Diagnostic polymerase chain reaction (PCR) is an extremely powerful, rapid method for diagnosis of microbial infections and genetic diseases, as well as for detecting microorganisms in environmental and food samples. However, the usefulness of diagnostic PCR is limited, in part, by the presence of inhibitory substances in complex biological samples, which reduce or even block the amplification capacity of PCR in comparison with pure solutions of nucleic acids . In general, diagnostic PCR may be divided into four steps: (1) sampling, (2) sample preparation, (3) nucleic acid amplification, and (4) detection of PCR products

2. Polymerase chain
reaction (PCR)
By
Romissaa Aly Esmail
Assistant lecturer of Oral
Medicine, Periodontology,
Diagnosis and Dental Radiology
(Al-Azhar University)

3. • Contents:
• Components of PCR
• Steps of PCR
• Polymerase Chain Reaction (PCR) Applications

4. Diagnostic polymerase chain reaction
(PCR) is an extremely powerful, rapid
method for diagnosis of microbial
infections and genetic diseases, as
well as for detecting microorganisms
in environmental and food samples.
However, the usefulness of diagnostic
PCR is limited, in part, by the presence
of inhibitory substances in complex
biological samples, which reduce or
even block the amplification capacity
of PCR in comparison with pure
solutions of nucleic acids .
In general, diagnostic PCR may be
divided into four steps: (1) sampling,
(2) sample preparation, (3) nucleic acid
amplification, and (4) detection of PCR
products .

5. Components of PCR

6. 1. Nucleic Acid Template (Template DNA)
Template DNA is the sample DNA that contains the selected
nucleic acid sequence that needs to be amplified.
The template must be DNA only: Genomic DNA (gDNA),
complementary DNA (cDNA), and plasmid DNA.
Reverse transcriptase polymerase chain reaction (RT-PCR) uses
RNAs as starting materials, but RNAs are primarily converted to
complementary DNA (cDNA) before amplification.
The template DNA must be highly pure with an absorbance
ratio of ~1.8. A quantity of 0.1 to 200 μg can be used, with an
ideal quantity of 30 μg to 50 μg.

9. 2. DNA Polymerase
DNA polymerases are enzymes that
catalyze the synthesis of
complementary DNA strands by
assembling the nucleotides
sequentially according to the
template strand. Simply, it is the
enzyme that synthesizes DNA; hence
plays a key role in DNA replication.
Taq DNA polymerase, the DNA
polymerase enzyme extracted from
the bacterium Thermus aquaticus, is
the most widely and the best–known
DNA polymerase used in PCR since its
establishment.
Taq DNA polymerase is thermally
stable and continues its activity after
the repeated heating and cooling
cycle.

11. It is stable up to 95°C and
shows the most effective
reaction at around 72°C to
78°C incorporating about 60
bases per second.
In a 50 L reaction mixture,
around 1 to 2 units of Taq
polymerase is sufficient for
amplification.
Recently, two other
thermostable DNA
polymerase enzymes are
available, viz., the Vent
enzyme isolated
from Thermococcus litoralis,
and the Pfu enzyme isolated
from Pyrococcus furiosus.

12. 3. Primers
Primers are artificially
synthesized short single-stranded
sequences of oligonucleotides
that are complementary to the
target nucleic acid sequence in
the template DNA.
They are short sequences of
around 15 to 30 bases that act as
starting point for DNA synthesis.
They anneal at their
complementary position in a
single-stranded template DNA
strand.
The DNA polymerase enzyme
then extends this primer from its
3’ OH- end forming a new
complementary strand.
Usually, 10 to 12 pMol of each
primer is sufficient for a PCR
reaction.
PCR primers are of 2 types;
forward and reverse primers.

14. • The forward primers are complementary to the antisense strand
(template strand from 3’ to 5’ direction) and are responsible for the
amplification of the antisense strand. They are also called 5’ primers.
• The reverse primers are complementary to the sense strand (template
strand from 5’ to 3’ direction) and are responsible for the amplification of
the sense strand. They are also called 3’ primers.

