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

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

8. Taqman – Probe Detection:

9. Types of rt-PCR probes:
SYBR Green Dye:

10. Taqman Probe:

11. 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.

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

13. 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.

14. 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).

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

16. Thank You