This document contain Real time PCR (RT-PCR) for gene regulation, it describes every part like principle, Mechanism , advantages , disadvantages etc

1. Real time PCR for Gene
Regulation
Submitted by- Sk Aziz uddin
Submitted to- Prof. Sandhya Bawa
Course- M.Pharm
Department- Pharmaceutical Analysis.
Batch- 2020-2022
College Name- Jamia Hamdard

2. Index
• Gene Regulation( Definition and its types )
• Real time PCR
• Principle of RT- PCR
• Mechanisms of RT-PCR
• Instrumentation of Real- time PCR
• Application
• Advantages of RT-PCR
• Disadvantages of RT-PCR
• Acronyms
• References

3. Gene regulation
• Definition-: The regulation of the expression of genes is
absolutely essential for the growth, development,
differentiation and the very existence of an organism,
there are two types of gene regulation.
1. Positive regulation- The gene regulation is said to be
positive when its expression is increased by a
regulatory element (positive regulator).
2. Negative regulation- A decrease in the gene expression
due to the presence of a regulatory element(negative
regulator) is referred to as negative regulation.
NOTE- That the double negative effect on gene regulation
results in a positive phenomenon.

4. Real time-PCR( RT-PCR)
• A real- time polymerase chain reaction is a
laboratory technique of molecular biology based
on the polymerase chain reaction(PCR). It
monitors the amplification of a target DNA
molecule during the PCR i.e. In real time and not
at its end, as in conventional PCR.
• It is also called as qPCR
• q PCR- q PCR stands for quantitative
polymerase chain reaction and is a technology
used for measuring DNA using PCR.

5. Principle of real time PCR
• The principle of real-time PCR: The principle of
real-time PCR relies on the uses of fluorescent
dye. The amount of the nucleic acid present into
the sample is quantified using fluorescent dye or
using the fluorescent labelled oligos.
• By measuring the reaction in real- time(how
many templates amplified during each reaction)
the amount of gene expression can be measured.

6. Why real- time PCR is more advanced
over the conventional PCR.
• The reason is simply clear, we can calculate or
measure the amount of amplification which is
not possible in the conventional PCR. And this
the reason the technique is also named
‘Quantitative PCR’.
• In the conventional PCR , we have to do agarose
gel electrophoresis that is also not required in
case of the real-time PCR. By doing the melting
curve analysis one can get results.

7. Continue....
• By running parallel reactions we can monitor
and choose which reaction is performing well.
Only ,that reaction can be monitored. Therefore
the chances of errors is minimum in quantitative
real-time PCR.
• Quantitative analysis of gene expression is not
possible in conventional PCR. By monitoring the
reaction in real-time the amount of gene
expression can be measured.

8. Mechanisms of Real time PCR
• Real-time PCR uses commercially available
fluorescence-detecting thermocyclers to amplify
specific nucleic-acid sequences and measure
their concentration simultaneously. Target
sequences are amplified and quantitated in the
same PCR machine.
• 2 common detection methods in Real-time PCR
1. By using non-specific fluorescent dyes.
2. By using sequence-specific fluorescent probes.

9. Types of RT- PCR

10. Non-specific fluorescent dye
• ds DNA binding dyes-:
• SYBR green I
• SYBR green II
• EVA green
• LC green
• YO-PRO
• SYTO family

11. DNA binding dye;
• The dye has its own fluorescence. Once the dye
bind to the double- stranded DNA the
fluorescence emitted by the dye increases 100to
1000 fold than the original signal.
• However , the original dye fluorescence is taken
as the baseline for the detection.
• The method is rapid, quick, reliable and cost
effective. Also, the chance of error in the
experiments are less and the reaction set up is
simple and easy to use.

12. Continue:-
• The result of the experiment depends on the
specificity of the primers used in PCR reaction.
• Because even through, the primers are bind non-
specifically, the DNA binding dye binds to the non-
specific sequence and gives the fluorescent signals.
• Because the dye detects the double- stranded DNA
to binds, even if the dsDNA is non- specific, the dye
most bind to it. Therefore the chance of non-
specific detection is high in the SYBR green dye-
based method. The SYBR green is one of the most
popular dyes used in real time PCR.

13. SYBR green I

15. SYBR green fluorescence chart
produced in real-time PCR

16. Some terms used in previous graph
• Baseline- The initial cycles of PCR when the
fluorescent signal shows no or little change.
• CT value- Threshold cycle (CT) indicates the cycle
number where the reaction fluorescence crosses the
threshold. This reflects the point in reaction when
sufficient amplicons have been generated to give
significant fluorescent signal over the baseline.
• Standard curve- A curve consisting of CT values
plotted against the log of standard concentration of
unknown samples are extrapolated from the
standard.