15. 4. Nucleotides (Deoxynucleotide
triphosphates)
Deoxynucleotide triphosphates
(dNTPs) are artificially synthesized
nucleotides that act as building
blocks of new DNA strands.
There are 4 different dNTPs used in
the PCR; deoxyadenosine
triphosphate (dATP),
deoxyguanosine triphosphate
(dGTP), deoxythymidine
triphosphate (dTTP), and
Deoxycytidine triphosphate (dCTP).
These four dNTPs are sequentially
added to the annealed primer by the
DNA polymerase enzyme generating
a new strand of DNA complementary
to the template strand.

16. 5. PCR Buffers and Other
Chemicals
The whole process needs to be
carried out in a Tris – HCl based
buffer system of pH 8.0 to 9.5.
The common buffer system used is
a 10X buffer with additional
MgCl2.
Common components of PCR
buffers are dimethyl sulfoxide
(DMSO), ammonium sulfate
((NH4)2SO4), nonionic detergents,
polyethylene glycon(PEG), N,N,N-
trimethyl glycine, potassium
chloride (KCl),
magnesium chloride (MgCl2), tetra
methyl ammonium chloride, Tris –
HCl, Ethylenediaminetetraacetic
acid (EDTA), 7-deaza-2′-
deoxyguanosine 5’-
triphosphate, glycerol,
formamide, serum albumin, etc.
The buffer system increases the
reaction’s efficiency and
specificity and prevents inhibition
and secondary structure
formation during the process.

18. 6. Thermocycler
Also known as the
PCR machine, the
thermocycler is
simply an electric
heating device that
regulates the
temperature as per
need during each
stage of the PCR
process.
This machine
increases the
temperature during
the denaturation
step and lowers it
during the annealing
and again increases it
during the elongation
step
This process of
increasing and
decreasing the
temperature occurs
in a cyclic manner
according to the pre-
programmed setup or
instruction by the
user prior to
operating.

20. Steps of PCR

21. • 1. Pre-preparation
• It is the initial step before the actual polymerase chain reaction
takes place inside the thermocycler.
• In this step, one must prepare a reaction mixture and load it on a
pre-programmed thermocycler in order to amplify the target DNA or
RNA segment.

22. Sample DNAs or RNAs are extracted from the
sample and stored (pre-extracted nucleic acids can
be used).
All materials are arranged, safety measures are
taken, the PCR reaction preparation area is cleaned,
all the reagents are brought to working
temperature, the sample is extracted or brought
from storage, the PCR reaction mixture is prepared,
the thermocycler is programmed, and the reaction
mixture is loaded on the thermocycler.

30. 2. Amplification
It is the main reaction process
occurring in PCR.
The amplification step
includes denaturation,
annealing, and elongation
occurring orderly in a cyclic
manner one after another for
a certain number of cycles
pre-programmed by the user.

31. Step I: Denaturation
It is the 1st step of the amplification reaction where the
double-stranded DNA is thermally denatured into two
single-stranded DNA templates.
Temperature is raised to about 94°C (90 to 95°C) for
about 30 to 90 seconds.
At this temperature, the thermal energy overcomes the
weak hydrogen bonds holding the two DNA strands
together, allowing them to separate.
dsDNA → 2 ssDNA templates

33. Step II: Annealing
Denaturation is followed by the annealing step,
where the primer anneals the ssDNA templates at
their complementary sites.
The forward primer anneals at the complementary
site of the antisense strand, and the reverse
primer anneals at the complementary site of the
sense strand of the template DNA.
For annealing to occur, the temperature is reduced
to 55°C-70°C (the annealing temperature differs
based on the GC content of the primer).
About 30 to 60 seconds are enough for annealing
in most of the PCR processes.
ssDNA + Forward and reverse primers → ssDNA
with annealed primers

35. Step III: Elongation
It is the final step in the
amplification reaction
where the temperature is
raised to 72°C so that the
Taq DNA polymerase
enzyme begins
synthesizing new DNA
strands in the 5’ to 3’
direction.
The DNA polymerase
enzyme adds nucleotides
from the reaction mixture
to the 3’ OH- end of the
annealed primer forming
a new complementary
strand.

