INTRODUCTION

According to the Wikipedia published journal; polymerase chain reaction (PCR) is a method
widely used to rapidly make millions to billions of copies (complete or partial) of a specific
DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of
it) to a large enough amount to study in detail. PCR was invented in 1983 by the American
biochemist Kary Mullis at Cetus Corporation; Mullis and biochemist Michael Smith, who had
developed other essential ways of manipulating DNA, were jointly awarded the Nobel Prize in
Chemistry in 1993.
PCR is fundamental to many of the procedures used in genetic testing and research, including
analysis of ancient samples of DNA and identification of infectious agents. Using PCR, copies of
very small amounts of DNA sequences are exponentially amplified in a series of cycles of
temperature changes. PCR is now a common and often indispensable technique used in medical
laboratory research for a broad variety of applications including biomedical research and
criminal forensics (Widefox 2023).

APPLICATIONS OF PCR IN HUMANS

Selective DNA isolation
PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a
specific region of DNA. This use of PCR augments many ways, such as generating hybridization
probes for Southern or northern hybridization and DNA cloning, which require larger amounts of
DNA, representing a specific DNA region. PCR supplies these techniques with high amounts of
pure DNA, enabling analysis of DNA samples even from very small amounts of starting
material. Other applications of PCR include DNA sequencing to determine unknown PCR-
amplified sequences in which one of the amplification primers may be used in Sanger
sequencing, isolation of a DNA sequence to expedite recombinant DNA technologies involving
the insertion of a DNA sequence into a plasmid, phage, or cosmid (depending on size) or the
genetic material of another organism. Bacterial colonies (such as E. coli) can be rapidly screened
by PCR for correct DNA vector constructs. PCR may also be used for genetic fingerprinting; a
forensic technique used to identify a person or organism by comparing experimental DNAs
through different PCR-based methods (Widefox 2023).
Medical and diagnostic applications
Prospective parents can be tested for being genetic carriers, or their children might be tested for
actually being affected by a disease. DNA samples for prenatal testing can be obtained by
amniocentesis, chorionic villus sampling, or even by the analysis of rare fetal cells circulating in
the mother's bloodstream. PCR analysis is also essential to preimplantation genetic diagnosis,
where individual cells of a developing embryo are tested for mutations (Widefox 2023).
 PCR can also be used as part of a sensitive test for tissue typing, vital to organ
transplantation. As of 2008, there is even a proposal to replace the traditional antibody-
based tests for blood type with PCR-based tests.
 Many forms of cancer involve alterations to oncogenes. By using PCR-based tests to
study these mutations, therapy regimens can sometimes be individually customized to a
patient. PCR permits early diagnosis of malignant diseases such as leukemia and
lymphomas, which is currently the highest-developed in cancer research and is already
being used routinely. PCR assays can be performed directly on genomic DNA samples to
detect translocation-specific malignant cells at a sensitivity that is at least 10,000 fold
higher than that of other methods.
 PCR is very useful in the medical field since it allows for the isolation and amplification
of tumor suppressors. Quantitative PCR for example, can be used to quantify and analyze
single cells, as well as recognize DNA, mRNA and protein confirmations and
combinations.
Infectious disease applications
PCR allows for rapid and highly specific diagnosis of infectious diseases, including those caused
by bacteria or viruses. PCR also permits identification of non-cultivatable or slow-growing
microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue culture assays
and animal models. The basis for PCR diagnostic applications in microbiology is the detection of
infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of
specific genes (Salis, 2009). Characterization and detection of infectious disease organisms have
been revolutionized by PCR in the following ways:
 The human immunodeficiency virus (or HIV), is a difficult target to find and eradicate.
The earliest tests for infection relied on the presence of antibodies to the virus circulating
 in the bloodstream. However, antibodies don't appear until many weeks after infection,
maternal antibodies mask the infection of a newborn, and therapeutic agents to fight the
infection don't affect the antibodies. PCR tests have been developed that can detect as
little as one viral genome among the DNA of over 50,000 host cells. Infections can be
detected earlier, donated blood can be screened directly for the virus, newborns can be
immediately tested for infection, and the effects of antiviral treatments can be quantified .
 Some disease organisms, such as that for tuberculosis, are difficult to sample from
patients and slow to be grown in the laboratory. PCR-based tests have allowed detection
of small numbers of disease organisms (both live and dead), in convenient samples.
Detailed genetic analysis can also be used to detect antibiotic resistance, allowing
immediate and effective therapy. The effects of therapy can also be immediately
evaluated.
 The spread of a disease organism through populations of domestic or wild animals can be
monitored by PCR testing. In many cases, the appearance of new virulent sub-types can
be detected and monitored. The sub-types of an organism that were responsible for earlier
epidemics can also be determined by PCR analysis.
 Viral DNA can be detected by PCR. The primers used must be specific to the targeted
sequences in the DNA of a virus, and PCR can be used for diagnostic analyses or DNA
sequencing of the viral genome. The high sensitivity of PCR permits virus detection soon
after infection and even before the onset of disease. Such early detection may give
physicians a significant lead time in treatment. The amount of virus ("viral load") in a
patient can also be quantified by PCR-based DNA quantitation techniques (see below). A
variant of PCR (RT-PCR) is used for detecting viral RNA rather than DNA: in this test
the enzyme reverse transcriptase is used to generate a DNA sequence which matches the
viral RNA; this DNA is then amplified as per the usual PCR method. RT-PCR is widely
used to detect the SARS-CoV-2 viral genome.
 Diseases such as pertussis (or whooping cough) are caused by the bacteria Bordetella
pertussis. This bacteria is marked by a serious acute respiratory infection that affects
various animals and humans and has led to the deaths of many young children. The
pertussis toxin is a protein exotoxin that binds to cell receptors by two dimers and reacts
with different cell types such as T lymphocytes which play a role in cell immunity. [40]
 PCR is an important testing tool that can detect sequences within the gene for the
pertussis toxin. Because PCR has a high sensitivity for the toxin and a rapid turnaround
time, it is very efficient for diagnosing pertussis when compared to culture.

