Reverse Transcriptase PCR (RT-PCR) is a variation of the polymerase chain reaction that amplifies target RNA. Addition of reverse transcriptase (RT) enzyme prior to PCR makes it possible to amplify and detect RNA targets. Reverse transcriptase enzyme transcribes the template RNA and forms complementary DNA (cDNA). Single-stranded cDNA is converted into double-stranded DNA using DNA polymerase. These DNA molecules can now be used as templates for a PCR reaction

1. Vipin singh
Department of biochemistry,
(IMS )
Banaras Hindu University

2. What is PCR
 PCR is technique that takes specific sequence of
DNA of small amount and amplifies it to be used for
further testing.
 It is in vitro technique
purpose
To amplify a lot of double-stranded DNA molecules (fragment)
with same (identical) size and sequence by enzymatic method
and cycling condition.

3. Short History of PCR
• 1983 Kary Mullis developed PCR.
• 1985 First publication of PCR by Cetus Corporation appears in
science
• 1986 Purified Taq polymerase is first used in PCR.
• 1990 amplification and detection of specific DNA sequences
using a fluorescent DNA- binding dye , laying the foundation
for future “real-time PCR.

4. PCR VS RT-PCR
• In the traditional PCR method after the
amplification, the PCR products or the amplicon are
run on the agarose gel or PAGE to detect the
presence or absence of DNA amplification.
• But in the real-time PCR monitor amplification after
each PCR cycle in a real-time manner.

5. What is Real-Time PCR?
The Polymerase Chain Reaction (PCR) is a process
for the amplification of specific fragments of
DNA.
Real-Time PCR a specialized technique that
allows a PCR reaction to be visualized “in real
time” as the reaction progresses.
All real -time PCR system rely upon the detection
and quantitation of a fluorescent reporter.
The signal of which increases in direct proportion
to the amount of PCR product in a reaction

6. The principle of real-time PCR:
The principle of real-time PCR relies on the use of fluorescent
dye. In general, the principle…
 “The amount of the nucleic acid present
into the sample is quantified using the
fluorescent dye or using the fluorescent-
labeled oligos.”
When a dye or probe binds with the target template, it
releases a fluorochrome which resultantly emits
fluorescence for the detector to detect. The detector
captures a signal as a positive template amplification.

7. Chemistry of RT-PCR
Two types of chemistry are
available for the real-time
quantitative PCR:
I. DNA binding dye
(Intercalating dye-based
method)
II. Sequence-specific probe
(Hydrolysis Probe-based
detection method)

8. 1.DNA binding dye

9. DNA binding dye
 DNA binding dye method is the best technique for real-
time detection.
 Dye binds to the DNA the fluorescence emitted by the dye
increases 100 to 1000 fold than the original signal.
 The method is rapid, quick, reliable and cost-effective.
Also, the chance of error in the experiments is less and the
reaction setup is simple & easy to use.

10.  The result of the experiment depends on the specificity of the
primers used in the PCR reaction. Because even though the
primers remain bound non-specifically, the DNA binding dye
binds to the non-specific sequence and gives the fluorescent
signals.
 As the dye detects the double-stranded DNA to bind, even if
the dsDNA is non-specific, the dye binds 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.

11. Is it suitable for the determination of sensitive
templates?
The answer is Yes,
A melting curve analysis helps to
identify non-specific bindings during
the reaction.

12. Melting curve analysis:
• During melting, at a high temperature, the template starts
denaturing which consequence dye dissociation and reduce
fluorescence.
• Varied heat transition reported shows the amount of non-specific
products while the gradual decrease in fluorescence shows the
presence of specific amplification product.
Put simply, the story tells that,
A larger sequence need more time and higher
temperature for melting while non-specific
amplicons needs lower and varied
temperature to melt and so gives more
shorter curves in a graph.

13. you can see the fluorescence vs melting temperature
graph below,
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 presence of
primer-dimer and dissociation curve
Pictorial representation of graphs
of two different samples with
many different amplicons

14. 2.Probe-based detection
method:

15. Linear probe:
 Linear probes are the TaqMan probe, which relies
on the activity of Taq DNA polymerase.
 The probes structurally consist of labeled short
single-stranded sequence-specific DNA molecules
that are radio or fluorescent-labeled.
Here the probe is labeled with the fluorescent dye described as a
reporter molecule, situated at the 3’ end. The other 5’ end has the
quencher dye which is in close proximity to the reporter dye and
quenches the fluorescence of the reporter dye.
Once the probe dissociates the reporter molecules emitted
fluorescent light. Because, if the DNA (the sequence of our interest)
is amplified, the reporter molecule is unquenched and releases the
fluorescence

16. The amount of
fluorescence
released
during each
run is directly
proportional
to the amount
of DNA
amplified
during the
reaction.

17. Advantage of the probe-based method
The main advantage of the probe-based method is that
we can use multiple probes for multiple template
DNA sequences. This 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.

18. From a technical point of view,
face one problem, with this! which indeed limits the use of
the probe-based technique.
.
It’s a big problem for many reactions.
However, the use of lower extension
temperature can help.
The same annealing temperature is not possible
for both- primer as well as a probe.

19.  At 72°C extension, the Taq pol. 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.
Annealing and the extension step are combined at
60°C. After the denaturation, the probe hybridization,
primer binds and extension is done at a single
temperature.

20. Molecular Beacon
Molecular Beacon are short segments of single stranded DNA
that forms a hairpin in its free form.
Structurally,
 the complementary sequences
present on both ends of the
hairpin loop-like structure helps
to prevent non-specificity.
 on the other hand, 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.