36. The time required
for elongation
depends on the
sample nucleic acid
sequence length
and the DNA
polymerase
activity.
Generally,
elongation takes
place at the rate of
1 kbp per 0.5 to 1
minute.
At the end of
elongation, two
new dsDNA will be
formed from a
single dsDNA
template at the
beginning of the
reaction.
2 ssDNA with
annealed primers +
dNTPs → 2 new
dsDNAs

37. 3. Product Analysis Phase
It is the phase after completion of the PCR where the reaction
mixture subjected to PCR is analyzed to confirm that desired
amplification is achieved.
For this, mostly agarose gel electrophoresis is employed in order
to check for amplified DNAs or RNAs. However, no additional step
is required in some types of PCR, like real-time PCR.

39. • In microarray analysis, a sample of tissue might be compared to a
control sample in order to determine the differences in expression
level between the two. During microarray analysis, a fluorescent dye
is attached to small fragments of cDNA previously generated from the
experimental and control samples.

40. Red dye is used to label
experimental cDNA and
green dye is used to label
control cDNA.
The process takes place
on a chip that has
thousands of
complementary DNA
fragments to both the
experimental and control.
The mixture of
fluorescently labeled
control and experimental
cDNA fragments are
applied to the chip and
hybridize to its
complementary strand.

42. Bridge PCR is
another method
used to amplify
sequences prior
to NGS.
Here, two types
of oligos are
fixed to a flow
cell. Each oligo
is
complimentary
to each adaptor
flanking the
DNA fragment.
The flanking
adaptors allow a
bridge to form
between the
two types of
oligos.
After each copy
is denatured,
the single
strands bridge
to the oligos
and the process
gets repeated

44. Here, each bead acts
as a microreactor for
PCR, each containing
one strand of DNA.
This type of PCR uses
bead surfaces, water
and oil. Emulsion PCR
allows simultaneous
amplification of each
sequence without
risk of contamination.
Emulsion PCR (ePCR)
is another variation
of PCR typically used
for amplification prior
to NGS.

47. What is DNA
methylation and
methylation analysis:
The process in which a
methyl group (CH3) is
added to DNA is called
DNA methylation.
Methylation helps
regulate gene
expression by repressing
transcription.
This activity changes
genetic function without
altering DNA sequences
and is one of many
epigenetic mechanisms.

55. Multiplex PCR
Multiplex PCR is a type of PCR
technique which allows an
amplification of many target
sequences concurrently in the
same reaction mixture.
A single reaction mixture
includes sets of primer pairs for
different DNA targets.
It reduces the consumption
of PCR reagents, and, at the
same time, imposes restrictions
on used primers.
To work properly within one
reaction, sets of primers must
be optimized.
They must have similar
annealing temperatures and
produce amplicons of different
sizes to form distinct gel
electrophoresis bands for the
followed PCR analysis.

59. Nested PCR
• Nested PCR is used to increase the specificity of a DNA
amplification reducing unspecific products. This technique utilizes
two sets of primers.
• The first set allows a first polymerase chain reaction. The
product of this reaction serves as a source of target DNA to a
second PCR using the second set of primers.

61. hot-start PCR
This type of
polymerase chain
reaction serves to
reduce non-specific
amplification during
the initial set up
stages.
Hot-start PCR
technique keeps the
DNA polymerase in
an inactive state at
temperatures lower
than an annealing
temperature.
This modification
prevents the
amplification during
reaction setup when
primers bind to DNA
sequences with low
homology.

62. Two variants of this
technique are mechanical
and non-mechanical hot
start PCR.
Mechanical hot start
PCR performed by
heating the reaction
mixture to the DNA
melting temperature
before adding the Taq
polymerase.
Non-mechanical hot
start PCR uses specialized
enzyme systems which
inhibit an activation of
the DNA polymerase at
room temperature.

