SLIDE CONTAIN BREIF NOTE ON PCR. IT CONTAINS 21 SLIDES INCLUDING, WHAT IS PCR? COMPONENTS, WORKING MECHANISM, APPLICATIONS, CONCLUSION, AND SOME REFRENCES, HISTORY ALSO

1. POLYMERASE CHAIN
REACTION
MID-II ASSIGNMENT
SUBMITTEDBY:JYOTISINGH
Y22265011
M.Sc.IISEM
SUBMITTEDTO:DR.DEEEPALIJAT
DEPARTMENT OF ZOOLOGY
DR. HARISINGH GOUR
VISHWAVIDYALAY
SAGAR (M.P.),470002
(A Central University)

2. POLYMERASE
CHAIN
REACTION :

3. CONTENT
1- INTRODUCTION
2-HISTORY
3-WHAT IS PCR .
4- BASIC COMPONENTS OF PCR .
5-WORKING MECHANISM OF PCR
 -DENATURATION
 ANNEALING
 EXTENSION
6-APPLICATIONS
7-ADVANTAGES
8-CONCLUSION
9-REFRENCES

4. Introduction
Polymerase chain reaction (PCR) is a new,
popular molecular biology technique for
enzymatically replicating DNA without using a
living organism, such as E. coli or yeast. The
technique allows a small amount of the DNA
molecule to be amplified many times, in an
exponential manner.
PCR is commonly used in medical and biological
research labs for a variety of tasks, such as the
detection of
hereditary diseases, the identification of genetic,
fingerprints, the diagnosis of infectious diseases,
the cloning of genes.
4
Today, the polymerase chain reaction makes the
amplification of short DNA fragments possible without
cloning, but cloning is still widely used for amplifying large
DNA fragments and for other manipulations of DNA
sequences.

5. HISTORY
The polymerase chain reaction (PCR), first developed
in 1985 by Kary Mullis, allows DNA fragments to be
amplified a billionfold within just a few hours. It can
be used with extremely small amounts of original
DNA, even a single molecule.
Kary Banks Mullis (December 28, 1944 – August 7,
2019) was an American biochemist. In recognition of
his role in the invention of the polymerase chain
reaction (PCR) technique, he shared the 1993 Nobel
Prize in Chemistry with Michael Smith and was
awarded the Japan Prize in the same year.

6. WHAT IS PCR .
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• PCRisbestdefinedastheDNAreplicationinvitro.
• PCRisbasedontheabilityofDNApolymerasetosynthesizenew
strandofDNAcomplementarytotheofferedtemplatestrand.
• Sometimescalled"molecularphotocopying,“
• PCRreliesonathermostableDNApolymerase,Taqpolymerase,and
requiresDNAprimersdesignedspecificallyfortheDNAregionof
interest.
• InPCR,thereactionisrepeatedlycycledthrough aseriesof
temperaturechanges,whichallowmanycopiesofthetargetregionto
beproduced.

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BasiccomponentsofPCRare :
• DNA template, or cDNA which contains
the region of the DNA fragment to be
amplified
• Two primers, which determine the
beginning and end of the region to be
amplified
• Taq polymerase, which copies the region
to be amplified
• Nucleotides, from which the DNA-
Polymerase form new DNA
• Buffer, which provides a suitable chemical
environment for the DNA-Polymerase.

8. WORKING MECHANISM OF PCR
1-denaturation
2- annealing
3-extension (polymerization).
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A single PCR amplification cycle involves three basic steps

9. 1-DENATURATION ; (Melting of Target DNA). In the denaturation
step , the target DNA is heated to a high temperature (usually 94 °C- 96
°C), resulting in the separation of the two strands . Each single strand of
the target DNA then acts as a template for DNA synthesis. This usually
takes between 15-30 seconds.

