A Review of PCR, Principle, and Its Applications

International Journal of Pharmaceutical Research and Applications
Volume 8, Issue 3 May-June 2023, pp: 3502-3513 www.ijprajournal.com ISSN: 2249-7781
A Review of PCR, Principle, and Its Applications

Laura S. L1., Rukmanidevi D2.
1
Associate Professor, Department of Pharmaceutics, K. K. College of Pharmacy, Chennai 600128.
2
Department of Pharmaceutical Analysis, K. K. College of Pharmacy, Chennai 600128.
Corresponding Author: Laura S. L.,Associate Professor, Department of Pharmaceutics, K. K. College of
Pharmacy, Chennai 600128.
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Submitted: 25-06-2023 Accepted: 05-07-2023
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ABSTRACT: PCR (Polymerase Chain Reaction) This is a process DNA sequences are continuously
is a laboratory method for laboriously creating replicated in vitro, at the same time producing
millions to billions of copies of a certain DNA millions of copies. There are many different
region. In PCR, a section of the genome to be methods that are used to amplify gene sequences.
amplified is chosen using short synthetic DNA The first developed and most widely used is
fragments called primers, and the segment is polymerase chain reaction (PCR). First introduced
subsequently amplified by numerous rounds of in 1985. The applications of this technique have
DNA synthesis. PCR was first developed in the increased over the past several years to the point
middle of the 1980s, about at the same time as the where it may well revolutionize diagnostic
Human Genome Project was being discussed and medicine [2].
then launched. Since then, PCR has been incredibly In the history of biological and medical
essential to a lot of biology and biomedical sciences, the development of the polymerase chain
research. It was a key technological advancement reaction (PCR) has been a turning point. In addition
in the early stages of the Human Genome Project. to completely revolutionizing research in the fields
And it continues to play a significant role in of molecular genetics, animal and plant
biological and medical research today. This review biotechnology, and PCR applications, these
article discusses the main uses of PCR technology techniques have also demonstrated their
in the areas of disease diagnosis, medical research, applicability and relevance in a number of other
food and drug regulations, microbiologic fields, including forensic sciences, molecular
properties, forensic science, epidemiology, systematics, molecular epidemiology, archaeology,
evolutionary genetics, anthropology, etc. It also anthropology, and evolutionary genetics. The
discusses the types of conventional PCR, such as development of RT-PCR, qPCR, and combination
RT-PCR (Real-time - polymerase chain reaction), RT-PCR/q-PCR was facilitated by conventional
qPCR (Quantitative polymerase chain reaction), PCR.
[3]
and combined RT-PCR/qPCR. By enabling the amplification and
KEYWORDS: Real-Time PCR, Molecular sequencing of human genes, PCR has also made it
biology, PCR, Applications. feasible to successfully complete the "human
genome project," which has created the
I. INTRODUCTION: groundwork for genetic engineering and now even
A technique for amplifying DNA in vitro makes it possible to make meaningful alterations to
is the polymerase chain reaction (PCR). Since its an organism's genome. Most of the most recent
invention in 1985, the PCR has found widespread advancements in modern sciences have effectively
use in a variety of applications in clinical and utilized PCR variations.
[4]
criminal investigations as well as scientific The study on the diagnosis of
[1]
research .The discovery of the molecular structure respiratory tract disease concluded that RT-PCR
of DNA over half a century ago marks the assays gave a significant improvement in the
introduction to the modern era of biotechnology. diagnosis since the time taken is less and can work
Research at those times uncovered numerous facts efficiently in managing and controlling of the
about the molecular processes that take place inside emergence these important neglected diseases.
[5]
the living cell. At present using the principles of High throughput screening, improved
the PCR diagnosis and treatment for human disease precision, and the integration of robotic setup
or carried out. The most significant application of technologies with real-time quantitative PCR
modern-erabiotechnology is gene amplification. (qPCR) are revolutionizing the field of
DOI: 10.35629/7781-080335023513 | Impact Factor value 7.429 | ISO 9001: 2008 Certified Journal Page 3502
biotechnology. Applications of qPCR in efficient whole genome amplification (WGA)

biotechnology are reviewed, with a focus on techniques, and even Single-cell comparative
genetic stability testing and biosafety in particular: genomic hybridization will make it possible for the
the biodistribution of gene therapy vectors in genetic study of individual cells to become
animals, determination of the amount of leftover standard practice, creating new opportunities for
DNA in therapeutic products, the discovery of research and diagnosis.
