Polymerase Chain Reaction

Polymerase chain reaction (PCR) is a technique used for exponentially amplifying DNA sequences. This article discusses the components, stages, and applications of PCR.

Running head: POLYMERASE CHAIN REACTION
POLYMERASE CHAIN REACTION
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Polymerase Chain Reaction
Polymerase chain reaction (PCR) is considered as the technique that is used for
exponentially amplifying the explicit target sequence of deoxy-ribonucleic acid (DNA)
permitting for the extraction, sequencing or replicating of the single DNA sequences into
many other sequences. This technique was developed by Kary Mullis, a Nobel Prize winner,
in 1983, for his impeccable invention in the modern biotechnology (Lab tests online, 2019).
Through this technique, smallest DNA samples can be cloned and amplified into millions of
other DNA sequence copy and hence it is includes primer facilitated enzymatic augmentation
of DNA. There are different types of PCR that is commonly used and it majorly relies on the
process of thermal cycling that comprise of repeated heating and cooling cycles that will
trigger the DNA melting reaction and enzymatic replication. There are various application of
polymerase chain reaction (PCR) in human, plants and animals sectors. PCR is currently the
most common and frequently used technique in the clinical and medical laboratory
examination for a comprehensive variability of applications that comprises of criminal
forensics and biomedical research (Poornima et al., 2016).

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So
urce: (Lab tests online, 2019)
Components of PCR
The accomplishment of PCR reaction depends on different factors, where the reaction
components play a critical role in amplifying the DNA sequences. The components of PCR
reaction are as follows (Jalali, Zaborowska and Jalali, 2017):
 DNA Template- DNA template is the most crucial component namely double
stranded deoxy-ribonucleic acid (DNA) that is removed from the sample.
 DNA polymerase- DNA polymerase are considered as the crucial players of
replication and amplification of target DNA. Thermostable (Taq) DNA polymerase
are most widely used in PCR reaction because they have a little longer half-life (40
minutes) and can easily withstand the high temperature (98 °C) and function
effectively.
 Oligonucleotide primers- The short fragments of single stranded DNA are used for
the PCR reaction of approximately 20-30 base pairs. The primer sequence are
complementary to the target DNA sequence.
Samles (Cultered
cells, RNA and
DNA)
Detection Method Electrophorectic
Method
Amplification
Method
Polymerase Chain
Reaction (PCR)
Sequencing

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 Deoxynucleotide triphosphates (dNTPs) - dNTPs included four different
nucleotides namely dATP, dCTP, dGTP, and dTTP that acts the building blocks
of DNA and provide required energy for performing the DBA synthesis.
 Buffer system- Buffer system includes the potassium and magnesium ions that act as
cofactors the enable the incorporation of different dNTPs during the polymerization
reaction. It also provides optimum condition of effective DNA renaturation and
denaturation. The buffer system is also crucial for carrying out the polymerase,
activity and maintaining the fidelity and stability.
Stages of PCR
The efficiency and reaction rate of PCR are majorly influenced by the limiting factors
and hence the complete PCR procedure are classified into three different stages. The stages
are classified on the basis of reaction progress and are as follows (Green and Sambrook,
2019):
 Exponential amplification stage
 Leveling off stage
 Plateau stage
Methodology of PCR
The complete methodology of polymerase chain reaction (PCR) is completed in 20-40
repeated cycles, where each cycle has a significant role in amplification of DNA sequences.
The cycle of PCR initiates with a fixed temperature (>90 °C) known as hold temperature.
There are 5 different stages of PCR that includes (Heuvel 2019):
 Initialization step- It is considered as the primary step that comprise of increasing the
reaction temperature to 96-98 °C and practice of thermostable (Hot-start) polymerase
that can withstand the high reaction temperature and the holding time is 1-9 minutes

