Real time PCR -A deatiled explanation of working principle of real time PCR with advantages over traditional PCR methods.

1. Presented by -Kumari Jyoti
Msc in biotechnology and
diploma in forensic science
Vinoba bhave university
Topics- Real time PCR

2. Need of different types of pcr-
Real time pcr
Rt-pcr .
Inverse pcr
Multiplex pcr
Pcr in forensic science-AmpFLP.
Nested pcr.
Primed pcr
Hot-start pcr.
In situ pcr.
Pcr Elisa.
ALU pcr

3. Real time polymerase chain
reaction
What is Real time PCR?
Materials and method
Applications
Advantages of real time PCR over traditional pcr.

4. It detect pcr amplification during the early phases of
reaction.Real time PCR system provide fast,precise and
accurate results.
In this pcr no need of post pcr methods for
quantitation .
Real time pcr detects the accumulation of amplicon
during the reaction.
In real time PCR the data is measured at exponential
phase.
Introduction of Real time
PCR

5. DNA extraction
from blood
Purified
DNA
Template DNA
is ready to PCR
Sample preparation steps

6. PCR tube-
buffer+dntps+primer+probe+polymerase
enzyme+ddH2o+template DNA+Mgcl2

7. Generally PCR works in three main steps -
1. Denaturation
2. Annealing
3. Extinction

8. Temperature required in these step's are
different and important to performe PCR.

9. Represents the Real time PCR process

10. Graph-it show the phase of pcr .
Exponential- doubling of product.
Linear- the raction is slow.
Plateau-The reaction had stopped,no more products are
being made.

11. Detection in Real Time PCR -
• Using the syber green
dye
•Taq man probe

12. It represents 5'nuclease assay (left) and
Syber green dye binding to DNA (right)

13. Syber green binds the DNA minor groove.

14. Advantages of syber
green-
Cheap
Sensitive
Effective
Easy to handle
Disadvantages-
It is non specific

15. Flouresence working
principle.

16. Taq man probe

17. FRET-It occurs when blue
light emitting flourescent
dye is in close proximity to a
black light -emitting
flourescent dye.
FRET does not occur
when the two
flourescent dyes are
not in close.
So reporter dye can't be
detected in presence of
quencher
Reporter dye is detectable
because quencher dye is not
close to reporter

18. Detector detect the flourescent
activity and it plot the results in a
graph -

20. Applications
• In array verification
• Drug therapy efficacy
• In DNA damage measurement
• Pathogen detection
• Genotyping
• Viral quantitation

21. Traditional pcr
disadvantage/limitations-
• End point gel detection.
• Poor sensitivity
• Short dynamic range <2fold
• Low resolution
• Size based description only.
• Results are not expressed as
number
• Ethidium bromide for staining
is not very qualitative.
Real time PCR advantage-
• Real time PCR detect
amplicons in reaction .
• More sensitive
• High dynamic range
•
• High resolution
• Not only sized based
description
• Results are expressed as no.
• Syber green or TaqMan
probe is used which is
quantitative

22. Traditional PCR and Real time PCR

23. Hard to differentiate between
the 10 copies or 50 copies of
DNA sample in gel in
traditional PCR.
In real time it is easy to
differentiate copies of
DNA i.e it capable to
detect 2 fold change.

24. Video link for Real time PCR
https://youtu.be/ThG_02miq-4
https://youtu.be/wUDysO8bFbA

25. Thankyou