. Advantages & Disadvantages of Taqman probe: Advantages: Stronger specificity Perform multiplex qPCR . More suitable to absolute qPCR Disadvantages: Higher cost . Complex operation.

11. Advantages of rt - PCR: High technical sensitivity (<5copies) High precision No post-PCR steps like running of gel or sequencing etc. Non – specific amplification can be detected . Minimized risk of cross contamination. Saves lot of time.

12. Disadvantages of rt - PCR PCR product increases exponentially cannot monitor amplicon size. Can give incorrect quantity if DNA sample is degraded. Maximum of four simultaneous reactions. Non- specific binding (SYBR Green analysis).

13. Applications: Diagnostic uses Microbiological uses Detection of phytopathogens Detection of genetically modified organisms Clinical quantification and genotyping

14. Thank You