[9]
bacterial and viral nucleic acids in compromised Prenatal diagnosis is now a common
cell banks and finished goods, measurement of the practice in obstetric care. Prenatal genetic
degree of virus elimination in process validation diagnoses are made by taking fetal genetic samples
viral clearance tests, very sensitive and specific using invasive techniques such as amniocentesis or
detection of retroviral RT activity in vaccines, and chorionic villus sampling, which carry a small risk
measurement of gene copy number for tracking of fetal loss. Consequently, intriguing opportunities
genetic stability during manufacturing. for noninvasive prenatal diagnostics have emerged
[6]
Nowadays, most illness diagnoses are as a result of the recent discovery of fetal DNA in
based on lab testing. Recent developments in maternal plasma. Since then, many applications for
medical science and molecular biology have made the study of circulating fetal DNA have been
it possible to diagnose the majority of rare, reported. The proper pre-analytical processing of
complex, or unique illness presentations, with maternal blood samples, a successful fetal DNA
molecular diagnostics remaining one of the most extraction protocol, and a sensitive and precise
effective diagnostic modalities. Today, a variety of detection technology are all necessary for accurate
molecular methods are in use all around the world, fetal DNA assessment from maternal plasma.
[10]
including PCR, flow cytometry, tissue microarray, It is becoming increasingly clear that
various blots, and genetic diagnostics. One of these answering scientific problems in molecular
diagnostic methods with the highest specificity and toxicology, molecular epidemiology, and human
sensitivity for accurate diagnosis is PCR, which is risk assessment requires a quantitative study of
the most well-acknowledged and often utilized. changes in molecular targets. Real-time and
[7]
The common cystic fibrosis mutation quantitative (RTAQ) polymerase chain reaction is
(AF) was discovered in an Irish family using allele- one of the cutting-edge methods being employed to
specific PCR. Based on the resolution of allelic examine these molecular targets (PCR).
[11]
amplifications on an agarose gel, it was determined The use of polymerase chain reaction
that each member's genomic status was either (PCR), a nucleic acid amplification technique, has
heterozygous (carrier) or homozygous (affected) completely changed how we detect, treat, and study
for the mutation. Five staphylococci were treated to diseases. Real-time PCR, a recent advancement in
PCR intended to amplify a DNA fragment from the PCR technique, has grown in prominence due to its
S. aureus protein A gene (spa). These examples many clinical and basic research applications. Real-
have been chosen to highlight the adaptability of time PCR combines the chemistry of PCR with the
this method and some of its possible uses. capacity to identify and continually track the
[8]
With the invention of the polymerase buildup of reaction products (amplicons) following
chain reaction (PCR), molecular scientists may each amplification cycle. Real-time PCR has the
now amplify and analyze minuscule amounts of capacity to identify whether there are any sequence
uncommon genetic material. This capability has alterations as well as the presence and quantity of
been extensively investigated, particularly in nucleic acid sequences (target nucleic acids). By
prenatal diagnostics, since it has opened the detecting amplified PCR products using
possibility of pre-implantation genetic analysis and fluorescent-tagged oligonucleotide probes, real-
the use of enriched fetal cells from the blood of time PCR can be made more precise, useful, and
pregnant women for the evaluation of single-gene specific. In the same procedure, nucleic acid
Mendelian illnesses. Single-cell PCR has been amplification and detection are performed closed
demonstrated to be of great value to basic vessel, which offers rapid, continuous data
scientists, addressing many immunological, collection and reduced time requirements for
neurological, and developmental problems aside findings while minimizing the likelihood of cross-
from diagnostic applications. where the expression contamination (amplified product carryover)
patterns of messenger RNA and the genome were between tests.
[12]
studied. Additionally, recent developments More and more scientists are using
including single-cell complementary DNA, molecular techniques to identify, count, and
analyze microbial communities in food or during preparing the DNA significantly slows down the
food processing. PCR-based approaches have PCR process and delays the results.
