Polymerase Chain Reaction (PCR) and Its Medical Applications

This report discusses the Polymerase Chain Reaction (PCR) and its medical applications. It covers the stages of PCR, the construction of DNA sequences, and the limitations of PCR. Additionally, it explores various medical applications of PCR, including gene expression, genotyping, cloning, mutagenesis, methylation analysis, sequencing, medical forensic, and applied sciences.

POLYMERASE
CHAIN REACTION

Table of Contents
INTRODUCTION...........................................................................................................................3
MAIN BODY...................................................................................................................................3
CONCLUSION................................................................................................................................6
REFERENCES................................................................................................................................7

INTRODUCTION
The polymerase chain reaction is defined as a laboratory technique that is helpful in order
to amplify the sequences of DNA. This method usually involves using the short DNA sequences
that are called primers in order to select the portion of the genome which is ready for
amplification. In addition to this, the temperature of the sample is associated with the repetitive
manner that raises and lowered to help the DNA replication enzyme that copies the target DNA
sequences (Rauch and et. al., 2021). Moreover, this technique is useful for the production of
billions of copies of the target sequence within the DNA. In this report, the discussion is based
on the polymerase chain reaction processes that emphasize in order to design the primer and their
aspect of construction. In addition to this, the stages of polymerase chain reaction and their
medical applications are discussed by taking the comparative study of traditional and quantitative
polymerase chain reactions (Bin and et. al., 2019).
MAIN BODY
The polymerase chain reaction is usually called a molecular photocopy, where the
polymerase chain reaction is the fast and cheapest method in order to amplifier the copy of a
small segment of DNA. Therefore, a significant amount of the sample which is associated with
the DNA is necessary for molecular and genetic analysis. In this, the studies are related to the
isolation of species that is related to the DNA and they are nearly impossible without polymerase
chain reaction amplification. The polymerase chain reaction very significant and scientific study
that shows the advanced level of molecular biology (Bonny and et. al., 2020).
There are some of the components which are useful for the process and construction of
polymerase chain reaction. The DNA template is the major component that play role in the
polymerase chain reaction. As per this, the sample DNA usually contains the target sequence. In
addition to this, at the initial stage of reaction, the high temperature is applied to the original
double-stranded DNA molecule to make them separate from each other. In this, the DNA
polymerase plays a scientific role by involving itself as an enzyme that is commonly useful for
the synthesis of new strands of DNA complementary to the target sequence (Tzanetakis and et.
al., 2021). The first and most commonly used of this enzyme is Taq DNA polymerase, whereas
PfuDNA polymerase is widely used because they have high flexibility when they are working in
the copying of DNA. In addition to this, these enzymes are different and they can complete the

reaction and the suitable way for the polymerase chain reaction. They also play a role by
formulating new strands of DNA by using the primer which is a DNA template and they are heat
resistant. The polymerase chain reaction is started with the synthesis of new DNA nucleotides or
deoxy-nucleotide triphosphate from the end of primers. The single unit of RT-PCR acts as a
basis that includes A, T, G, and C which is an essential component that works as a building block
for the new DNA strand (Deng and et. al., 2021).
There are some of the limitations which is associated with the polymerase chain reaction and
RT-PCR. Moreover, the polymerase chain reaction initiated to generate the copy of DNA
sequences during the exponential phases of the polymerase chain reaction. It is essential to
extrapolate back to identify the initial quantity of the DNA sequence which is contained within
the sample. In this, the polymerase chain reaction is also available in the sample that creates
reagent limitation, accumulation of pyrophosphate molecules, and self-annealing of the product
that is accumulated. However, the polymerase chain reaction is usually ceasing the amplification
of target sequences at the exponential rate that creates the plateau effect. In addition to this, the
endpoint quantification of polymerase chain reaction has a product that is not reliable and
validated. This is defined as an attribute of polymerase chain reaction that makes real-time
quantitative RT-PCR which is very necessary for the for the study.
The polymerase chain reaction usually contained a series of 20-40 repeated temperature
changes which is also called as a thermal cycle. They are usually contained different types of
temperature sets the cycling is usually processed by the single temperature set at a different high
temperature which is more than 90 degrees Celsius that is followed by one hold at a time for the
final product extension are the storage. There are different stages of polymerase chain reaction
that are helpful for the application of DNA sequences. The first step is known as initialization
where the requirement for DNA polymerases is initiated that require heat activation by hot-start
PCR. It usually consists of heating the reaction chamber at a temperature of 94 to 96 degrees
Celsius. They are extremely thermostable polymerase that is used which is usually held for 5 to
10 minutes. The second step is called denaturation, this step is the regular cycling event and they
contain a heating reaction chamber, where the temperature is 94 to 98 degrees Celsius for 30
seconds. They are responsible for DNA melting are also called denaturation by breaking the
bond between complementary bases that are made up of hydrogen bonds (Jalil and et. al., 2021).