64. Touchdown PCR
Touchdown PCR is another technique to
reduce nonspecific amplification.
It is achieved by raising the annealing
temperature above the melting temperature
of the used primers in the initial cycles and
lowering in the later cycles.
The higher temperatures during the initial
cycles help primers to bind to DNA templates
with greater specificity while the lower
temperatures allow more efficient
amplification from the produced amplicons

65. Ligase Chain Reaction
(LCR)
This type of PCR
technique uses four
primers for DNA
amplification (two
primers for each
strand of the DNA
target).
Ligase Chain Reaction
primers are much
longer than usual
PCR primers and
designed to cover the
entire sequence to be
amplified.

67. During the first annealing step,
primers are sealed by a
thermostable DNA ligase.
This generates a fragment that
is as long as the total length of
each pair of primers which
serves as the DNA templates
for subsequent cycles.

68. Quantitative PCR (qPCR)
The amount of product that is
synthesized during a set number
of cycles of a polymerase chain
reaction depends on the number
of DNA molecules that are present
in the starting mixture.
This enables PCR to be used to
quantify the number of DNA
molecules present in an extract.
In quantitative PCR the amount of
product synthesized during a test
PCR is compared with the
amounts synthesized during PCRs
with known quantities of starting
DNA.

71. Real-time PCR
Today, quantification is carried
out by real-time PCR - a
modification of the standard PCR
technique in which synthesis of
the product is measured over
time.
More frequently this
method is used to
measure RNA amounts.

73. For example, to determine the
expression of a particular gene
in cancerous cells. This method
allows monitoring the
development of cancer

74. Reverse transcription PCR
To carry out polymerase chain
reaction where RNA is the
starting material this method
uses reverse transcriptase, a
process called RT–PCR (reverse
transcriptase polymerase
chain reaction).
The first step in this
method is to convert the
RNA molecules into single-
stranded complementary
DNA (cDNA.
After this step, the
experiment proceeds as in
the standard technique.
Some thermostable
polymerases, such as Tth, have
a reverse transcriptase activity
under certain buffer conditions
and able to make DNA copies
of both RNA and DNA
molecules

75. Figure 1. Reverse transcription polymerase chain reaction (RT-PCR). RT =
reverse transcription, RTase = reverse transcriptase.

76. TaqMan PCR
TaqMan PCR is
one of the real-
time PCR
techniques.
It uses an
oligonucleotide
probe which is
complementary
to an internal
sequence within
the amplified
strands.
It has a
fluorescent
group at one
end and a
quencher at
another end.
As long as both
fluorophore and
quencher stay
within the
oligonucleotide
probe, no
fluorescence is
emitted.

77. During DNA amplification,
the oligonucleotide probe,
and the primers will bind
to newly synthesized
strands.
The polymerase will
destroy the probe due to
the intrinsic 5′→3′
exonuclease activity and
release the fluorophore.
The intensity of the
fluorescence is
proportional to the
amount of generated
product.

79. Assembly PCR
Assembly PCR or
Polymerase Cycling
Assembly was developed
to produce novel long
nucleic acid sequences.
The main difference from
traditional polymerase
chain reaction is the
length and quantity of
primers.
To synthesize artificial
oligonucleotide,
assembly PCR is
performed on long, up to
50 nucleotides, primers..

80. These primers have short overlapping
segments and alternate between
sense and antisense directions
covering the entire target sequence.
During successive cycles, the primers
hybridize by complementary segments
and then polymerase increases the
length of fragments producing the
final long nucleic acid sequence

83. Polymerase Chain
Reaction (PCR)
Applications

84. Identification
and
Classification of
Organism
PCR is used
widely in
identifying
microorganisms
up to the level of
subspecies and
strains.
This has reduced
the time
required for
microbial
identification
from days to a
few hours.
Additionally,
larger animals
can also be
identified and
systematically
classified using
PCR.
DNA isolated
from fossilized
animals are also
amplified and
studied to relate
them with
animals that are
still living on the
Earth.

