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PCR

The document provides a comprehensive overview of Polymerase Chain Reaction (PCR), detailing its history, principles, steps, components, and applications. PCR is a technique used to amplify DNA, enabling the detection of specific pathogens and genetic analysis. It highlights the advantages and limitations of PCR testing, including sensitivity to contamination and the inability to distinguish between viable and nonviable organisms.

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Aira Black
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4 views

PCR

The document provides a comprehensive overview of Polymerase Chain Reaction (PCR), detailing its history, principles, steps, components, and applications. PCR is a technique used to amplify DNA, enabling the detection of specific pathogens and genetic analysis. It highlights the advantages and limitations of PCR testing, including sensitivity to contamination and the inability to distinguish between viable and nonviable organisms.

Uploaded by

Aira Black
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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Polymerase Chain

Reaction (PCR)
Contents
 History
 What is PCR?
 Principle
 DNA extraction
 DNA identification
 Steps of PCR
 Components of PCR
 Instrument
 Reaction conditions for PCR
 Experimental protocol
 Applications
 Advantages of PCR testing
 Limitations
 Interpreting results
History
PCR is not a discovery but
1 an invention

Invented by Kary Mullis in


2 1983

He was awarded nobel prize


3 in chemistry in 1993 for
PCR invention.
What is PCR?
 It is a wide technique used in cellular and molecular
biology to amplify a single copy or few copies of a
segment of a DNA and generating thousands to millions
of copies of a particular DNA sequence.
 This procedure is whole carried out biochemically that is
in vitro.
Principle
• PCR makes it possible to obtain, by in vitro replication,
multiple copies of a DNA fragment from an extract. Matrix
DNA can be genomic DNA as well as complementary DNA.
• It is a technique for obtaining large amounts of a specific
DNA sequence from a DNA sample.
• This amplification is based on the replication of a double-
stranded DNA template.
DNA Extraction
DNA can be extract from two ways.

1. Pure strain
that is already
2. From any
isolated in
environmental
laboratory
sample like soil,
wastewater etc.
DNA Identification
1. After extracting our target DNA then we have to
identify if our targeted material is DNA or not.
2. This is followed by Gel Electrophoresis procedure.
3. The gel used in it is Agarose that separates DNA
fragments ranging from 100bp to 25 kb.
4. After this agarose gel electrophoresis florescent
bands of the DNA will show up on software attached
with it by passing through gel documentation system
with UV light in it.
Steps of PCR
• PCR has three major steps 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 or hybridization
3. Elongation or extension
1. Denaturation
The DNA template is heated
to 94° C for 1 min and it is
called denaturation
temperature.
This breaks the weak
hydrogen bonds that hold
DNA strands together in a
helix, allowing the strands to
separate creating single
stranded DNA.
2. Annealing or hybridization
The denatured mixture is cooled
to anywhere from 50-70° C for
0.5-2 min and it is called primer
hybridization temperature.
This allows the primers to bind
(anneal) to their complementary
sequence in the template DNA.
3. Elongation or extension
The reaction is then heated to
72° C, the optimal temperature
for DNA polymerase to act.
At 72°C, Taq polymerase binds
to primed single-stranded DNAs
and catalyzes replication using
the deoxyribonucleoside
triphosphates present in the
reaction mixture.
Components of PCR
DNA template Buffer (containing
2 primers Mg++)

Taq DNA
Nucleotides dNTPs PCR tube
polymerase
Instrument
A thermocycler or PCR machine is
a laboratory apparatus used for
PCR. The device has a thermal
block with holes where tubes with
the PCR reaction mixtures can be
inserted. The cycler then rises and
lowers the temperature of the
block in discrete, pre-programmed
steps.
Reaction conditions for PCR
 Denaturing conditions are best at 94-95ºC for
30-60 seconds.
 Lower temperature may result in incomplete
denaturation of target template and PCR
products.
 Higher temperatures and a longer amount of
time can lead to enzyme activity loss.
Experimental protocol
In a traditional PCR protocol, reaction components are assembled as
described below. The final volume should be 50 µL.

o Thaw all reagents on ice.


o Assemble reaction mix into 50 µL volume in a thin walled 0.2 mL PCR
tubes.
o Add reagents in following order: water, buffer, dNTPs, Mg CL2, template
primers, Taq polymerase.
o Gently mix by tapping tube. Briefly centrifuge to settle tube contents.

Now followed by advance protocol, a master mix containing all reagents are
available and we just have to add it in tube with template DNA in the same
amount given in protocol by the company.
Applications
Amplification of DNA or RNA In analysis of mutations

Genetic fingerprinting and


In cloning of genes
forensics

Detection of any viral diseases


e.g. Covid 19, HIV
Advantages of PCR testing
 Valuable for detecting specific pathogens that
are difficult to culture in vitro or require a
 long cultivation period
 Quickly performed in just 4-8 hours time
period
 High sensitivity compared to culture and
staining
 It has the ability to test anti-microbial samples
Limitations
1. PCR is very sensitive to contamination. A small
amount of contaminating DNA could result in
misleading or ambiguous results
2. It cannot discriminate between viable and
nonviable and infectious and noninfectious cells or
viruses
3. It is subjected to inhibition by chemicals in
environmental samples like humic substances and
metals
4. False positive and false negative results
Interpreting results

Negative results Positive results


A positive result
A negative result
indicates detection of
means that there is DNA or RNA and
no evidence of DNA confirms infection, but
or RNA of the target does not necessarily
organism in the mean viable organism
specimen tested of interest is present
Thanks !

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