polymerase chain reaction,

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

2. 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.

3. 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 .

4. 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.

7. 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.

10. 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.

11. 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.

13.  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.

14. 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.

15. 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.

17. 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.

18. 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).

19. 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.

20. 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),

21. 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.

23. 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

24. . 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.

26. Steps of PCR

27. 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.

28. 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.

35. 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.

36. 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

38. 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.

39. 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

41. 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.

42. 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

43. 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.

45. 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.

46. 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.

48. 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

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

53. 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.

61. 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.

62. 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 have to have similar annealing temperatures and produce
amplicons of different sizes to form distinct gel electrophoresis
bands for the followed PCR analysis.

66. 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.

68. 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.

69. 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.

71. 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.

72. 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.

73. 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.

75. 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.

76. 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.

79. 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.

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

82. 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.

83. 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

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

85. 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.

86. 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.

88. 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..

89. 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

92. Polymerase Chain Reaction (PCR) Applications

93. 1. 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.

94. 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

95. 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.

97. 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.

98. 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.

99. 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.

102. 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.

105. Figure 1.
The
developm
ent of the
PCR
system
and its
applicatio
ns.

106. 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.

107. 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.

108. REFERANCEs:
1.Valasek MA, Repa JJ. The power of real-time PCR. Adv Physiol Educ. 2005
Sep;29(3):151-9. doi: 10.1152/advan.00019.2005. PMID: 16109794.
2.Mackay IM. Real-time PCR in the microbiology laboratory. Clin Microbiol
Infect. 2004 Mar;10(3):190-212. doi: 10.1111/j.1198-743x.2004.00722.x.
PMID: 15008940
3. Zhu H, Zhang H, Xu Y, Laššáková S, Korabečná M, Neužil P. PCR past,
present and future. Biotechniques. 2020 Jul;69(4):317-25.
4.Rådström P, Knutsson R, Wolffs P, Lövenklev M, Löfström C. Pre-PCR
processing: strategies to generate PCR-compatible samples. Mol Biotechnol.
2004 Feb;26(2):133-46. doi: 10.1385/MB:26:2:133. PMID: 14764939