10. 2. ANNEALING: In this step , the two oligo-nucleotide primers anneal
to each of the single strand template DNA , since the sequence of the
primers is complementary to the 3’ ends of the template DNA . This step
is carried out at a lower temperature (usually 40 °C- 60) depending on
the length and sequence of the primers.This step usually takes about 10-
30 seconds.

11. 3.EXTENSION (polymerisation): The final step is extension ,
wherein Taq DNA polymerase ( of a thermophilic bacterium Thermus
aquaticus ) synthesizes the DNA region between the primers , using
DNTPs ( deoxynucleoside triphosphate ) and Mg 2+ .It means the
primers are extended towards each other so that the DNA segment lying
between the two primers is copied .The optimum temperature for this
polymerisation step is 72°C.

12. 20XX presentation title 12

13. -The duration of this step depends on the length of DNA sequence being
amplified but usually takes around one minute to copy 1,000 DNA bases
(1Kb).
-These three processes of thermal cycling are repeated 20-40 times to
produce lots of copies of the DNA sequence of interest.
-The new fragments of DNA that are made during PCR also serve as
templates to which the DNA polymerase enzyme can attach and start
making DNA.
-The result is a huge number of copies of the specific DNA segment
produced in a relatively short period of time.

15. APPLICATIONS
1-DNA Sequencing: PCR is a crucial step in DNA sequencing methods like Sanger sequencing and
Next Generation Sequencing (NGS). It helps in amplifying the target DNA region for further analysis.
2-Diagnostic Testing:

Infectious Diseases: PCR is extensively used for detecting pathogens like viruses (e.g., COVID-
19, HIV), bacteria, and fungi. It allows for highly specific and sensitive identification of the
causative agent.

Genetic Disorders: PCR can be used to identify mutations associated with genetic diseases,
allowing for early diagnosis and treatment.

Forensic Science: PCR can be employed in DNA profiling for criminal investigations and paternity
testing.
3- Reverse Transcription PCR (RT-PCR): This is used to quantify the amount of mRNA in a sample,
giving insight into gene expression.
4-Genetic Engineering and Cloning:
PCR is used to amplify genes of interest for further manipulation or insertion into vectors for cloning..

17. 5-Phylogenetic Analysis:
PCR can be used in combination with sequencing to study genetic
relationships between different organisms, helping in the reconstruction
of evolutionary trees.
6-Environmental Microbiology:
PCR can be used to detect and quantify microorganisms in
environmental samples, aiding in studies of microbial ecology.
7- Food Safety Testing:
PCR can be used to detect pathogens or contaminants in food products,
ensuring food safety.
8-Paternity and Relationship Testing:
PCR-based DNA fingerprinting is commonly used in paternity testing
and establishing familial relationships.
9-Paleontology and Archeology:
PCR can be used to amplify and analyze ancient DNA from preserved
specimens, helping in studies of extinct organisms and ancient human
populations.

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Advantages of PCR
1- Relatively fast results
2- Highly sensitive method
3-Can amplify specific DNA targets
4-Requires minimal starting material

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CONCLUSION
In conclusion, Polymerase Chain Reaction (PCR) is a powerful molecular biology
technique that enables the rapid and precise amplification of specific DNA
segments.
Its applications span a wide range of fields, from diagnostics and genetic research
to forensics and environmental studies.
PCR has revolutionized genetic analysis, allowing for highly sensitive and specific
detection of pathogens, genetic mutations, and gene expression levels.
 Its impact on various industries, including healthcare, biotechnology, and
forensic science, underscores its crucial role in advancing scientific knowledge
and improving human health and safety.

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REFRENCES
1-https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3768498/
2-https://www.khanacademy.org/science/ap-biology/gene-expression-and-
regulation/biotechnology/a/polymerase-chain-reaction-pcr
3-(PDF) Polymerase Chain Reaction (PCR): A Short Review (researchgate.net)
4- TRUEMAN’S ELEMENTARY BIOLOGY
5-Benjamin Pierce - Genetics_ A Conceptual Approach, 4th Edition -W.H.
Freeman (2010)

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