85. 2. Infectious Disease Diagnosis
The use of PCR in the identification of pathogens has
led to the quick and accurate diagnosis of infections.
Not only diagnosis but parallel identification of
antimicrobial resistant genes in the pathogen is also
possible, allowing choosing of appropriate
antimicrobial treatment option.
HIV, SARS CoV – 2, human T – cell leukemia virus
(HTLV type I and II), Tuberculosis, Hepatitis Virus,
Enterovirus, Sexually Transmitted Diseases (STDs),
etc., are diagnosed using PCR

86. 3. Detection of Gene Mutation and
Genetic Disorders
Mutation in any segment of a gene
can be detected using PCR. Knowing
this mutation, we can confirm a
genetic disorder. DNA polymorphism
can also be analyzed, which can also
relate to a genetic disorder of some
kind.

88. • In medical science, PCR is used as one of the most important tools to diagnose
congenital diseases, genetic disorders, and any mutation leading to a negative
health problem and behavioral change in the prenatal stage.
• Detection of cancerous cells is another very important application of PCR in
medicine.

89. 4. DNA Fingerprinting
In forensics, PCR is used for DNA
fingerprinting. DNA fingerprinting
is used for the identification of
criminals or individuals and for
confirming parents.
5. Gene Sequencing
For gene sequencing, a gene
must be amplified into a large
number using techniques like
PCR. All the sequencing methods
use PCR as their important step.
6. DNA and RNA Quantification
PCR can also be used for the
quantification of sample DNA and
RNA. Quantitative Real-Time PCR
(RT – qPCR) is one common type
of PCR used for the quantification
of sample DNA.

90. 7. As a Tool in Genetic
Engineering
PCR is used in genetic
engineering for analyzing
modified DNAs and amplifying
target or vector DNA. Desired
genes are amplified using PCR
and applied in the required
process.
8. Gene Expression Analysis and
Genetic Imprinting
PCR of RNA (Reverse
Transcription PCR) is used in
gene expression analysis, study
genetic imprinting, etc.
9. It is also used in drug and
vaccine discovery, human
genome projects, paleontology,
and evolutionary biology.

93. • 8. It is not suitable for very long DNA molecules.
Very long DNA needs to be cut into smaller
fragments. Usually, from 0.1 kbp up to 10 kbp or 40
kbp can be used.
• 9. RNA needs to be first converted to DNA using
reverse-transcriptase enzyme before its
amplification.
• 10.Most types of PCR processes require additional
steps for product analysis.

98. Figure 2. Current examples of commercially available techniques:
quantitative PCR, droplet-based digital PCR, crystal digital PCR (cdPCR),
PCR,
bridge PCR for next-generation sequencing and sequencing of mRNA
from individual cells using microfluidics. (A) A comparison of end-point
PCR,
qPCR and ddPCR. (B) Schematic representation of the principle of solid
phase bridge DNA amplification. (C) Different techniques for splitting of
samples. (D) Crystal droplet PCR – formation of droplet crystals. (E) PCR
and droplet-based library generation for single-cell RNA sequencing.
ddPCR: Droplet-based digital PCR; qPCR: Quantitative PCR.

100. Figure 3. Applications of microfluidics into massively
parallel and handheld point-of-care systems. (A)
Chip-based integrated real-time reverse
transcription PCR platform for the analysis of the
immunomagnetic exosomal RNA. (B) Droplet-based
quantitative PCR for a single cell to mRNA
purification and gene expression analysis. (C) Chip-
based digital RT-PCR for absolutequantification of
mRNA in single cells. (D) Droplet-based dPCR for
miRNA quantitation assay. (E) Paper-based LAMP
system made by polydimethylsiloxane for molecular
diagnostics. (F) Forensic science, DNA profiles
on a chip. (G) BioFire, detection of bacteria and
viruses on a chip.

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