about pcr

1. Polymerase Chain
Reaction
Arifa Akram Barna
MD (VIROLOGY)
IEDCR, DGHS compound
BSMMU,DHAKA.
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2. The polymerase chain reaction (PCR) is a rapid, in
expensive and simple way of copying specific DNA
fragments from minute quantities of source DNA
material.
 Also called as “PEOPLE CHOICE REACTION”
 Developed in 1983 by Kary Mullis & he received the
Nobel Prize in chemistry in 1993, for his invention.
Purpose:
To amplify a lot of double-stranded DNA molecules
(fragments) with same (identical) size and sequence by
enzymatic method and cycling condition
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3. Chemical components:
•DNA template that contains the DNA region (target) to be
amplified.
•Two primers that are complementary to the 3' (three prime) ends of
each of the sense and anti-sense strand of the DNA target.
•Taq polymerase or another DNA polymerase with a temperature
optimum at around 70 °C.
•Deoxynucleoside triphosphates (dNTPs) nucleotides containing
triphosphate groups), the building-blocks from which the DNA
polymerase synthesizes a new DNA strand.
•Buffer solution, providing a suitable chemical environment for
optimum activity and stability of the DNA polymerase.
•Divalent cations, magnesium or manganese ions; generally Mg2+ is
used, but Mn2+ can be utilized for PCR-mediated DNA
mutagenesis, as higher Mn2+ concentration increases the error rate
during DNA synthesis[7]
•Monovalent cation potassium ions.
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5. DNA Extraction : It is the removal of DNA from the cells or
viruses in which it normally resides.The purpose of DNA extraction
is To obtain DNA in a relatively purified form which can be used
for further investigations, i.e. PCR, sequencing, etc.
Blood Cigarette
butts
Semen Chewing Gum
Saliva Finger Nails
Urine Bite Marks
Hair Envelopes &
Stamps
Teeth Faeces
Bone Clippings
Tissue
Source of DNA
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6. Cell lysis: The first step involves disrupting the
cell to access the DNA. This can be done using
chemical or physical methods.
Lipid removal: Removal or separation of membrane
lipids and cell debris. This is usually done by using
detergents such as sodium dodecyl sulfate, and by
centrifugation
Deproteinize cell extract: Protein denaturation is done
using a protease such as pronase and proteinase K.
Subsequently denatured protein is separated from the
cell extract.
RNA removal: Removal of RNA is done by adding an
RNase, which rapidly degrades RNA into
ribonucleotide subunits. This step is usually optional
within most kits
DNA precipitation/aggregation/elution:
Basic steps of DNA extraction
The basic steps involved in all DNA extraction methods consist of
the following-
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7. Procedures for DNA Extraction
1.Organic Extraction:
This method has been widely used and is the conventional method for
DNA extraction. In the first step, cells are lysed and cell debris is
usually removed by centrifugation. Then the proteins are denatured
using a protease.In the organic extraction method, after denaturation,
the proteins are removed using organic solvents such as phenol, or 1:1
mixture of phenol and chloroform.
a) Phenol–chloroform extraction: It is a liquid-liquid extraction
technique in biochemistry and molecular biology for purifying nucleic
acids and eliminating proteins. In brief, aqueous samples are mixed
with equal volumes of a phenol:chloroform mixture. The proteins will
partition into the organic phase while the nucleic acids (as well as
other contaminants such as salts, sugars, etc.) remain in the
aqueous phase. If the mixture is acidic, DNA partitions into the
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8. organic phase while RNA remains in the aqueous phase. DNA is
usually recovered from the aqueous phase by alcohol
precipitation. This is a time-consuming. Phenol is highly corrosive
and can cause severe burns. DNA isolated using this method may
contain residual phenol and/or chloroform, which can inhibit
enzyme reactions in downstream applications, and therefore may
not be sufficiently pure for sensitive downstream applications such
as PCR.
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9. b)Ethanol precipitation: The technique is rapid and is quantitative
even with nanogram amounts of DNA. The most widely used
method for concentrating DNA is precipitation with ethanol. The
precipitate of nucleic acid, forms in the presence of moderate
concentrations of monovalent cations (Salt, such as Na+), is
recovered by centrifugation and redissolved in an appropriate buffer
such as TE.usually by ice-cold ethanol or isopropanol. Since DNA is
insoluble in these alcohols, it will aggregate together, giving a
pellet upon centrifugation.
100% _alcohol:: aggregation
70%_purification
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10. Advantage of organic process:
•yields relatively pure, high molecular weight DNA
•DNA is double stranded – good for RFLP
Disadvantage:
•Time consuming
•Requires sample to be transferred to multiple tubes – increases risk of
contamination
•Involves use of hazardous (and smelly!) chemicals
2.Non organic method
Does not use organic reagents such as phenol or chloroform.Digested proteins are
removed by salting out with high concentrations of LiCl.
