PCR & ITS TYPES
Submitted to: Sachin Malik
Submitted by: Sameeksha Dubey
Roll: 19001435028
DD-Msc Biotechnology
CONTENT
Principle of PCR
What is PCR?
Steps of PCR
Types of PCR
Conclusion
References
PRINCIPLE OF PCR
The PCR technique is based on the enzymatic replication
of DNA. In PCR, a short segment of DNA is amplified
using primer mediated enzymes. DNA Polymerase
synthesizes new strands of DNA complementary to the
template DNA. The DNA polymerase can add a nucleotide
to the pre-existing 3’-OH group only. Therefore, a primer
is required. Thus, more nucleotides are added to the 3’
prime end of the DNA polymerase.
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WHAT IS PCR?
PCR or Polymerase Chain Reaction is a technique used in
molecular biology to create several copies of a certain DNA
segment. This technique was developed in 1983 by Kary
Mullis, an American biochemist. PCR has made it possible
to generate millions of copies of a small segment of DNA.
This tool is commonly used in the molecular biology and
biotechnology labs.
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WHAT IS PCR?
Components Of PCR constitutes the following:
[Link] Template– The DNA of interest from the sample.
[Link] Polymerase– Taq Polymerase is used. It is
thermostable and does not denature at very high
temperatures.
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WHAT IS PCR?
[Link] Primers- These are the short stretches of single-
stranded DNA complementary to the 3’ ends of sense and anti-sense
strands.
[Link] triphosphate– These provide energy for
polymerization and are the building blocks for the synthesis of DNA.
These are single units of bases.
5. Taq polymerase- PCR relies on a thermostable DNA polymerase, Taq
polymerase, and requires DNA primers designed specifically for the DNA
region of interest.
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WHAT IS PCR?
[Link] System– Magnesium and Potassium provide
optimum conditions for DNA denaturation and
renaturation. It is also important for fidelity, polymerase
activity, and stability.
Presentation title 8
STEPS OF PCR
PCR consists of three basic steps:
• Denaturation at 94-96 °C
• Annealing at ~68°C
• Elongation at Ca. 72 °C
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STEPS OF PCR
1. Denaturation:
Two strand of DNA separates (melt down) to form single stranded DNA
This step is generally carried out at 92C-96C for 2 minutes.
Denaturation occurs when the reaction mixture is heated to 94 ℃ for about 0.5 to 2
minutes. This breaks the hydrogen bonds between the two strands of DNA and converts
it into a single-stranded DNA.
The single strands now act as a template for the production of new strands of DNA. The
temperature should be provided for a longer time to ensure the separation of the two
strands.
STEPS OF PCR
2. Annealing:
Annealing of primer to each strand is carried out at 54C-60C.
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.
The two separated strands run in the opposite direction and consequently there are two
primers- a forward primer and a reverse primer.
STEPS OF PCR
3. Extension:
DNA polymerase adds dNTPs complementary to templates strands at 3’end of primer.
It is carried out at temperature of 72C.
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.
These three steps are repeated 20-40 times in order to obtain a number of sequences of DNA
of interest in a very short time period.
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TYPES OF PCR
Real-time PCR:
In this type, the DNA amplification is detected in real-time
with the help of a fluorescent reporter. The signal strength of
the fluorescent reporter is directly proportional to the number
of amplified DNA molecules.
Nested PCR:
This was designed to improve sensitivity and specificity. They
reduce the non-specific binding of products due to the
amplification of unexpected primer binding sites.
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TYPES OF PCR
Multiplex PCR:
This is used for the amplification of multiple targets in a
single PCR experiment. It amplifies many different DNA
sequences simultaneously.
Quantitative PCR:
It uses the DNA amplification linearity to detect, characterize
and quantify a known sequence in a sample.
Arbitrary Primed PCR:
It is a DNA fingerprinting technique based on PCR. It uses
primers the DNA sequence of which is chosen arbitrarily.
REAL TIME PCR 14
Real-time polymerase chain reaction (real-time
PCR) is commonly used to measure gene
expression. It is more sensitive than
microarrays in detecting small changes in
expression but requires more input RNA and is
less adaptable to high-throughput studies (1). It
is best suited for studies of small subsets of
genes.
NESTED PCR 15
Nested PCR is a modification of PCR that was
designed to improve sensitivity and specificity.
Nested PCR involves the use of two primer sets
and two successive PCR reactions. The first set
of primers are designed to anneal to sequences
upstream from the second set of primers and
are used in an initial PCR reaction.
MULTIPLEX PCR 16
The multiplex polymerase chain reaction
(Multiplex PCR) refers to the use of PCR to
amplify several different DNA sequences
simultaneously using multiple primers in one
tube and one amplification program for all
amplicons.
QUANTITATIVE PCR 17
Quantitative PCR (qPCR), also called real-time
PCR or quantitative real-time PCR, is a PCR-
based technique that couples amplification of a
target DNA sequence with quantification of the
concentration of that DNA species in the
reaction.
ARBITRARY PRIMED PCR 18
RNA-arbitrarily-primed PCR (RAP-PCR) can
be used to detect changes in gene expression in
organisms for which only minimal genomic
information is available. In this study, RAP-
PCR was used to detect modification of mRNA
expression in the freshwater.
APPLICATION OF PCR
Using PCR, a DNA sequence can be amplified millions or billions of
times, producing enough DNA copies to be analyzed using other
techniques. For instance, the DNA may be visualized by gel
electrophoresis, sent for sequencing, or digested with restriction enzymes
and cloned into a plasmid.
PCR is used in many research labs, and it also has practical applications
in forensics, genetic testing, and diagnostics. For instance, PCR is used to
amplify genes associated with genetic disorders from the DNA of patients
(or from fetal DNA, in the case of prenatal testing). PCR can also be used
to test for a bacterium or DNA virus in a patient's body: if the pathogen
is present, it may be possible to amplify regions of its DNA from a blood
or tissue sample.
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