The document describes the polymerase chain reaction (PCR) technique. PCR is used to make millions of copies of a specific DNA sequence. It involves repeated cycles of heating and cooling DNA in the presence of primers and a polymerase enzyme. During each cycle, the DNA strand is separated from its complement by heating, then the primers bind to the template and the polymerase synthesizes the complementary strand. This results in exponential amplification of the target DNA sequence. PCR is widely used in research, forensics, medicine and other fields due to its ability to rapidly produce large amounts of DNA.

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2. POLYMERASE CHAIN
REACTION
PRESENTED BY
DR. QURESHI JALIB
ROLL NO 04
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3. INTRODUCTION
 A method of making multiple copies of a DNA sequence,
involving repeated reactions with a polymerase.
 Polymerase chain reaction (PCR) is a common
laboratory technique used to make many copies (millions
or billions) of a particular region of DNA.
 This DNA region can be anything the experimenter is
interested in.
 For example, it might be a gene whose function a
researcher wants to understand, or a genetic marker
used by forensic scientists to match crime scene DNA
with suspects.
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4.  Typically, the goal of PCR is to make enough of the target DNA
region that it can be analyzed or used in some other way.
 For instance, DNA amplified by PCR may be sent for sequencing,
visualized by gel electrophoresis, or cloned into a plasmid for
further experiments.
 PCR is used in many areas of biology and medicine, including
molecular biology research, medical diagnostics, and even some
branches of ecology.
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5. HISTORY
 Basic principle of replicating ,Gobind Khorana in 1971.
 Polymerase Chain Reaction was developed in 1984 by
the American biochemist, Kary Mullis.
 Mullis received the Nobel Prize for developing PCR in
1993.
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6. PRINCIPLE
 it is a chain reaction,
 a small fragment of the DNA section of interest needs to
be identified which serves as the template , separated
by high temperature, primers that initiate the reaction,
polymerase elongate the primer.
 One DNA molecule is used to produce two copies, then
four, then eight and so forth.
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7. COMPONENTS
 The key ingredients of a PCR reaction are:
 template DNA
 Taq polymerase
 primers
 nucleotides (DNA building blocks).
 Buffer
 The ingredients are assembled in a tube, along with
cofactors needed by the enzyme, and are put through
repeated cycles of heating and cooling that allow DNA to
be synthesized.
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9. TEMPLATE DNA
 particular DNA sequence which is to be copied.
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10. POLYMERASE
 good activity rate around 75°C.
 withstand temperatures of 95-100°.
 first to be discovered with these characteristics
was Taq polymerase.
 Thermus aquaticus, a thermophilic eubacterium found in hot
springs.
 adds DNA bases to the single strand one-by-one in the 5’ to 3’
direction.
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11. NUCLEOTIDES
 building blocks of nucleic acids; they are composed of
three subunit molecules: a nitrogenous base, a five-carbon
sugar (ribose or deoxyribose), and at least one phosphate
group
 four different deoxyribonucleotide triphosphates (dNTPs);
 adenine (A) dATP
 guanine (G) dGTP
 cytosine (C) dCTP
 Thymine (T) dTTP
 A---------T C-------G
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12. PRIMER
 short oligonucleotides of DNA, usually around 8-60 base
pairs in length.
 starting point for DNA synthesis
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13. BUFFER
 reaction buffer is used to provide a stable pH.
 Mg2+ plays a vital role in the PCR reaction.
 acting as a co-factor for Taq polymerase and thereby
influencing enzyme activity.
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14. INSTRUMENT
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15. STEPS OF PCR
 Initialization:
 This step is only required for DNA polymerases that require
heat activation by hot start PCR.
 It consists of heating the reaction chamber to a temperature
of 94–96 °C (201–205 °F), or 98 °C (208 °F) if extremely
thermostable polymerases are used, which is then held for 1–
10 minutes.
 Denaturation:
 Heat the reaction strongly to separate, or denature, the DNA
strands. This provides single-stranded template for the next
step.
 first regular cycling event.
 heating the reaction chamber to 94–98 °C (201–208 °F) for
20–30 seconds
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16.  double-stranded DNA template by breaking the
hydrogen bonds between complementary bases, yielding
two single-stranded DNA molecules.
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17.  Annealing
 50–65 °C (122–149 °F
 for 20–40 seconds
 Cool the reaction so the primers can bind to their complementary
sequences on the single-stranded template DNA.
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18.  Stable hydrogen bonds between complementary bases are formed
only when the primer sequence very closely matches the template
sequence. During this step, the polymerase binds to the primer-
template hybrid and begins DNA formation.
 This temperature must be low enough to allow for hybridization of the
primer to the strand, but high enough for the hybridization to be
specific, i.e., the primer should bind only to a perfectly
complementary part of the strand, and nowhere else.
 If the temperature is too low, the primer may bind imperfectly.
 If it is too high, the primer may not bind at all.
 A typical annealing temperature is about 3–5 °C below the Tm of the
primers used.
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19.  Extension
 (72°C): Raise the reaction temperatures so Taq polymerase
extends the primers, synthesizing new strands of DNA.
