2. Polymerase Chain Reaction Methodology
A Milestone in Biomedical History
He had the idea to use a
pair of primers to bracket
the desired DNA sequence
and to copy it using DNA
polymerase, a technique
which would allow a small
strand of DNA to be
copied almost an infinite
number of times. Cetus
took Mullis off his usual
projects to concentrate on
PCR full-time
3. How DNA Works
DNA usually exists as a
double-stranded structure,
with both strands coiled
together to form the
characteristic double-helix.
Each single strand of DNA
is a chain of four types of
nucleotides: adenine,
cytosine, guanine, and
thymine.
4. DNA – RNA - DNA
In Molecular biology, the
polymerase chain
reaction (PCR) is a
technique to amplify a
single or few copies of a
piece of DNA across several
orders of magnitude,
generating millions or more
copies of a particular DNA
sequence.
5. Create primers
To begin synthesis of a
new strand, a short
fragment of DNA or
RNA, called a primer,
must be created and
paired with the template
strand before DNA
polymerase can
synthesize new DNA.
7. A Thermocycler is the
Backbone of PCR methodology
The method relies on
thermal cycling,
consisting of cycles of
repeated heating and
cooling of the reaction
for DNA melting and
enzymatic replication of
the DNA
8. Primers
Primers (short DNA
fragments) containing
sequences complementary
to the target region along
with a Primers after which
the method is named) are
key components to enable
selective and repeated
amplification.
9. PCR a Chain reaction
As PCR progresses, the
DNA generated is itself
used as a template for
replication, setting in motion
a chain reaction in which
the DNA template is
exponentially amplified.
PCR can be extensively
modified to perform a wide
array of genetic
manipulations.
11. Thermus aquaticus is a bacterium that lives in
hot springs and hydrothermal vents, and Taq
polymerase was identified as an enzyme able
to withstand the protein-denaturing conditions
(high temperature) required during PCR. It
replaced the DNA polymerase from E.coli
originally used in PCR . Taq's optimum
temperature for activity is 75-80°C, with a
halflife of 9 minutes at 97.5°C, and can
replicate a 1000 base pair strand of DNA in
less than 10 seconds at 72°C.
Taq polymerase fuels the
Reaction
12. Taq polymerase is a thermostable DNA
polymerase named after the
thermophilic bacterium Thermus
aquaticus from which it was originally
isolated by Thomas D. Brock in 1965. It
is often abbreviated to "Taq Pol" (or
simply "Taq"), and is frequently used in
polymerase chain reaction (PCR),
methods for greatly amplifying short
segments of DNA
Taq polymerase
13. One of Taq's drawbacks is its relatively
low replication fidelity. It lacks a 3' to 5'
exonuclease proofreading activity, and
has an error rate measured at about 1
in 9,000 nucleotides. Some thermos
table DNA polymerases have been
isolated from other thermophilic bacteria
and archaea, such as Pfu DNA
polymerase, possessing a proofreading
activity, and are being used instead of
(or in combination with) Taq for high-
fidelity amplification.
Drawbacks of Taq
polymerase
14. Taq Pol lacks 3’ to 5’ exonuclease proof reading
activity, commonly present in other polymerases.
Taq mis-incorporates 1 base in 104.
A 400 bp target will contain an error in 33% of
molecules after 20 cycles.
Error distribution will be random.
Disadvantages of
Taq Pol
16. What does PCR need?
• Template (the DNA you are exploring)
• Sequence-specific primers flanking the
target sequence, Forward & Reverse.
• Polymerases
• Nucleotides (dATP, dCTP, dGTP,
dTTP)
• Magnesium chloride (enzyme cofactor)
• Buffer
• Water, mineral oil
17. Create primers
To begin synthesis of a
new strand, a short
fragment of DNA or
RNA, called a primer,
must be created and
paired with the template
strand before DNA
polymerase can
synthesize new DNA.
20. Denaturation 93 to 95°C 1min
Annealing 50 to 55°C 45sec
Elongation 70 to 75°C 1-2min
Steps in PCR
21. How does PCR work?
• Heat (94oC) to denature DNA strands
• Cool (54oC) to anneal primers to template
• Warm (72oC) to activateTaq Polymerase,
which extends primers and replicates DNA
• Repeat multiple cycles
26. The target product is
made in the third cycle
3’
5’
3’
5’
3’
5’
5’
3’
3’
5’
5’
3’
5’
3’
5’
3’
Cycle 1
Cycle 2
Cycle 3
3’
3’
3’
3’
5’
5’
5’
5’
27. Denaturation: 94°- 95°C
Primer Annealing: 55°- 65°C
Elongation of DNA: 72°
Number of Cycles: 25-40
No target products are made until the third cycle.