17. Sequence specific Fluorescent probe
method
• Based on sequence specific oligonucleotide
probes.
• Fluorogenic( reporter dye) at 5’ end and
quencher dye at 3’ end.
• Hybridizes to the target gene either in between
the primers or as a part of one of the
oligonucleotide primer.
• FRET( Fluorescence Resonance Energy
Transfer).

19. Probes- based detection method;
• The method used the single short sequence- specific
probes which are of two types;
1. Liner probe
2. Molecular beacons
• Linear probe- Linear probe are the TaqMan probe,
relies on the activity of Taq DNA polymerase. The
probes are the labelled short single stranded sequence-
specific DNA molecules which are radio or fluorescent
labelled. Here the probe is labelled with the fluorescent
dye called a reporter molecule, situated at the 3’ end.
The other 5’ end has the quencher dye which is in the
close proximity to the reporter dye and quenches the
fluorescence of the reporter dye.

20. Linear probe contd...
• The Taq DNA polymerase used in the real-time
PCR has the 5’ to 3’ exonuclease activity, which
removes the probe by extending the DNA.
• Once the probe is dissociated the reported molecules
emitted fluorescent. Because if the DNA( the
sequence of our interest) is amplified, reported
molecule unquenched and releases the fluorescence.
• N.B-The amount of fluorescence released during
each run is directly proportional to the amount of
DNA amplified.

21. Advantages of Probe-based method
• The main advantages of the probe-based method is
that we can use multiple probes for multiple
template DNA sequences. Which means we can
amplify multiple templates in a single reaction
efficiently.
• TAMRA and Black Hole quencher are two widely
used quencher dyes. While FAM is the most popular
dye. Here one problem occurred which limits the
TaqMan probe-based technique, The same
annealing temperature is not possible for both-
primer as well as a probe. By using lower extension
temperature the problem can be resolved.

22. Molecular beacons
• The molecular beacons operated on the
mechanism of the thermodynamics in which a
molecule remains in such a condition where the
majority of its energy can be saved. Here instead
of binding non-specifically, the molecular
beacon remains in a hair pin structure. A linear
probe can cause non-specific bindings which can
be prevented by using a beacon. Beacons are the
hairpin loop-like structure of the
oligonucleotides which has complimentary
sequences on both the ends.

23. Molecular beacons contd...
• The central loop is complementary to the target
sequences. One end of the hairpin loop has the
quencher dye and one end has the reporter
fluorescent dye. Here when the two ends of the
hairpin stem are in close proximity with each
other, the reporter molecule is quenched and
cannot generate fluorescence. But when it binds
to the complementary sequence , the two ends of
the hairpin separated with each other, the
quencher is blocked, the reporter dye released
and emits the fluorescence.

25. Scorpion probes
• Scorpion probes are other types of a probe or we
say it is a type of molecular beacons in which
instead of two different probes and primer, the
hairpin loop is incorporated directly at 5’ end of
the primer. The 3’ end contains the
complementary sequence to our target DNA.
• The scorpion probe is even more specific than
the molecular beacons.

28. Applications of Real-Time PCR
▫ Diagnostic uses- To diagnose infectious diseases.
▫ Microbiological uses- Used by microbiologists in
the fields safety, food spoilage and fermentation
and for the microbial risk assessment of water
quality.
▫ Used in research- used to provide quantitative
measurements of gene transcription.
▫ Detection of genetically modified organisms.
▫ Used in microbiology, oncology, and gene therapy.

29. Advantages of Real- Time PCR
• Wide dynamic range of quantification(7-8 log
decades)
• High technical sensitivity
• High precision
• No post PCR steps like running of gel or sequencing
etc.
• Non-specific amplification can be detected by melt
curve analysis of PCR products.
• Minimized risk of cross-contamination
• High throughput
• Multiplex approach possible
• Saves lot of time

30. Disadvantages of Real time- PCR
• At very low or high levels of DNA precision may
suffer.
• Can give the incorrect quantity if DNA sample is
degraded.
• qPCR assumes the sample is quantified at same
efficiency as the calibrant sample.
• Results are calculated based on the calibrants. If
the calibrants are made up wrong then the
quantities of the samples will be wrong.

31. Acronyms & references
• Acronyms
• RT-PCR- Real time polymerase chain reaction.
• DNA- de-oxy ribo nucleic acid
• CT value- threshold cycle
• TAMRA- tetramethylrhodamine.
• FAM- 6-carboxyfluorescein
• References
• You tube channel- shomus biology
• Book – bio technology satyanarayan
• Google web page-https://geneticeducation.co.in/real-
time-pcr-principle-procedure-advantages-limitations-
and-applications/
• Google slide share.

32. THANK YOU