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for such polymerase. This step is significant for activating the DNA polymerase that
will be used in the PCR reaction (Davies and Dalton 2017).
 Denaturation Step- This step includes increasing the reaction temperature to 94-98
°C for 30 seconds. This step will result in breaking of hydrogen bonds among the
complementary bases that will ultimately lead to single-stranded DNA molecule.
Denaturation step is repeated for 15-40 times depending in the application.
 Annealing Step- In this step, the mixture cooled and the reaction temperature is
reduced to 50-65 °C and further centrifuged for subsequent 20-40 seconds that will
assist in annealing the primers to single-stranded DNA template. Stable DNA-DNA
hydrogen binds are formed, when the sequence of primer matches with the template
sequence which will allow the annealing process of primer sequence to the
complementary sequence of DNA. The total duration of annealing step is 1 minutes
and is repeated 15-40 times (Dorado et al. 2019).
 Extension stage- In this step, the reaction temperature is increased to 80 °C and the
polymerase will initiate the proof reading (5´-3´ direction) and copy the template
DNA (3´-5´direction). The high temperature used in this step depends on the type of
DNA polymerase used and determining the specificity of the reaction. Based on
thumb rule, the optimum temperature of chosen polymerase will initiate the
polymerization reaction for thousand bases per minute, which leads to the
amplification of explicit DNA fragment.
 Final elongation and hold- This step is conducted at the optimum temperature and
condition of 70-74 °C for maximum 15 minutes. In this step, the DNA polymerase
completes the proof reading and reduce any truncated copies of final product. The
final hold step signifies that the PCR reaction is complete and the temperature is
reduced to 4 °C for short-term storage (Belmont 2019).

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Source: (Dorado et al. 2019)
Application of PCR
There are various sectors in modern and conventional biotechnology, where PCR are
greatly used and applied. PCR is most widely used in the field of human specifically in
medical and diagnostic application. The parents could be tested using the PCR application of
identifying if the parents are genetic carrier of any disease or not and even the small children
can be tested, if they are affected by any kind of disease or not (Luthra, Singh and Patel
2016). The DNA samples can be acquired by chorionic villus sampling or amniocentesis for
performing prenatal testing. PCR analysis is crucial for performing preimplantation genetic
diagnosis in which the specific cells of evolving embryo are verified for mutation. PCR-
founded test are also used for testing mutation or presence of cancer in an individual. PCR
test can also be helpful; for performing and identifying the early detection of malignant
condition like lymphomas and leukaemia (Kuypers and Jerome 2017).
The target
sequnce is
identified
Denaturation
at 94-98 °C
Anealing step
at 50-65 °C
Extension
step at 75-
78 °C
Multiple
copies
produced

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The other sectors of PCR applications includes:
Source: (Kuypers and Jerome 2017)
PCR
Application
Infectious disease
apllication (HIv
testing)
Forensic
Application
(genetic
fingerprinting and
paternity testing)
Research
Application (DNA
cloning, Sequence-
atgged sites)
Monitoring the
genes during
the gene
therapy

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References
Belmont, J.W., 2019. Molecular methods. In Clinical Immunology (pp. 1297-1310). Content
Repository Only!.
Davies, M. and Dalton, T., Stokes Bio Ltd, 2017. Methods and devices for digital PCR. U.S.
Patent 9,631,230.
Dorado, G., Besnard, G., Unver, T. and Hernández Molina, P., 2019. Polymerase chain
reaction (PCR).
Green, M.R. and Sambrook, J., 2019. Nested polymerase chain reaction (PCR). Cold Spring
Harbor Protocols, 2019(2), pp.pdb-prot095182.
Heuvel, J.P.V., 2019. PCR Protocols in Molecular Toxicology. CRC Press.
Jalali, M., Zaborowska, J. and Jalali, M., 2017. The Polymerase Chain Reaction: PCR, qPCR,
and RT-PCR. In Basic Science Methods for Clinical Researchers (pp. 1-18). Academic Press.
Kuypers, J. and Jerome, K.R., 2017. Applications of digital PCR for clinical microbiology.
Journal of clinical microbiology, 55(6), pp.1621-1628.
Lab tests online (2019). Polymerase Chain Reaction (PCR). [online] Labtestsonline.org.au.
Available at: https://www.labtestsonline.org.au/inside-the-lab/laboratory-methods/pcr-(1)
[Accessed 31 Aug. 2019].
Luthra, R., Singh, R.R. and Patel, K.P. eds., 2016. Clinical applications of PCR. Humana
Press.
Poornima, M., Rajan, J.S., Kumar, S. and Otta, S.K., 2016. Polymerase Chain Reaction
(PCR)–General Principles and Practices. Molecular Diagnosis of Shrimp Diseases, p.20.