A)Minicolumn purification/Spin column-based nucleic acid purification:
Column-based nucleic acid purification is a solid phase extraction method to
quickly purify nucleic acids.This method relies on the fact that the nucleic acid
may bind (adsorption) to the solid phase (silica or other) depending on the pH and
the salt content of the buffer, which may be a Tris-EDTA (TE) buffer or
Phosphate buffer.
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11. B)Magnetic separation
C)Anion exchange
technology
D)Chelex (Ion Exchange
Resin) Extraction
E)FTA Paper
F)Salting-out methods
G) Cesium chloride
density gradients
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12. Reagents used for Extraction
 Proteinase K:
 digest the contaminating proteins
 protects the nucleic acid from degradation by degrading nucleases.
 SDS:
It is a strong anionic detergent that will help the cell mb & nuclear
envelopes to break down & expose the chromosomes that contain
DNA.
 TRIS:
It interacts with the lipopolysaccharides on outer mb& helps to
permeabilize the mb.
 Lysis buffer:
The function of lysis buffer is to aid in the breaking of the cell mb to
release the DNA
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13.  NaCl:
It provides Na+ ions that will block negative charge from
phosphate on DNA, neutralize the negative charges & allowing the
DNA molecules to come together.
 EDTA (Ethylenediaminetetraacetic disodium salt) is a chelating
agent of divalent cations such as Mg2+. Mg2+is a cofactor for
Dnase nucleases. If the Mg2+is bound up by EDTA, nucleases are
inactivated.
 Ethanol:
It is a dehydrating agent & it is used for precipitation of
DNA molecules
 Wash buffer:
Towards the end of DNA extraction,it is necessary to wash away
reagents containing salts in order to obtain the clean DNA for further
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14. Principle of PCR
1.Denaturation at 94°C : the
double strand melts open to
single stranded DNA.
2.Annealing at 54°C:
formation of hydrogen bonds
between single stranded primer
and single stranded bases .
3.Extension at 72°C : after the
primers attach, the Taq
polymerase begins to add
nucleotide to form
complementary strand .
4.Amplification/Quantificatio
n
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15. The PCR process can be divided into three stages:-
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 need to be present.
Levelling 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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16. 1.REAL TIME PCR : Real-time PCR, also called quantitative PCR
or qPCR, can provide a simple and elegant method to actually view
the amount of a target sequence or gene that is present in a sample.
Several different types of real-time PCR are being marketed :
SYBR Green (non- specific detection)
Taq Man real-time PCR
Molecular beacon Specific detection
Scorpion primer
TYPES OF PCR
TaqMan real-time PCR: TaqMan probe consists of two types of
fluorophores.While the probe is attached or unattached to the
template DNA and before the polymerase acts, the quencher (Q)
fluorophore (usually a long-wavelength colored dye, such as red)
reduces the fluorescence from the reporter (R) fluorophore
(usually a short-wavelength coloered dye, such as green). It does
this by the use of Fluorescenc Resonance Energy Transfer(FRET),
which is the inhibition of one dye caused by another without
emission of a proton.The reporter dye is found on the 5’ endBarna 16

17. Cont.
of the probe and the quencher at the 3’
end. The Taq Man probe binds to the
target DNA, and the primer binds as
well. Because the primer is bound,Taq
polymerase then adds nucleotides
and removes the Taqman probe from
the template DNA. This separates the
quencher from the reporter, and allows
the reporter to give off its emit its
energy. This is then quantified using a
computer. The more times the
denaturing and annealing takes place,
the more opportunities there are for the
Taqman probe to bind and, in turn, the
more emitted light is detected.
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18. The reporter dye is released from the extending double-stranded
DNA created by the Taq polymerase. Away from the quenching dye,
the light emitted from the reporter dye in an excited state can now
be observed.
Quantification:The light emitted from the dye in the excited state is
received by a computer and shown on a graph display, such as this,
showing PCR cycles on the X-axis and a logarithmic indication
of intensity on the Y-axis.
2.REVERSE TRANSCRIPTASE PCR :
Based on process of reverse transcription, which reverse transcribes
RNA into DNA initially isolated from retroviruses.
´Formation of cDNA from RNA using oligo dT primers which
stores the sequence of RNA (such as messenger RNA, mRNA) in
the more stable form of nucleic acid ie., DNA is the first step- called
“first strand reaction”
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19. Cont.
• RNaseH is added (an
RNA digestion enzyme)
which digests the RNA
away from the RNA-
cDNA hybrid.
• ´Standard PCR is
conducted using DNA
oligo primers specific for
sequence of interest. This
second step is referred to
as the "second strand
reaction”.
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20. APPLICATIONS
o Cancer diagnosis
o RT-PCR can be used to diagnose genetic disease such as Lesch–
Nyhan syndrome.
o Research Purpose: Allow expression of genes.
o Used2clone mRNA sequence in form of cDNA allowing libraries
of cDNA2 b created which contain all mrna sequence of gene
expressed in a cell.