[14,15]
received a lot of attention among these techniques, The most well-characterized
and ISO guidelines have been developed for the amplification technology is still PCR. There has
identification of food-borne infections. More been a lot of work put into increasing amplification
specifically, real-time quantitative PCR (qPCR) is fidelity,specificity, and addressing the carryover
regarded as the preferred technique for the contamination issue. The creation of novel
identification and measurement of microorganisms. thermophilic DNA polymerases was revealed by
Its ability to be quicker than traditional culture- researchers at Roche Molecular Systems. The
based procedures is one of its main advantages. UITma DNA Polymerase, which has proofreading
Additionally, it allows for the simultaneous activity and allowed for the cloning of high-fidelity
identification of many microbes and is highly amplification products, was one of the most
sensitive and specific. Contrary to the usage of intriguing enzymes ever described.The variety of
qPCR, reverse transcription-qPCR (RT-qPCR) is stated new technologies has the prospect of
just now being applied to research population significantly improving PCR specificity. The
dynamics and activities by quantification of gene inherent challenges in identifying low target copy
expression in food. As long as the studies contain numbers in diseases like HIV-1 have sparked this
sufficient controls, Gene and gene expression can research. The so-called "hot start," which involves
be quantified using qPCR and RT-qPCR, which injecting a specific enzyme at an ideal temperature,
appear to be very accurate and reliable methods. is promoted by Applied Biosystems. This
This review discusses several crucial technical straightforward method directs polymerase activity
issues to consider while employing these strategies. toward primer-dimer synthesis and non-specific
The most recent uses of qPCR and RT-qPCR for product amplification. Hot starts can be performed
food microbiology are presented. There have been either manually or automatically using wax beads
reports of some intriguing uses, including risk that have been carefully prepared to melt at the
assessments and research into how industrial ideal PCR temperature and physically release the
processes affect microbial activity and gene enzyme.
[16,17]
expression. The wax approach is replaced by
[13]
The polymerase chain reaction (PCR), a several novel reagents that offer chemical hot-start
potent method with a large and expanding variety options. A method used by Life Technologies and
of uses, has emerged as a crucial research and PerkinElmer involves the enzyme uracyl-N-
diagnostic tool. DNA extraction from biological glycosylase (UNG) with PCR primers that have 3'-
materials, which should produce the substrate for terminal deoxyuridine residues. The dU residues
the amplification reaction assuming a minimal are preferentially integrated into the PCR product
level of quality and quantity is met, is the from dUTP, therefore removing nonspecific
foundation of PCR. For this goal, a number of products. Additionally, specificity advantages can
quick techniques that mostly depend on the sample be obtained from single-stranded DNA-binding
and microorganisms to be studied have been proteins.Eastman Kodak developed one of the best
proposed in recent years. As the temperature hot-start strategies. The business developed a
increases to 95 °C for 4-5 minutes during the first monoclonal antibody that inhibits Taq DNA
denaturation stage, cells in the colony that is added polymerase to offer hot-start advantages. The
immediately to the reaction mix are disturbed. This antibody denatures when the reaction mixture gets
approach has been demonstrated to be effective for hot, which permits amplification to start.
isolated colonies of yeast, certain Lactobacillus
species, and gram-negative bacteria. Cell lysis with II. PRINCIPLE:
[18-21]
lytic enzymes and detergents has been utilized for The principle of PCR (Polymerase
refractory microorganisms because this procedure Chain Reaction) is a widely used molecular biology
is frequently ineffective. This is particularly true technique that amplifies a specific segment of DNA
for gram-positive bacteria, for which colony (deoxyribonucleic acid) in vitro. It was developed
extraction requires digestion with mutanolysin, cell by Kary B. Mullis in the 1980s and has since
disruption with Triton X-100, and boiling. The become an essential tool in various areas of
entire procedure takes around 45 minutes. biological research, diagnostics, and forensic
Therefore. The lengthy process of extracting and analysis.
The PCR process involves a series of strands, it is heated to a high temperature (usually
temperature cycles that enable the replication of the approximately 95°C). As a result of this phase,
target DNA sequence exponentially. The primary DNA becomes denaturized because the hydrogen
PCR stages are listed below: connections between the complementary base pairs
are broken.
Denaturation:To divide the double-stranded DNA
sample carrying the target sequence into two single
Figure 1: Step 1- Denaturation
Annealing: The temperature is lowered (typically target DNA sequence. These primers are designed
around 50-60°C) to allow short DNA primers to to be specific to the target sequence and serve as
bind to the complementary regions flanking the starting points for DNA synthesis.
Figure 2: Step 2- Annealing
Extension: The temperature is raised (typically The DNA polymerase extends the primers by
around 72°C), and a heat-stable DNA polymerase adding nucleotides complementary to the template
enzyme (e.g., Taq polymerase) synthesizes new DNA strand.
DNA strands using the primers as a starting point.
