GROUP #09

PCR (Polymerase Chain Reaction)

Represented to: Dr. Shamim Akhtar

Represented by: Alina Fatima 17361506-091
Khadija Nisar 17361506-092
Faryal Fatima 17361506-093
  Polymerase chain reaction ( PCR), a technique
used to make numerous copies of a specific segment
of DNA quickly and accurately.

 The polymerase chain reaction enables investigators

PCR to obtain the large quantities of DNA that are

required for various experiments and procedures
in molecular biology, forensic analysis,
 evolutionary biology, and medical diagnostics.

• PCR is used in molecular biology to make many

copies of (amplify) small sections of DNA or a gene.
 • Using PCR it is possible to generate thousands to
millions of copies of a particular section of DNA
from a very small amount of DNA.

• PCR is a common tool used in medical and

biological research labs. It is used in the early
Cont… stages of processing DNA for sequencing, for
detecting the presence or absence of a gene to help
identify pathogens during infection, and when
generating forensic DNA profiles from tiny
samples of DNA.
  PCR was developed in 1983 by Kary B. Mullis, an
American biochemist who won the Nobel Prize for
Chemistry in 1993 for his invention.

 Before the development of PCR, the methods used

to amplify, or generate copies of, recombinant DNA
History fragments were time-consuming and labour-
intensive.

 In contrast, a machine designed to carry out PCR

reactions can complete many rounds of replication,
producing billions of copies of a DNA fragment, in
only a few hours.
  The PCR technique is based on the natural processes a cell
uses to replicate a new DNA strand. Only a few biological
ingredients are needed for PCR.

 The integral component is the template DNA—i.e., the DNA

that contains the region to be copied, such as a gene. As little
as one DNA molecule can serve as a template.
Cont…
 The only information needed for this fragment to be replicated
is the sequence of two short regions of nucleotides (the
subunits of DNA) at either end of the region of interest.

 These two short template sequences must be known so that

two primers—short stretches of nucleotides that correspond to
the template sequences—can be synthesized.
  The primers bind, or anneal, to the template at their
complementary sites and serve as the starting point
for copying. DNA synthesis at one primer is
directed toward the other, resulting in replication of
the desired intervening sequence.
Cont…
 Also needed are free nucleotides used to build the
new DNA strands and a DNA polymerase,
an enzyme that does the building by sequentially
adding on free nucleotides according to the
instructions of the template.
 • The principles behind every PCR, whatever the sample of
DNA, are the same.

• Five core ‘ingredients’ are required to set up a PCR. We will

explain exactly what each of these do as we go along. These
are:
How does PCR
• the DNA template to be copied
Work? • primers, short stretches of DNA that initiate the PCR
reaction, designed to bind to either side of the section of
DNA you want to copy
 DNA nucleotide bases (also known as dNTPs). DNA
bases (A, C, G and T) are the building blocks of DNA
and are needed to construct the new strand of DNA
 • Taq polymerase enzyme to add in the new DNA
bases
• buffer to ensure the right conditions for the
Cont…
reaction.

• PCR involves a process of heating and cooling called

thermal cycling which is carried out by machine.
 • These three stages are repeated 20-40 times,
doubling the number of DNA copies each time.

• A complete PCR reaction can be performed in a

few hours, or even less than an hour with certain
Cont… high-speed machines.

• After PCR has been completed, a method called

electrophoresis can be used to check the quantity
and size of the DNA fragments produced.
  Components Of PCR constitutes the
following:

1.DNA Template– The DNA of interest from the

sample.

Components of 2.DNA Polymerase– Taq Polymerase is used. It is

PCR 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.
 1.Deoxyribonucleotide triphosphate– These provide
energy for polymerization and are the building
blocks for the synthesis of DNA. These are single
units of bases.
Cont…. 2.Buffer System– Magnesium and Potassium provide
optimum conditions for DNA denaturation and
renaturation. It is also important for fidelity,
polymerase activity, and stability.
  PCR is of the following types:

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.
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.
Cont… Arbitrary Primed PCR
It is a DNA fingerprinting technique based on PCR.
It uses primers of DNA sequence of which is chosen
arbitrarily.
Principle of PCR
Purpose
Condition
Components
Purpose
To amplify a lot of double-stranded DNA molecules
(fragments) with same (identical) size and sequence by
enzymatic method and cycling condition.
Condition
Denaturation of ds DNA template
Annealing of primers
Extension of ds DNA molecules
Denaturation
Temperature: 92-94C
Double stranded DNA melts single stranded DNA
Annealing
Temperature: ~50-70C (dependant on the melting
temperature of the expected duplex)
 Primers bind to their complementary sequences 5’ 3’
Forward primer Reverse primer 3’ 5’
Extension
Temperature: ~72C
Time: 0.5-3min
DNA polymerase binds to the annealed primers and extends
DNA at the 3’ end of the chain Taq 5’ Taq 3’ 5’
Extension at 72°C
The reaction mixture is heated to 72°C which is the ideal
working temperature for the Taq polymerase. The
polymerase adds nucleotide (dNTP's) complimentary to
template on 3’ –OH of primers thereby extending the new
strand.
Three Aspects of PCR
Specificity
Efficiency
Fidelity
Standard PCR Protocol

A standard polymerase chain reaction (PCR) setup consists

of four steps:
Add required reagents or mastermix and template to PCR
tubes.

