INTRODUCTION TO REAL TIME PCR IS GIVEN, basic principle of realtime pcr, along with the process of operating this, diagrammatic representation of the process, advantages and disadvantages o f reatimem pcr, applications of the same is also there

1. Real Time PCR
Presented by
Swati Suman
A35204418013
B.Sc Biotech – 5th sem

2. Introduction
• A real-time polymerase chain reaction (real-
time PCR), also known as quantitative
polymerase chain reaction (qPCR), 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 not
at its end, as in conventional PCR. Real-time
PCR can be used quantitatively and semi-
quantitatively.

3. Principle of real time PCR
• The principle of real-time PCR relies on the
use of fluorescent dye. In general, the
principle of the present method is :-
• “The amount of the nucleic acid present into the
sample is quantified using the fluorescent dye or
using the fluorescent labeled oligos.”

4. Methods of detection in real time PCR
• Two types of chemistry are available for the
real-time quantitative PCR:
• DNA binding dye (Intercalating dye-based
method)
• Sequence-specific probe (Hydrolysis Probe-based
detection method)

6. DNA binding dye
• The DNA binding dye method is the best technique for real-
time detection.
• The dye has its own fluorescence. Once the dye bind to the
double-stranded DNA the fluorescence emitted by the dye
increases 100 to 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.
• The primers are bind non-specifically, the DNA binding dye
binds to the non-specific sequence and gives the
fluorescent signals.

7. • The dye detects the double-stranded DNA to binds,
even if the dsDNA is non-specific, the dye must bind to
it.
• Therefore the chance of the 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.
• The sensitivity of the experiment is limited.

8.  Melting curve analysis
• Once the amplification reaction is completed and the
fluorescence signals are recorded, the template is
melted for the determination of the non-specific
bindings.
• The template is melted using heating, the dye
dissociates and the fluorescence signals are reduced.
• The decreased transition of a wide range fluorescence
is reported for the specific product while different heat
transition recorded for different short non-specific
bands.
• SYBR green and EvaGreen are two main dye used in the
quantitative real-time PCR. The experiments are used
in the validation of the assays such as DNA microarray.

9. • The fluorescence vs melting temperature graph is also
called a dissociation curve and the method is called a
dissociation curve analysis.
• The image shows the dissociation curve for specific
product and primer dimers while another image
shows different dissociation curves for two
homozygous and a heterozygous.

10. Probe-based detection method
• The method used the single short sequence-specific
probes which are of two types:
• Linear probe
• Molecular beacons
• The main advantage 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
reporter dye.

11. • Here, the annealing and the extension step is
combined at 60°C. After the denaturation, the probe
hybridization, primer hybridization and extension are
done at a single temperature.
• At 72°C extension, the Taq will be at its highest activity
therefore, instead of removing the probe it facilitates
strand displacement of a probe.
• That is why the annealing and the extension in the
linear probe-based real-time PCR are done at a single
temperature.

13.  Linear probe
• Linear probes 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.
• A linear probe can cause non-specific bindings which
can be prevented by using a beacon.

14. 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 hairpin structure.
• Beacons are the hairpin loop-like structure of the
oligonucleotides which has complementary sequences
on both the ends.
• 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.

15. Molecular Beacon

16. • Here, interestingly, 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 reported dye released and emits
the fluorescence.
• The emission is recorded by the detector.

17. Steps and procedure of real-time PCR
• The quantification is achieved by amplifying and monitoring the
DNA or RNA present into the sample. For the quantification of the
gene expression, the RNA is quantified into the real-time PCR.
• If DNA is present into the sample in higher quantity, amplification
and quantification start at the early stage of the reaction otherwise,
the amplification starts in late stage.
• As like the conventional PCR, there are three main steps in real-time
PCR;
• Denaturation
• Annealing
• Extension

18. • Denaturation occurs at 94°C where
the double-stranded DNA is
denatured and two single-stranded
DNA is generated. The DNA is
melted.
• This single-stranded DNA is the sight
of the annealing for the primers in
the later step of the amplification.
• Annealing occurs at 55°C to 66°C in
which the sequence-specific primer
bind to the single-stranded DNA.
Along with it, the fluorescent dye or
the probe binds to the DNA
sequence too.
• Extension occurs at 72°C at which
the Taq DNA polymerase activated
highest. In this step, the Taq adds
dNTPs to the growing DNA strand

20. ADVANTAGES LIMITATION
The method is cost-effective.
It is time-efficient.
More sensitivity and specificity
Fewer templates required
Melting curve analysis
The instrument is itself is too costly as
compared with the conventional PCR
Also, the multiplexing is still limited in the
Real-time PCR.
Kits are not available for all kind of genes
and disorders.
The technical and standardized protocols are
limited.