In this slide contains contents, steps, different and application of PCR and RT-PCR Presented by: RAMYA NAGARAJU GARI (Department of pharmacology). RIPER, anantapur

1. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1
PCR and RT-PCR
A Seminar as a part of curricular requirement
for M. Pharmacy, I Year - I semester
Presented by
N. Ramya
(20L81S0110)
PHARMACOLOGY
Under the guidance of
A. Sudheer, M. Pharm
Associate Professor Dept. of pharmacology

2. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 2
RT-PCR
PCR
Contents of PCR
Steps in PCR
PCR VS RT-PCR
Applications
References
contents

3. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 3
• principle
• In RT- PCR, the RNA template is first converted into a
Complementary DNA (cDNA) using a reverse transcriptase (RT).
• The cDNA is then used as a template for exponential
amplification using PCR.
• The two technique use the same process except that RT-PCR has
an added step of reverse transcription of RNA to DNA to allow
for amplification.
RT-PCR:

4. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 4

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5
• optimized one- step RT- PCR condition, supports the reverse
transcription of the RNA from unpurified or crude samples, such as
whole blood and serum However, the starting RNA
• However, the starting RNA templates are prone to degradation in the
one- step approach, and the use of this approach is not recommended when
repeated assays from the same sample is required.

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6
Polymerase Chain Reaction
• Polymerase chain reaction (PCR) is a technique used in
molecular biology.
• To amplify a single copy or a few
copies of a segment of DNA generating
thousands to millions of copies of a
particular DNA sequence.

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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7
• Components Of PCR constitutes the following:
1.DNA Template– The DNA of interest from the sample.
2.DNA Polymerase– Taq Polymerase is used. It is thermostable and does not
denature at very high temperatures.
3.Oligonucleotide Primers- These are the short stretches of single-stranded
DNA complementary to the 3’ ends of sense and anti-sense strands.
Components Of PCR

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4.Deoxyribonucleotide triphosphate– These provide energy for polymerization
and are the building blocks for the synthesis of DNA. These are single units
of bases.
5.Buffer System– Magnesium and Potassium provide optimum conditions for
DNA denaturation and renaturation. It is also important for fidelity,
polymerase activity, and stability.

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• There are three major steps in a PCR, which are repeated
for 30 or 40 cycles.
• This is done on an automated cycler, which can heat and cool the
tubes with the reaction mixture in a very short time.
1. Denaturation.
2. Annealing.
3. Extension.
Procedure:

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Denaturation at 94°C :
• During the denaturation, the reaction
mixture is heated
to 94°C for 1 min, which causes
separation of DNA double stranded.
• Now, each strand acts as template
for synthesis of complimentary strand.

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Annealing at 54°C :
• The reaction temperature is lowered to 54-60℃ for around 20-
40 seconds. Here, the primers bind to their complementary
sequences on the template DNA.
• Primers are single-strand sequences of DNA or RNA around 20
to 30 bases in length.
• They serve as the starting point for the synthesis of DNA.

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• The two separated strands run in the opposite direction
and consequently there are two primers- a forward primer
and a reverse primer.

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• At this step, the temperature is raised to 72-80℃.
• The bases are added to the 3’ end of the primer by the Taq polymerase
enzyme.
• This elongates the DNA in the 5’ to 3’ direction. The DNA polymerase adds
about 1000bp/minute under optimum conditions.
• Taq Polymerase can tolerate very high temperatures. It attaches to the
primer and adds DNA bases to the single strand. As a result, a double-
stranded DNA molecule is obtained.
Extension:

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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 15
PCR RT-PCR
PCR is a technique to amplify a segment
of DNA, generating millions of copies of a
DNA Sequence.
RT-PCR is a variant of PCR used in
detection of gene expression in
molecular biology.
Denaturation, annealing, and extension
are the three steps.
RT-PCR is followed by PCR.
A double-standard DNA molecule serve
as the template.
A single standard RNA molecule is the
template for the reverse transcription.
DNA Polymerase is used as the
enzyme.
Reverse transcriptase and DNA
polymerase are used as enzyme.
Used in functional analysis of
genes, diagnosis, and monitoring
of heredity diseases, DNA Cloning,
DNA Sequencing.
Used in detection of gene
expression.

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• Gene Insertion: RT-PCR can also be very useful in the insertion of
eukaryotic genes into prokaryotes.
• Genetic Insertion Diagnosis: RT-PCR can be used to diagnose genetic
diseases such as Lesch -Nyhan symdrome.
• Cancer Detection: scientists are working on ways to use RT-PCR in cancer
detection to help improve prognosis and monitor response to therapy .
Applications

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• Julia Bachman. Reverse Transcription PCR. Elsevier. 2013,
530. 67-74.
• Freeman WM, Walker SJ. Quantitative RT-PCR. Elsevier.
2012, 26(1): 124-125.
• Alexander A. Morley. Digital PCR. Elsevier. 2014, 272.
12-13.
References