3.HOT START PCR : reversibly inactivated It is a modified form of
PCR which avoids a non -specific amplification of DNA by inactivating
the taq polymerase at lower temperature. In this dsDNA is denatured by
heating the sample at its denaturing temperature and then the
temperature is suddenly reduced to 55 degree C at which primer and
Taq-polymerase is added, but here the difference arises i.e. specific
antibodies are used to block this Taq-polymerase at annealing
temperature. Now when the temperature raises for amplification to 72
degrees, the specific antibody detaches from Taq- polymerase and the
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21. amplification with greater specificity starts. In conventional
PCR, the Taq DNA polymerase is active at room temperature
and to a lesser degree, even on ice.
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22. 4. INVERSE PCR
Inverse PCR is a variant of the
polymerase chain reaction that is
used to amplify DNA with only
one known sequence. One
limitation of conventional PCR is
that it requires primers
complementary to both termini of
the target DNA, but this method
allows PCR to be carried out even
if only one sequence is available
from which primers may be
designed. Is commonly used to
identify the flanking sequences
around genomic inserts.
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23. 5.NESTED PCR
In the first round external primer are
used(short DNA sequences that fit the
outer end of the piece of DNA to be
amplified) & an internal set of
primers is used for the second
round.Nested PCR is often used if the
target DNA is not abundantly present
& to avoid the contaminations with
products that are amplified.
The advantage of nested PCR is that
if the wrong PCR fragment was
amplified the probability is quite low
that the region would be amplified a
second time by the second set of
primers.Thus, Nested PCR is a very
specific PCR amplification.
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24. 6.MULTIPLEX PCR : Consists of multiple primer sets
within a single PCR mixture to produce amplicons of varying
sizes that are specific to different DNA sequences. By targeting
multiple genes at once, additional information may be gained from a
single test-run that otherwise would require several times the
reagents and more time to perform. Annealing temperatures for
each of the primer sets must be optimized to work correctly
within a single reaction, and amplicon sizes. That is, their base pair
length should be different enough to form distinct bands when
visualized by gel electrophoresis.
7.ASYMMETRIC PCR
8.ANCHORED PCR
9.TOUCH DOWN PCR
10.MINIPRIMER PCR Barna 24

25. Real-Time Vs Traditional PCR
Real-Time PCR is identical to a simple PCR except that the
progress of the reaction in monitored by a camera or detector in
“real-time. Traditional methods use Agarose gels for detection of
PCR amplification at the final phase or end-point of the PCR
reaction.This is advantageous because the efficiency and rate of
the reaction can be seen. There is also no need to run the PCR
product out on a gel after the reaction.
Traditional PCR may be semiQuantitative-
though comparing the intensity of the amplified band on a
gel to standards of a known concentration can give 'semi-
quantitative' results.
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26. One step:
 Easier & faster to set up.
 Costs are higher.
Two step:
 More time,PCR master mixes made separately.
 Greater sensitivity.
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27. Advantages of using Real-Time PCR:
Traditional PCR is measured at End-Point (plateau), while Real-
Time PCR collects data in the exponential growth phase.
An increase in Reporter fluorescent signal is directly proportional
to the number of amplicons generated.
The cleaved probe provides a permanent record amplification of
an Amplicon.
Increase dynamic range of detection.
No-post PCR processing.
Disadvantages of real-time PCR:
 Due to its extremely high sensitivity,may be high deviations of
the same experiment, thus, the use of internal control genes is a
recommended (in gene expression experiments).
 Requires expensive equipments and reagent.
 Requires high technical skill & support.
 DNA contamination.
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28. Limitations of End-Point PCR:
 Agarose gel results are obtained from the end point of the
reaction. Endpoint detection is very time consuming. Results
may not be obtained for days.Agarose Gel resolution is very
poor, about 10 fold. Real-TimePCR can detect as little as a
two-fold change Some of the problems with End-Point
Detection:
 Poor Precision
 Low sensitivity
 Short dynamic range
 Low resolution
 Non - Automated
 Size-based discrimination only
 Results are not expressed as numbers
 Ethidium bromide for staining is not very quantitative
 Post PCR processing
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29. Power supply
Gel tank
Casting tray
Gel combs
Electrical leads
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30. Applications of PCR
1) Cloning a gene encoding a known protein
2) Rapid amplification of cDNA
3) Detecting bacterial or viral infection
4) Early Diagonose of Cancer like leukemia,
Lymphoma
5) Genetic diagnosis
a. Diagnosing inherited disorders
* Cystic fibrosis
* Muscular dystrophy
* Haemophilia A and B
b. Diagnosing cancer
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31. c. Blood group typing
d. Prenatal diagnosis - such as determining the sex of foetuses
for those at risk of X-linked disorders
 6.Medicolegal use:
 Criminal identification
 Paternity test
 Dead body identification.
 7.Biosynthesis of hormone, vaccine and other proteins.
 8.Tissue typing by PCR and detection of genetic variants
especially of MHC class II alleles.
 9.Cancer research purpose.
 10.Drug therapy efficacy.
 11.DNA damage measurement
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32. ThankBarna 32