Add mineral oil to prevent evaporation in a thermal cycler

without a heated lid.
Amplify per thermo cycler and primer parameters.
Evaluate amplified DNA by agarose gel electrophoresis
followed by ethidium bromide staining.
Cont..
These steps are presented below in greater detail along with
materials and reagent selection tips. This is a basic PCR
protocol using Taq DNA polymerase.
Find additional protocols for other polymerases or advanced
PCR techniques in the Protocol section of our PCR
Technologies Guide.
Learn more about standard PCR, including what it is, on
our PCR basic page.
Reagents: What Is Needed for PCR
Procedure: Steps of PCR

The optimal conditions for the concentration of Taq DNA

polymerase, template DNA, primers, and MgCl2 will depend
on the system being utilized.
It may be necessary to determine the optimal conditions for
each individual component.
This is especially true for the Taq DNA polymerase, cycling
parameters, and the MgCl2 concentration. It is recommended
the enzyme and the MgCl2 be titrated to determine the optimal
efficiency.
1.Add the reagents to an appropriately sized
tube
in the order provided in the table. (Select appropriate
table for reaction setup: standard or readymix
reagent.)
For a large number of reactions, a matermix without
the template should be set up and aliquoted into
reaction tubes. At the end, template should be added
to appropriate tubes.
2.Mix gently by vortex and briefly centrifuge
To collect all components to the bottom of the tube.
Note: Add 50 µL of mineral oil to the top of each tube to
prevent evaporation if using a thermal cycler without a heated
lid.
3.Amplify
The amplification parameters will vary depending on the
primers and the thermal cycler used. It may be necessary
to optimize the system for individual primers, template,
and thermal cycler.
Typical Cycling Parameters
25-30 cycles of amplification are recommended.
Uses Applications advantages
and disadvantages limitations
presented To Mam shamium Akhtar
Presented by syeda Faryal Fatima
Course code Bot-410
Roll No 17361506-093
 Uses of PCR
• PCR is a common tool used in medical and
biological research labs.
• It is used in the early stages of processing
DNA for sequencing.
• It is for detecting the presence or absence of a
gene to help identify pathogens during
infection, and when generating forensic DNA
profiles from tiny samples of DNA
 Uses of PCR
• Quantitative PCR (qPCR) is only one type of
technique used for genotyping and is used to
identify single nucleotide polymorphisms.
• The polymerase chain reaction (PCR) is used to
make millions of copies of a target piece of
DNA.
• It is an indispensable tool in modern molecular
biology and has transformed scientific research
and diagnostic medicine.
 Applications of PCR
• Genetic Disease
• Primers can be created that will only bind and amplify certain alleles of
genes or mutations of genes.
• This is the basis of genetic counseling and PCR is used as part of the
diagnostic tests for genetic diseases.
• Some diseases that can be diagnosed with the help of PCR:
• Huntington's disease
• Cystic fibrosis
• Human immunodeficiency virus
 PCR Applications
• Fingerprinting
• PCR can be used as a tool in
genetic fingerprinting. This
technology can identify any
one person from millions of
others. For example, tiny
samples of DNA isolated
from a crime scene can be
compared with DNA from
suspects, or compared with
a DNA database
 Classification of organisms
• PCR product of the 16S gene
is obtained, sequenced, and
aligned
against bacterial DNA data
base, then the bacterium
can be identified. ... A
selected PCR band from
each of 40 isolates was
sequenced and
the bacterium identified to
species or genus level using
BLAST
 Genotyping
• Quantitative PCR (qPCR) is
only one type of technique
used for genotyping and is
used to identify single
nucleotide polymorphisms
(SNPs)
 PCR for genotyping usually
involves designing primers
specific to the mutation or
allele being studied.
 Mutation screening
• PCR, mutations producing
single-gene
disorders can be detected
by several different
methods, including
endonuclease digestion
and gel electrophoresis.
• Hence a genetic screen is
a type of
phenotypic screen
 Drug Discovery
• PCR is also used in molecular diagnostics and
biochemical analyses.
• Among other things, these techniques can
be used in drug development, especially in
measuring the efficacy of drug therapy
and research into cancer detection and
treatment.
 Sequencing

• Bioinformatics

• Genomic cloning

• Human Genome project

 Bioinformatics
• Bioinformatics is now being used for a vast
array of other important tasks.
• Including analysis of gene variation and
expression, analysis and prediction of gene
and protein structure and function, prediction
and detection of gene regulation networks,
simulation environments
 Genomic Cloning
An alternative to cloning, called (PCR), can be used to directly
amplify rare specific DNA sequences in a complex mixture
when the ends of the sequence are known Genomic DNA.

• (PCR) is a laboratory technique used to amplify DNA

sequences. The method involves using short DNA
sequences called primers to select the portion of the
genome to be amplified.
• The technique can produce a billion copies of the target
sequence in just a few hours.
 Human Genome project
• Human Genome Project
(HGP) relied on PCR. PCR
is also valuable in a
number of laboratory and
clinical techniques,
including DNA
fingerprinting, detection
of bacteria or viruses
(particularly AIDS), and
diagnosis of genetic
disorders
 Advantages
• High sensitivity compared to
culture and staining.
• Ability to test for anti-
microbial resistance.
• Quickly performed in 4-8
hours.
• Shown to be more cost-
effective with selective use
than culture and staining.
Disadvantages
• Potentially lower specificity
compared to culture and
staining.
• Need for narrow list of causative
agents to use specific primers.
• Possibility of amplifying normal
flora from corneal scrapings.
• Becomes less cost-effective
when performed with a multi-
organism PCR approach.
 Limitations of PCR Testing

• PCR testing alone may be limited as a diagnostic tool .

• Still need culture for testing for drug/antibiotic

susceptibility and genetic typing
• Post treatment diagnosis may be challenging
• PCR detects dead organisms that may be shed for
weeks after the patient stops showing symptoms.
 Limitations

• Unclear regarding persistence of infection.

• Detecting dead organisms at this stage may
have no clinical relevance .
• PCR results should not be used as the sole basis
of a patient treatment management decision.
THANKYOU FOR ATTENTION