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20. polymerase
primer
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21.  The precise time required for elongation depends both on the DNA
polymerase used and on the length of the DNA target region to
amplify.
 With each successive cycle, the original template strands plus all newly
generated strands become template strands for the next round of
elongation, leading to exponential (geometric) amplification of the
specific DNA target region.
 The formula used to calculate the number of DNA copies formed after
a given number of cycles is 2n, where n is the number of cycles. Thus,
a reaction set for 30 cycles results in 230, or 1073741824, copies of
the original double-stranded DNA target region.
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24.  This cycle repeats 25 - 35 times in a typical PCR
reaction, which generally takes 2 - 4hours, depending on
the length of the DNA region being copied. If the
reaction is efficient (works well), the target region can
go from just one or a few copies to billions.
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25. STAGES
 the reaction rate and efficiency of PCR are affected by limiting factors.
Thus, the entire PCR process can further be divided into three stages
based on reaction progress
 Exponential amplification: At every cycle, the amount of product is
doubled (assuming 100% reaction efficiency). The reaction is very
sensitive: only minute quantities of DNA must be present.
 Leveling off stage: The reaction slows as the DNA polymerase loses
activity and as consumption of reagents such as dNTPs and primers
causes them to become limiting.
 Plateau: No more product accumulates due to exhaustion of reagents
and enzyme.
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26. ADVANTAGES
 It is fairly simple to understand and
 Easy to use and produces results rapidly.
 The technique is highly sensitive with the potential to
produce millions to billions of copies of a specific product
for sequencing, cloning, and analysis.
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27. APPLICATIONS OF PCR
 PCR is used in research laboratories in DNA cloning procedures,,
DNA sequencing, recombinant DNA technology.
 The role of PCR in genetic engineering
These cloned DNA fragments can then be inserted into the target organism,
including microorganisms, plants or animals, using vectors such as bacteria and
viruses.
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28.  Detection and diagnosis of infectious disease
PCR can detect infectious disease before standard serological laboratory
tests (tests to detect the presence of antibodies), so allowing treatment to
start much earlier.
PCR is also useful for screening donated blood for infections.
Infections that are difficult to culture in the laboratory, such as
tuberculosis.
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29.  Detection of infection in the environment
PCR is used to monitor and track the spread of infectious disease within an animal
or human population.
PCR can also be used to detect bacterial and viral DNA in the environment, for
example looking at pathogens in water supplies.
Many viruses contain RNA rather than DNA. In such cases the viral genome has to
be transcribed before PCR is performed, and RTPCR is therefore used.
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30.  Forensic science : Genetic fingerprints
PCR is very important for the identification of criminal.
The DNA fingerprinting technique is used in forensic science.
A single molecule of DNA ( stains of blood , hair etc. ) is enough for amplification
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31.  Genetic analysis based on PCR is also used in paternity
testing, and in tissue typing for organ transplantation.
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32.  Genomic studies / genotyping
compare the genotype of two organisms and identify the
difference between them when body characteristics alone
do not provide enough evidence.
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33.  Evolutionary studies:
 It plays an important role in phylogenetic analysis. Minute
quantities of DNA from any source such a fossilized material,
hair, bones, mummified tissues can be amplified using PCR
techniques
 to examine how the genome of an organism has
changed over the course of evolution
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34.  Personalized medicine
PCR is used in personalized medicine to select patients for certain
treatments, for example in cancer when patients have a genetic
change that makes a patient more or less likely to respond to a
certain treatment.
 PCR in medical research :
to amplify the DNA of a virus, such as HIV, to understand how it
infects humans, or to replicate the DNA of a hormone , such as
insulin, to understand how it functions.
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35.  PCR in research
PCR can be used to create copies of DNA for introduction into host
organisms such as Escherichia
PCR can be used in analysis of gene expression, for example
looking at levels of expression and when genes are switched on and
off in physiological processes, including in health and disease.
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36.  Other uses
PCR is used in archaeology, to identify human or animal
remains, including insects trapped in amber, and to track
human migration patterns; degraded DNA samples may be
able to be reconstructed during the early cycles of PCR. PCR
can be used to differentiate between similar microorganisms
such as ticks, or work out relationships between different
species.
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37. LIMITATION
 prior information about the target sequence.
 DNA polymerases are also prone to error, which in turn
causes mutations in the PCR fragments that are
generated.
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38. REFERENCES
 https://www.khanacademy.org/science/...dna...pcr.../polymeras
e-chain-reaction-pcr
 vlab.amrita.edu/?sub=3&brch=186&sim=321&cnt=1
 https://en.wikipedia.org/wiki/Polymerase_chain_reaction
 ssjournals.com/index.php/ijbr/article/view/640/636
 passel.unl.edu/pages/informationmodule.php?idinformationmod
ule...topicorder...
 https://www.yourgenome.org/facts/what-is-pcr-polymerase-
chain-reaction.
 https://xxpresspcr.com/the-applications-of-pcr/
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ANY
QUESTIONS

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