POLYMERASE CHAIN REACTION

Polymerase Chain Reaction
(PCR)
Prepared by: Meghana Patel
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Contents
 Definition
 Meaning of PCR.
 Purpose of PCR
 Components of PCR.
 Steps of PCR
 Advantages
 Disadvantages
 REAL TIME PCR
 Definition
 Detection methods in real time PCR
 Applications.
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Meghana Patel APC - Anand

Definition :
 Polymerase chain reaction is a technique used in molecular
biology to amplify a single copy or a few copies of a segment of
DNA ,generating thousands to millions of copies of a particular
DNA sequence.
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What is meaning of PCR ?
P stands for polymerase
because the only enzyme
used in this reaction is DNA
polymerase
R stands for reaction
because the reaction
takes place in process.
C stands for chain because
the products of the 1st
reaction become
substrates of the following
one, and so on
PCR
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Purpose:
 To amplify a lot of double stranded DNA molecules with same size
and sequence by enzymatic method and cycling condition.
 Developed in 1983 by Karry Mullis. In 1993 Mullis was awarded the
Nobel prize in chemistry for his work on PCR.
 PCR is now considered as a basic tool for the molecular biologist.
 As is a photocopier a basic requirement in an office, so is the PCR
machine in a molecular biology laboratory!
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Components of PCR
 DNA template: It is DNA segment to be amplified.
 Two primers: Primers are synthetic DNA strands of about 18 to 25
nucleotides complimentary to 3’ end of template strand.
 Forward primer: It is complimentary to the 3’ end of antisense
strand (3’-5’).
 Reverse primer: It is complimentary to the 3’ end of sense strand
(5’-3’).
 Taq polymerase: Taq polymerase adds nucleotides complimentary to
template strand and synthesis new strand of DNA. It is isolated from
Thermus aquaticus( heat resistant bacteria). It is used because of
high temperature stability.
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 Nucleotides(dNTPs): All types of nucleotides are “building
blocks’’ for new DNA strands and essential for reaction. It
includes Adenine(A), Guanine(G), Thymine(T), Cytosine(C).
 Buffer solution: providing a suitable chemical environment for
optimum activity and stability of the DNA polymerase.
 Divalent cations: Polymerases require free divalent cations
usually Mg+2 for activity. It act as cofactor in the catalytic
addition of dNTPs.
 Monovalent ions(K+): It promotes primer annealing.
 PCR Machine: a thermal cycler.
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Quantity of components
 Target DNA: <1 μg
 Primers: 0.1-0.5 μM
 Taq polymerase: 1-2.5 units
 dNTPs: 20-200 μM
 Buffers: pH 8.3-8.8
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Steps of PCR
 There are three major steps in a PCR, 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.
 The three major steps are as follows:
1) Denaturation at 94oC
2) Annealing at 540C
3) Extension at 72oC
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Denaturation at 94oC
 During the these step, the reaction mixture is heated to 94OC for
1 min, which causes separation of DNA double stranded. Now
each strand acts as template for synthesis of complimentary
strand.
 The Hydrogen bonds between the two strands breaks down and
the two strands separates.
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Annealing at 54oC
 This step consist of cooling of reaction mixture after denaturation
step to 54oC, which causes annealing of primers to separated strand of
DNA.
 The length and GC-content of the primer should be sufficient for
stable binding with template .Guanine pairs with cytosine with three
hydrogen bonds. Thus, higher GC content results in stronger binding.
 Time taken to anneal is 45 second.
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Extension at 72oC
 Taq polymerase binds to the template DNA and starts adding
nucleotides that are complementary to the first strand.
 This happens at 72oC as it is
the optimum temperature
for Taq polymerase.
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Advantages :
 Rapid and easy to perform.
 Small amount of DNA is required per test.
 Result obtained more quickly.
 Usually not necessary to use radioactive material for PCR.
 PCR can be used to detect point mutations
 Making it possible to amplify DNA from degraded samples.
 It is very accurate ,especially for determining various
diseases, leading to better diagnoses.
 It is most specific ,sensitive.
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Disadvantages :
 Target DNA sequence must be known.
 Errors made by polymerase.
 High degree of operator skill required.
 High equipment cost.
 High sterile environment should be provided.
 Chances of contamination.
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What is the need of real time PCR ?
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Real Time PCR
Definition:
 A real time polymerase chain reaction is a laboratory technique of
molecular biology based on the polymerase chain reaction. It monitors
the amplification of a targeted DNA molecule during the PCR, i.e. in real
time, and not at its end, as in conventional PCR.
 It is also called as quantitative real time polymerase chain reaction.
 PRINCIPLE: The amount of the nucleic acid present into the sample is
quantified using the fluorescent dye or using the fluorescent labeled
oligos.
 Real Time PCR is sensitive and reliable method for detection and
quantification of nucleic acid levels.
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Detection methods in Real Time PCR:
(1) By using sequence specific fluorescent probes
(2) By using non specific fluorescent dyes
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1) By using sequence specific fluorescent probes
 Fluorescent reporter probes detect only the DNA containing the probe
sequence ; therefore ,use of the reporter probe significantly increases
specificity.
 It is hydrolysis probe which bear a reporter dye, often fluorescein at its 5’
end and a quencher attached to the 3’ end of the oligonucleotide.
 Under normal condition, the probe remain coiled on itself bringing the
fluorescence dye near the quencher, which inhibits or quenches of
fluorescent single of the dye so it is also known as FRET ( fluorescence
resonance energy transfer).
 During the annealing stage of the rtPCR both probe and primers anneal to
separated strand of DNA.
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• As the taq-polymerase start to synthesize new DNA strand in the
extension stage, and once the polymerase reaches the probe ,its 5’-
3’exonuclease degrades the probe, physically separating the fluorescent
reporter from the quencher, resulting in an increase in fluorescence.
• The increase in PCR product is proportional to amount of fluorescence.
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2) By using non specific fluorescent dyes
 Non specific fluorescent dye binds to all double stranded DNA product
in real time PCR, causing fluorescence of the dye.
 An increase in DNA product during PCR therefore leads to an increase in
fluorescence intensity and is measured at each cycle, thus allowing DNA
concentrations to be quantified.
 SYBR Green 1 ,SYBR Green 2, EVA green, LC green dyes are used in these
process.
 SYBR Green is most widely used for its higher signal intensity.
 The reaction is prepared as usual, with the addition of fluorescent dsDNA
dye.
 The reaction is run in a real time PCR instrument, and after each the levels
of fluorescence are measured with a detector. 21