Figure 3: Step 3- Extension
Repeat cycles: Steps 1-3 are repeated multiple level. The amplified DNA can then be analyzed
times (typically 20-40 cycles) to achieve using various methods, such as gel electrophoresis
exponential amplification of the target DNA or DNA sequencing, depending on the specific
sequence. Each cycle doubles the amount of DNA, application.
resulting in a substantial increase in the number of PCR has revolutionized many areas of biology and
target DNA molecules. has numerous applications, including genetic
By the end of the PCR process, the desired research, medical diagnostics, forensic analysis,
DNA segment has been amplified to a detectable paternity testing, and infectious disease detection.
Figure 4- Steps in PCR
TYPES OF PCR: There are several types of PCR that have been
PCR (Polymerase Chain Reaction) is a developed for various applications.
widely used technique in molecular biology and 1) Conventional PCR: Also known as endpoint
genetics for amplifying specific DNA sequences. PCR, it is the original PCR method developed
by Kary Mullis in 1983. It involves cycles of to the target DNA regions of interest. The
DNA denaturation, primer annealing, and ligated probes are then amplified using PCR,
DNA extension using a heat-stable DNA and the resulting products are analyzed to
polymerase such as Taq polymerase. It determine the copy number status of specific
amplifies a specific DNA target region, and the DNA sequences.
results are analyzed after the PCR is complete. 8) Digital PCR (dPCR): Digital PCR is a method
2) Real-time PCR: Also called quantitative PCR that enables absolute quantification of DNA
(qPCR), this technique allows for the targets. It involves partitioning the PCR
quantification of DNA during the amplification reaction into thousands or millions of
process. It uses fluorescent DNA-binding dyes individual reactions, each containing a few
or specific probes (such as TaqMan probes) target DNA molecules or none at all. The
that emit a signal when bound to the amplified amplification is then carried out in each
DNA. Real-time PCR can measure the DNA partition, and the presence or absence of the
amount in real-time, providing information on target DNA is determined. Digital PCR
the initial quantity of the target DNA. provides higher precision and sensitivity for
3) Reverse Transcription PCR (RT-PCR): This detecting rare targets or determining precise
technique is used to amplify RNA molecules. target concentrations.
It involves the reverse transcription of RNA 9) Inverse PCR (iPCR): In iPCR, the DNA
into complementary DNA (cDNA) using fragment of interest is ligated to adaptors, and
reverse transcriptase enzyme. The cDNA is subsequent PCR amplification is performed
then amplified using conventional PCR or real- using primers that anneal to the adaptors rather
time PCR. RT-PCR is commonly used for than the target sequence. This technique is
gene expression analysis and studying RNA useful for amplifying DNA sequences flanking
viruses. known regions or for identifying unknown
4) Nested PCR: This PCR method involves two sequences adjacent to known DNA sequences.
rounds of amplification. In the first round, 10) Assembly PCR: Assembly PCR is employed to
external primers bind to the target DNA region generate large DNA fragments by assembling
and amplify a larger fragment. Then, a portion smaller overlapping DNA fragments. Each
of the product from the first round is used as a fragment contains complementary ends that
template in the second round with a different can anneal to adjacent fragments. PCR
set of internal primers. Nested PCR increases amplification is then performed using primers
specificity and sensitivity by reducing that hybridize to the outermost ends of the
nonspecific amplification. assembly. Assembly PCR is commonly used in
5) Multiplex PCR: This PCR technique allows for cloning and gene synthesis.
the amplification of multiple target DNA 11) Nested Reverse Transcription PCR (Nested
sequences in a single reaction. It involves the RT-PCR): This technique combines the
use of multiple primer pairs that target principles of nested PCR and reverse
different DNA regions. Multiplex PCR is transcription PCR. It is used for the detection
useful for detecting multiple pathogens and amplification of specific RNA sequences.
simultaneously and for genotyping studies. The first round involves reverse transcription
6) Hot Start PCR: This method includes of RNA into cDNA, followed by PCR
modifications to the PCR reaction mix to amplification using external primers. A portion
prevent nonspecific amplification. It involves of the first-round PCR product is then used as
using modified DNA polymerases that are a template for a second round of PCR with
inactive at lower temperatures, and they internal primers, increasing specificity.
become active only after an initial heat 12) Methylation-Specific PCR (MSP): MSP is
activation step. Hot Start PCR improves designed to detect DNA methylation patterns
specificity and sensitivity by reducing at specific CpG sites within a DNA region. It
background amplification. involves bisulfite treatment of DNA to convert
7) Multiplex Ligation-dependent Probe unmethylated cytosines to uracils, followed by
Amplification (MLPA): MLPA is a PCR- PCR amplification using primers specific to
based technique used for detecting copy methylated or unmethylated DNA sequences.