At 30 cycles there are 1,073,741,764 target
copies (~1×109).
PCR Cycles Review
28. Primers can have self-annealing regions within each primer (i.e. hairpin and
fold back loops)
A primer may be self-complementary and be able to fold into a hairpin:
5´-GTTGACTTGATA
||||| T
3´-GAACTCT
The 3´ end of the primer is base-paired,
preventing it annealing to the target DNA.
Primers That Form
Hairpins
30. Need for target DNA sequence information
Primer Designing for unexplored ones.
Boundary regions of DNA to be amplified must be known.
Infidelity of DNA replication.
Taq Pol – no Proof reading mech – Error 40% after 20
cycles
Short size and limiting amounts of PCR product
Up to 5kb can be easily amplified .
Up to 40kb can be amplified with some modifications.
Cannot amplify gene >100kb
Cannot be used in genome sequencing projects.
Limitations of PCR
31. • Pfu DNA Polymerase from Pyrococcus
furiosus possesses 3' to 5' exonuclease
proofreading activity.
• The error rate is only 3.5% after 20 cycles
• More amount of primer is added to avoid
primer dimering.
• For unexplored genes, primers used in closely
related species are used.
How to overcome
Difficulties?
33. The Real Time assays are proving to better
technologies
1 Rapid
2 Quantitative measurement
3 Lower contamination rate
4 Higher sensitivity
5 Higher specificity
6 Easy standardization
Now a new gold standard for rapid diagnosis of
virus infection in the acute phase samples.
New Technologies – Real
Time Assays
34. Real-time polymerase chain reaction, also called
quantitative real time polymerase chain reaction (Q-
PCR/qPCR) or kinetic polymerase chain reaction, is a
laboratory technique based on the polymerase chain
reaction, which is used to amplify and simultaneously
quantify a targeted DNA molecule. It enables both detection
and quantification (as absolute number of copies or relative
amount when normalized to DNA input or additional
normalizing genes) of a specific sequence in a DNA sample.
Real-time polymerase
(qPCR) chain reaction
35. All real time PCR systems rely upon the
detection and quantization of fluorescent
reporter, the signal of which increases in direct
proportion of the amount of PCR product in a
reaction.
Real Time Reporters
36. qPCR Syber Green
USING SYBER®
GREEN
The simplest and economical
format the reporter is the
double strand DNA specific
dye SYBR ® Green
Called as Molecular Probe.
37. How SYBR Green
works
SYBR green binds to
double stranded DNA
and upon excitation
emits light
Thus as PCR product
accumulates the
fluoresce increases
39. Other Alternatives
Two most popular
alternatives to SYBR green
are TaqMan® and
Molecular Beacons.
Both technologies depend
on hybridization probes
relying on fluorescence
resonance energy transfer.(
FRET) and quantization
43. Documentation of
Amplification
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.
45. Molecular Beacons –
qPCR
Molecular beacons are designed
to adopt a hairpin structure while
free in solution, brining the
fluorescent dye and quencher in
close proximity. When a molecular
beacon hybridizes to a target the
fluorescent dye emits light upon
irradiation, and rebind to target in
every cycle for signal
measurement.
46. qPCR Method
Molecular Beacons
Molecular beacons are
short segments of
single-stranded DNA .
The sequence of each
molecular beacon must
be customized to detect
the PCR product of
interest.
47. Quantitation of gene expression
Pathogen detection
Viral quantitation
Array verification
Drug therapy efficacy
DNA damage measurement
Quality control and assay validation
Genotyping
qPCR applications
48. Reverse transcription polymerase chain reaction
(RT-PCR) is a variant of polymerase chain reaction
(PCR), commonly used in molecular biology to
generate many copies of a DNA sequence, a
process termed "amplification". In RT-PCR,
however, RNA strand is first reverse transcribed into
its DNA complement using the enzyme reverse
transcriptase, and the resulting cDNA is amplified
using traditional or real-time PCR..
Reverse transcription
polymerase chain reaction
(RT-PCR)
49. Not to be confused with
qPCR
Reverse transcription
PCR is not to be
confused with real-time
polymerase chain
reaction (qPCR), which
is also sometimes
(incorrectly) abbreviated
as RT-PCR.
50. Multiplex PCR
TaqMan probes and
Molecular beacons
allow multiple DNA
species to be measured
in the same sample (
Multiplex PCR) since
fluorescent dyes with
different emission
spectra may be
attached to different
probes
51. Several viral infections can be detected in acute phase
serum samples.