Forensic applications
The development of PCR-based genetic (or DNA) fingerprinting protocols has seen widespread
application in forensics:
 In its most discriminating form, genetic fingerprinting can uniquely discriminate any one
person from the entire population of the world. Minute samples of DNA can be isolated
from a crime scene, and compared to that from suspects, or from a DNA database of
earlier evidence or convicts. Simpler versions of these tests are often used to rapidly rule
out suspects during a criminal investigation. Evidence from decades-old crimes can be
tested, confirming or exonerating the people originally convicted.
 Forensic DNA typing has been an effective way of identifying or exonerating criminal
suspects due to analysis of evidence discovered at a crime scene. The human genome has
many repetitive regions that can be found within gene sequences or in non-coding regions
of the genome. Specifically, up to 40% of human DNA is repetitive.[5] There are two
distinct categories for these repetitive, non-coding regions in the genome. The first
category is called variable number tandem repeats (VNTR), which are 10–100 base pairs
long and the second category is called short tandem repeats (STR) and these consist of
repeated 2–10 base pair sections. PCR is used to amplify several well-known VNTRs and
STRs using primers that flank each of the repetitive regions. The sizes of the fragments
obtained from any individual for each of the STRs will indicate which alleles are present.
By analyzing several STRs for an individual, a set of alleles for each person will be found
that statistically is likely to be unique.[5] Researchers have identified the complete
sequence of the human genome. This DNA samples are often taken at crime scenes and
analyzed by PCR. Sequence can be easily accessed through the NCBI website and is used
in many real-life applications.
APPLICATIONS OF PCR IN MALARIAL GENOME
Malaria is a mosquito-transmitted disease caused by apicomplexan parasites in the
genus Plasmodium. It is an important cause of morbidity and mortality worldwide, with the
World Health Organization (WHO) estimating 219 million cases and 435,000 malaria-related
deaths in 2017. Malaria is caused primarily by 4 species of the protozoa Plasmodium: P
falciparum, P vivax, P malariae, and P ovale. A fifth Plasmodium species, P knowlesi, is a
simian parasite that may be an important source of human infection in some regions of Southeast
Asia. Differentiating P falciparum and P knowlesi from other species is important since both can
cause life-threatening infections. In addition, P falciparum is typically resistant to many
commonly used antimalarial agents such as chloroquine.
PCR is an alternative method of malaria diagnosis that allows for sensitive and specific detection
of Plasmodium species DNA from peripheral blood. PCR may be more sensitive than
conventional microscopy in very low parasitemias, and is more specific for species
identification. It may be particularly useful when subjective microscopy does not permit certain
identification of the species present. Malaria PCR can be used in conjunction with a traditional
blood film or Babesia PCR when the clinical or morphologic differential includes both
babesiosis and malaria. Examination of the thin film also allows for calculation of percent
parasitemia, which can be used to predict prognosis and monitor response to treatment. This test
does not include blood smear examination or calculation of parasitemia.
REFERENCES
Salis AD (2009). "Applications in Clinical Microbiology". Real-Time PCR: Current Technology
and Applications. Caister Academic Press. ISBN 978-1-904455-39-4.
Widefox. (2023). Wikipedia https://en.wikipedia.org/w/index.php?
title=Polymerasechainreaction&oldid=1125785259.