21. • The molecular beacon probes are highly sequence-specific
and are the best choice for sensitive reactions. If the
molecular beacon cannot find its complementary sequence, it
remains in the hairpin loop form and prevents non-specific
bindings.
• In molecular beacon chemistry, the structure of the beacon
stem is very important. Designing the loop for the beacon is a
crucial step for getting specific amplification.
• Suppose, if the structure of the hairpin loop is too stable, it
can’t be separated, can’t unquench, can’t do hybridization
and fails reaction.

22. Scorpion probes:
 Scorpion probes are other types of probes or we say, a
type of molecular beacon in which instead of two
different probes and primer, the hairpin loop is
incorporated directly at the 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.

23. Components used into the real-time
Similar to conventional PCR, the real-time PCR reaction
contains almost the same components except for the
fluorescent dye or fluorescent-labeled probe.
I. dNTPs:
II. Taq DNA polymerase:
III. MgCl2:
IV. Template:
V. Primers:
VI. Water MASTERMIX

24. • dNTPs:
I. dNTPs are added during the synthesis of the growing
DNA strand by the Taq DNA polymerase.
II. The dNTPs remain the same as the conventional
PCR.
• Taq DNA polymerase:
I. Normal Taq cannot work efficiently for the real-time
PCR, instead always use the hot-start Taq DNA
polymerase.
II. The hot start Taq DNA polymerase is the best choice
for the quantification.

25. • MgCl2:
I. Magnesium ion also plays a crucial role in the
amplification during real-time PCR.
II. Concentration of the Mg2+ ions is different from the
conventional PCR.
III. Use 3 to 5mM of MgCl2 in the real-time PCR.
• Template:
I. 100pg to 1microgram template DNA is sufficient for real-
time PCR.
II. We required only 100 copies of genomic DNA/RNA
fragments for the amplification and to start the reaction.
III. The template DNA must be pure and free from any
contaminants.

26. Primers:
A primer set consists of short, single-stranded, target-
specific DNA sequences, provides the 3’ OH end and
facilitates DNA synthesis during PCR.
The length of the primer
The GC content
Annealing temperature
Complementation of each
primer
Start and end of primers
Hairpin formation in primers.
Properties of PCR primers:
The ideal PCR primer should have several properties or we
can say criteria in which it should fit. It relies on,

27. • Longer primers lead to non-specific amplification whereas shorter
primers can’t do amplification.
• primer is between 18 to 25 nucleotides. A ~20 nucleotides long
primer gives the best results in PCR.
• The ideal primer should have GC content between 40% to 60%.
• The annealing temperature should be 5ºC lower than the melting
temperature.
• The melting temperature of the primer is calculated using the
formula below,
• Tm= 4 (G + C) + 2 (A + T)
• Melting temperature difference between forward and reverse
primer between 5ºC.
• No repetitive and complementary bases.
• No complementary regions between forward and reverse primers.

28.  DNA primers are more temperature stable than
RNA primers.
 As the polymerization process is unidirectional,
RNA primers can’t be removed after the
completion of the reaction.
 DNA primers are easy to synthesize and use in
comparison to RNA primers.
 We are amplifying DNA not RNA so ideally it’s
recommended to use DNA primers.
Unlike the Replication process, the
primer used in the PCR is DNA primers
because of a couple of reasons,

29. What
Type
of
Instrument
are
used
with
Real-Time
PCR?
Real-time PCR instruments consist of
TWO main components:
• Thermal Cycler (PCR machine)
• Optical Module (to detect
fluorescence in the tubes during the
run)

30. How does real-time PCR work?

31. Denaturation
 Temperature : 92-94C
 Duoble stranded DNA
melts into single
standed

32. Annealing
 Temperature :50-60C
 Primer bind to their
complementary
sequences

33. Elongation
• Temperature : 72C
• Time 0.5-3min.
• DNA polymerase bind
to the annealed primers
and extends DNA at the
3’ end of the chain

34. What does Ct mean
 The Ct (cycle threshold) is defined as the number of cycles
required for the fluorescent signal to cross the threshold (ie
exceeds background level).
 Cts < 29 are strong positive reactions
 indicative of abundant target nucleic acid in the sample
 Cts of 30-37 are positive reactions indicative of moderate
amounts of target nucleic acid
 Cts of 38-40 are weak reactions indicative of minimal amounts
of target nucleic acid which could represent an infection state
or environmental contamination

35. How the signal of real time PCR is quantified
 The signal measured during these PCR
cycle is
used to plot the threshold
 The threshold is calculated as 10
times the stranded deviation of the
average signal of the baseline
fluorescent signal
 A fluorescent signal that is detected
above the threshold is considered a
real signal that can be used to define
the threshold cycle (Ct) for a sample
 The Ct is defined as the fractional
PCR cycle number at which the
fluorescent signal is greater than the
minimal detection level

37. Advantages of Real-time PCR:
• The method is cost-effective
• It is time-efficient
• The quantitative real-time PCR method is
more sensitive, specific and efficient
• Fewer templates required
• Melting curve analysis

38. Limitation of real-time PCR
• Although the advantages of the quantitative rt PCR are far
more than the conventional PCR, still the technology has
several limitations.
• The instrument itself is too costly as compared with
conventional PCR.
• multiplexing is still limited in Real-time PCR.
• Kits are not available for all kinds of genes and disorders. The
technical and standardized protocols are limited.
Furthermore, higher expertise and technical skills are required
for developing a novel qPCR assay.

39. What is
Real-
Time
PCR
used
for?
Real-Time PCR has become a
cornerstone of molecular biology:
• Gene expression analysis
– Medical research
– Drug research,Genotyping
• Disease diagnosis
– Viral quantification
• Food testing
– Percent GMO food
• Transgenic research
– Gene copy number