Increasing used in for early and accurate detection of
all most human viruses including
Measles, Mumps, Herpes simplex
viruses, Rota viruses Noro
virus,Influenzae virus type A and B,
Respiratory Syncitical virus, SARS,
Dengue Japanese Encephalitis,
Hepatitis B and C, West Nile,
Chikungunya,HIV, Avian flu virus,
Uses of Automated
qPCR
52. Multiplex PCR in Real Time
Multiplex real time
quantitative RT-PCR
assays have been
developed for simultaneous
detection identification and
quantification of HBV, HCV
and HIV-1 In plasma and
Serum samples.
53. Directly
Sybr green
Quality of primers critical
Indirectly
In addition to primers, add a
fluorescently labeled
hybridization probe
How are you going to
measure the PCR product
54. Negative control (no DNA)
checks reagents for contamination
No reverse transcriptase control
detects if signal from contaminating DNA
Positive control
checks that reagents and primers work
especially importance if trying to show absence of
expression of a gene
Importance of
controls
55. Same copy number in all cells
Expressed in all cells
Medium copy number advantageous
correction more accurate
Reasonably large introns
No pseudo gene
No alternate splicing in region you want to PCR
Standards
56. Loop mediated isothermal amplification is a simple,
rapid, specific and cost effective nucleic acid
amplification method characterized by use of 8
distinct regions on the target gene.
The amplification proceeds at a constant
temperature using strand displacement reaction.
Loop Mediated Isothermal
Amplification (LAMP)
57. LAMP
Amplification and detection
of gene can be completed in
a single step, by incubating
the mixture of samples,
DNA polymerase, primers
with strand displacement
activity and substrates at a
constant temperature of
630c.
58. LAMP (Loop mediated isothermal
amplification)
Originally reported by Notomi et al in 2000 of EIKEN Chemical Co. Ltd.,
Japan
(http://www.eiken.co.jp/en/)
As of 17th April 2013, PubMed database has listed more than 820 articles
on this topic
59. Bst DNA polymerase with strand displacement
activity at 65℃
The whole amplification to 109 – 1010 copies is
finished within 15 to 60 min at 65℃, isothermally
Amplification and detection of gene can be
completed in a single step
No need for a step to denature double stranded
into a single stranded form
60. The amplification efficiency is extremely high
Reduced total cost- not require special reagents
or sophisticated equipments
Amplified products have a structure consisting
of alternately inverted repeats of the target
sequence on the same strand
Amplification can be done with RNA templates
following the same procedure as with DNA
templates, simply through the addition of
reverse transcriptase (RT-LAMP)
61. LAMP
Compared with PCR, and
real time PCR, the LAMP
has advantages of reaction
simplicity and detection
sensitivity.
The higher sensitivity and
specificity of LAMP reaction
is attributed to continuous
amplification under
isothermal condition
employing six primers
recognizing eight distinct
regions of the target.
62. LAMP functions on isothermal amplification.
LAMP does not require any thermal cycler and thus
can be performed even with water bath/heating block
LAMP method does not require sophisticated
temperature control devices
Cost effective
Advantages of LAMP
63. Continued…
2- Amplification
Is of two types;
A. Non-cycling Amplification
B. Cycling Amplification
A. Non-cycling Amplification:
Generation of stem loop DNA with dumbbell-shaped structure at
both ends.
B. Cycling Amplification:
Dumbbell-shaped DNA is quickly amplified by the use of loop
primers.
66. Continued…
3- Detection
i. Visual Detection
Turbidity - Magnesium pyrophosphate
Fluorescence – Calcein
ii. Gel Electrophoresis
Lane 1 and 3 has target DNA.
Lane 2 and 4 has non-target DNA.
Lane M has DNA Ladder.
67. { {
LAMP vs. PCR
Isothermal Reaction.
Isothermal Temperature (60-
65⁰C).
Doesn’t require expensive
thermocycler.
Detection limit is greater.
Amplification specificity is
higher as uses 4/6
oligonucleotides.
Visualization of DNA could be
done through eyes, gel
electrophoresis and
turbidimeter.
Cyclic Reaction.
Variable Temperature.
Denaturation (95⁰C)
Annealing (50-60⁰C)
Polymerization (72⁰C)
• Require thermocycler.
• Detection limit is lower.
• Amplification specificity is
lower than that of LAMP.
• Visualization of DNA is done
through gel electrophoresis.
Loop-mediated Isothermal
Amplification - LAMP
Polymerase Chain Reaction - PCR
68. { {
Could be done using crude
DNA samples.