Detection :
 The detection is based on fluorescence technology.
 The specimen is first kept in proper well and subjected to thermal cycle
like in normal PCR.
 The machine, however ,in the real time PCR is subjected to tungsten or
halogen source that lead to fluoresce the marker added to the sample and
the signal is amplified with the amplification of copy number of sample
DNA.
 The emitted signal is detected by an detector and sent to computer after
conversion into digital signal that is displayed on screen.
 The signal can be detected when it comes up the threshold level.
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ct value: It is defined
as number of cycles
required for the
fluorescent signal to
cross the threshold.
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Applications :
 There are different applications for real time PCR as follows:
 The use of PCR in clinical settings can be broadly divided into three
categories:
1) To amplify human genes to check for mutations.
2) To amplify microbial genes in a sample.
3) To amplify human gene from a limited sample for creating a
complete DNA profile of an individual.
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Diagnosis of Virus
 The virus contain mRNA as a genetic material so that for detection of
virus is done by coupling of real time PCR with a procedure called
reverse transcription.
 In this method , RNA is first
transcribed into complementary
DNA(cDNA) by reverse transcriptase
from total mRNA. The cDNA is then
used as the template for the real
time PCR reaction.
It is also used in diagnosis of genetic
disease, cancer etc.
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Gene Expression
 It is most sensitive method for the detection and quantification of gene
expression levels.
 It is used for determining how the gene expression and gene changes
over time ,such as in:
1) The response of tissue and cell cultures to an administration of a
pharmacological agent
2) response to change in environmental conditions.
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Agriculture
 Detection of phytopathogens:
 The agricultural industry is constantly striving to produce plant
propagules that are free of pathogens in order to prevent economic
losses and safeguard health.
 Discrimination between the DNA of the pathogen and the plant is based
on the amplification of specific sequences in ribosomal RNA gene’s
coding area by real time PCR.
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Genetically modified organisms (GMO)
 A genetically modified organism(GMO) is a living organism e.g. a plant
,whose genetic composition has been altered by means of gene technology.
 The genetic modification usually involves insertion of a piece of DNA , into
the genome of the organism to be modified.
 These smaller piece of DNA is usually taken from other naturally occurring
organisms.
 Specific primers are that amplify the gene sequences used during the
process of engineering the vector.
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In Gene therapy:
 In gene therapy as a drug delivery system there are two important
parameter have to be analyzed :
1) The expression level of the therapeutic gene and
2) The distribution of the drug in different organs.
For the control of both parameters , real time PCR is used.
Microbiological uses:
used by microbiologists in the fields of food safety, food spoilage and
fermentation and for the microbial risk assessment of water quality.
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POLYMERASE CHAIN REACTION

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