The third step of the polymerase chain reaction is known as annealing where the reaction
temperature is minimized up to 50 to 60 degrees Celsius for 40 seconds. It is helpful because
they allow the annealing of the primers to each of the single-stranded DNA templates. Two
different primers are typically included for the reaction mixture. In this, one of each two single-
stranded complements containing their target area, where the primers are single-stranded
sequence but they are shorter as a comparison to their length for the target area. The
complimenting of very short sequences at the 3’ and of each strand is important. It is difficult to
identify the proper temperature for this step because the effectiveness is strongly affected by
temperature. Moreover, the temperature is set as they are low enough in order to allow for
hybridization of the primer to strand but high enough for the hybridization in order to make them
specific (Liang and et. al., 2018).
The extension is the fourth step which is associated with the polymerase chain reaction
which is also called an elongation, it depends on the DNA polymerase enzyme the activity of the
temperature for this step is thermostable DNA polymerase which is approximately 80 degrees
Celsius. Within this temperature, the polymerase enzyme is commonly used with this contrast
the DNA polymerases produce a new DNA strand complementary to the DNA template strand
by accumulating free dNTPs from the mixture of reaction which is complementary towards the
template in the 5’-3’ directions. The final elongation is the fifth step of PCR method which is
optional but they are usually performed at 70 to 75 degrees Celsius. They usually helpful to
ensure that the remaining single stranded DNA is fully elongated within the polymerase chain
reaction cycle. The last step of the polymerase chain reaction is known as final hold where the
reaction chamber at the temperature of 1 to 10 degrees Celsius for an indefinite time and may be
employed for the short-term storage of the product which is related to the polymerase chain
reaction (Mofrad and et. al., 2021).
The medical applications which are used by polymerase chain reaction on various fields
mainly include gene expression, genotyping, cloning, mutagenesis, methylation analysis,
sequencing, medical forensic, and applied sciences. Whereas, the polymerase chain reaction has
a vast range of applications that play a significant role within the different sectors in the gene
expression the application is associated with the variation in gene expression among the cell,
tissue, and the organism which is evaluated with the help of polymerase chain reaction.
Moreover, the genotyping is also an application of the polymerase chain reaction where the

detection of sequences variation in the specific cell or organism is analyzed and cloning is also
an aspect of the application that is associated with the polymerase chain reaction that is widely
used in DNA fragment of interest which is known as polymerase chain reaction cloning. As per
this, there is a various application that shows the approach in order to the treatment of cancer
cells and heredity with the help of genotyping by polymerase chain reaction with the aspect of
genetic analysis of mutations (Prabhu and et. al., 2018).
CONCLUSION
As per the above discussion, it is analyzed that the polymerase chain reaction is a method that is
widely used in the different sectors in order to make millions to billions of copies of DNA
samples and amplify it to a large enough amount to study in detail. Moreover, they usually
involve various processes and stages for the construction of DNA sequences. In this, the
applications of the polymerase chain reaction are widely used in the different sectors for the
treatment diagnosis amplification cloning, and many more.

REFERENCES
Books and Journals
Bin and et. al., 2019. Selection of reference genes for optimal normalization of quantitative real-
time polymerase chain reaction results for Diaphorina citri adults. Journal of economic
entomology, 112(1), pp.355-363.
Bonny and et. al., 2020. Current trends in polymerase chain reaction based detection of three
major human pathogenic vibrios. Critical Reviews in Food Science and Nutrition, pp.1-19.
Deng and et. al., 2021. Poly-l-lysine-functionalized magnetic beads combined with polymerase
chain reaction for the detection of Staphylococcus aureus and Escherichia coli O157: H7 in
milk. Journal of Dairy Science, 104(12), pp.12342-12352.
Jalil and et. al., 2021. Polymerase chain reaction technique for molecular detection of HPV16
infections among women with cervical cancer in Dhi-Qar Province. Materials Today:
Proceedings.
Liang and et. al., 2018. Development and application of a real‐time polymerase chain reaction
assay for detection of a novel gut bacteriophage (crAssphage). Journal of medical
virology, 90(3), pp.464-468.
Mofrad and et. al., 2021. Detection of Major Human Herpesviruses in Iranian Patients with
Suspected Encephalitis Using Multiplex Polymerase Chain Reaction. European Neurology,
pp.1-6.
Prabhu and et. al., 2018. Application and comparative evaluation of fluorescent antibody,
immunohistochemistry and reverse transcription polymerase chain reaction tests for the
detection of rabies virus antigen or nucleic acid in brain samples of animals suspected of
rabies in India. Veterinary sciences, 5(1), p.24.
Rauch and et. al., 2021. Comparison of Severe Acute Respiratory Syndrome Coronavirus 2
Screening Using Reverse Transcriptase–Quantitative Polymerase Chain Reaction or
CRISPR-Based Assays in Asymptomatic College Students. JAMA network open, 4(2),
pp.e2037129-e2037129.
Tzanetakis and et. al., 2021. Prevalence of Fungi in Primary Endodontic Infections of a Greek-
living Population Through Real-time Polymerase Chain Reaction and Matrix-assisted
Laser Desorption/Ionization Time-of-flight Mass Spectrometry. Journal of Endodontics.