Loop primers accelerate
reaction rate.
Need pure DNA samples
for amplification.
No loop primer.
LAMP vs. PCR
Loop-mediated Isothermal
Amplification - LAMP
Polymerase Chain Reaction - PCR
69. Lesser False Positives in
LAMP
In LAMP both amplification
and detection occur
simultaneously during the
exponential phase without
going through the plateau
phase where the non
spurious amplification leads
to lower sensitivity and false
positivity.
70. Loop Mediated Isothermal
Amplification in Clinical Diagnosis
LAMP technology proving to
be ideal in detection of DNA
or RNA of the pathogenic
organisms
Proving to be highly efficient
in diagnosis of Viral and
Bacterial infections,
LAMP is capable of
detecting the presence of
pathogenic agents earlier
than PCR
71. LAMP proving an efficient
Technology
A one step single tube real time
accelerated reverse transcription
loop mediated isothermal
amplification (RT-LAMP) assays
for rapid detection of some
recently emerged viral pathogen
eg West Nile, SARS, Dengue,
Japanese encephalitis
Chikungunya Norwalk, H5N1
highly pathogenic avian
influenza., and CMV,HPV,VZV
73. One Step RT-PCR Kit
The QIAGEN One Step
RT-PCR Kit is designed
for easy and sensitive
one-step RT-PCR using
any RNA template. A
unique enzyme
combination and specially
developed reaction buffer
ensure efficient reverse
transcription and PCR in
one tube.
74. RT-PCR in one step
The Robus™ T I Kit is base
RobusT RT-PCR Kits
perform cDNA
synthesis and PCR
amplification of cDNA
successively in a
single tube during a
continuous thermal
cycling
75. Clinical diagnostics: detection and
quantification of infectious microorganisms,
cancer cells and genetic disorders
Capable of amplifying long targets, up to 6.0 kb
One-tube system allows rapid, sensitive and
reproducible analysis of RNA with minimal risk
of sample contamination
Amplifies products from a wide variety of total
RNA or mRNA sources
Uses and Advantages in
Testing by PCR Methods
76. Advantages
Molecular methods
•High sensitivity and specificity
•Detects pathogen, not immune response
•Quick results
•High transport toleration
In-house (home-brew) PCR methods
•Cost effective
•High sensitivity
•High quality
•Fast implementation of scientific discoveries
•Customer friendly
R&D is absolutely necessary
77. Advantages
Molecular methods
•High sensitivity and specificity
•Detects pathogen, not immune response
•Quick results
•High transport toleration
In-house (home-brew) PCR methods
•Cost effective
•High sensitivity
•High quality
•Fast implementation of scientific discoveries
•Customer friendly
R&D is absolutely necessary
79. ESTABLISHMENT OF A PCR
LABORATORY
To perform PCR for the
repetitive detection of a
specific sequence, three
distinct laboratory areas
are required. The
specific technical
operations, reagents
,and personnel
considerations
80. PCR contamination be considered as a form of
infection. If standard sterile techniques that
would be applied to tissue culture or
microbiological manipulations are applied to PCR,
then the risk of contamination will be greatly
reduced. Above all else, common sense should
prevail.
Prevention of
Contamination in PCR
Laboratory
81. The single most important source of PCR
product contamination is the generation of
aerosols of PCR amplicons that is associated
with the post-PCR analysis. Methods for
eliminating this aerosol range from physical
design of laboratories and use of specific
pipettes to chemical and enzymatic
approaches. The choice of method is often
dependent on the frequency of amplification of
a target amplicon and the relative amounts and
concentrations of the amplicons created by the
PCR.
Avoiding
contamination
82. PCR laboratory
Sample handling
DNA preparation
Clean room
Stock solutions
Laboratory
Mixing site
Thermocycler
Amplification
Detection
Documentation
QC & QA
Quality control & assurance
R & D
(Research and development)
Alternatives: - commercial kits
- robots + kits
No alternative
85. Contamination
PCR allows the
production of more than
10 million copies of a
target DNA sequence from
only a few molecules of
DNA. The sensitivity of
PCR means that the
sample used for PCR
should not be
contaminated with any
other DNA’s that may
reside in the laboratory
environment.
86. PCR is not only vital in the clinical laboratory by
amplifying small amounts of DNA in
infectious diseases, but it is also important
for genetic predisposing for defects in
Genetic disorders.
The PCR technology can also be employed in
law enforcement, genetic testing of animal
stocks and vegetable hybrids, and drug
screening along with many more areas.
Conclusion