number variations (CNVs) in genomic DNA. It MSP is widely used in epigenetic studies and
involves the hybridization of multiple probes
can provide information on DNA methylation amplifying and studying DNA from preserved
patterns in various biological samples. remains, such as bones or teeth, scientists can gain
insights into evolutionary relationships, genetic
III. APPLICATIONS OF PCR: diversity, and ancient populations.
PCR (Polymerase Chain Reaction) is a
widely used molecular biology technique that  Environmental monitoring:
allows for the amplification of specific DNA PCR is employed in environmental
sequences. It has numerous applications in various science to detect and monitor organisms present in
fields. various environmental samples. For example, it can
be used to identify microbial species in soil, water,
 Genetic research: or air samples. PCR-based techniques like
PCR is extensively used in genetic quantitative PCR (qPCR) allow for the
research to study DNA sequences. It enables the quantification of target organisms, providing
amplification of specific DNA regions of interest, valuable information for environmental studies and
which can then be analyzed for genetic variations, monitoring.
mutations, or polymorphisms. PCR is employed in
fields like genomics, gene expression analysis, and  Oncology:
population genetics. PCR is utilized in cancer research and
diagnosis. It can detect genetic mutations or
 Disease diagnosis: alterations associated with cancer, enabling the
PCR plays a crucial role in medical identification of specific types of cancer and
diagnostics. It allows for the detection and guiding treatment decisions. PCR-based methods
identification of infectious agents such as bacteria, like allele-specific PCR and quantitative PCR are
viruses, and parasites. By amplifying and detecting used in cancer studies and monitoring.
the specific DNA or RNA sequences of pathogens,
PCR-based tests can diagnose various diseases,  Pharmacogenomics:
including viral infections (e.g., HIV, influenza), PCR plays a role in pharmacogenomic
bacterial infections (e.g., tuberculosis, Lyme studies, which investigate the relationship between
disease), and genetic disorders (e.g., cystic an individual’s genetic makeup and their response
fibrosis). to drugs. By amplifying and analyzing specific
genetic markers, PCR can help predict an
 Forensic analysis: individual’s likelihood of responding to certain
PCR is utilized in forensic science for medications or experiencing adverse drug
DNA profiling and identification purposes. The reactions.
technique allows for the amplification of small  Food safety and quality control:
DNA samples found at crime scenes, enabling PCR-based techniques are employed in
investigators to generate enough DNA material for the food industry to detect and identify pathogens,
analysis. PCR-based methods like short tandem allergens, and genetically modified organisms
repeat (STR) analysis and DNA fingerprinting are (GMOs) in food products. PCR allows for rapid
commonly used in forensic investigations. and accurate screening of food samples, ensuring
food safety and adherence to regulatory standards.
 Paternity testing:
PCR-based DNA testing is employed in  Veterinary medicine:
paternity testing to determine biological parentage. PCR is used in veterinary diagnostics for
By comparing specific DNA regions between a the detection and identification of pathogens that
child and potential parents,PCR can establish affect animals. It enables the diagnosis of
familial relationships. It is a reliable method used infectious diseases in livestock, pets, and wildlife,
in legal cases, immigration proceedings, and facilitating appropriate treatment and control
personal identification scenarios. measures.
 Archaeology and paleontology:  Ancient DNA studies:

PCR has revolutionized the fields of PCR has been instrumental in the field of
archaeology and paleontology by enabling DNA ancient DNA research. By amplifying and
analysis of ancient or extinct organisms. By sequencing DNA from ancient samples, including
fossils and preserved remains, scientists can APPLICATIONS OF PCR IN

reconstruct the genomes of extinct species, study TOXICOLOGY[20,21]:
evolutionary history, and explore genetic  Molecular Genetics:
relationships between ancient and modern Two distinct Taqman probes can be used
organisms. in conjunction with Taq DNA polymerase’s 5’
nuclease activity to produce allele-specific
 Microbial ecology: amplification detection (allelic discrimination),
PCR-based techniques like metagenomics which can then be used to quickly genotype
and microbial community profiling allow for the individual subjects. While real-time PCR analysis
study of microbial diversity and dynamics in of these assays helps in their creation and can aid in
various environments, includingsoil,oceans, and the the assessment of the allele specific amplification,
human microbiome. PCR amplification of specific these allelic discrimination assays simply need a
genetic markers helps identify and characterize terminal fluorescence analysis. Aldehyde
microbial communities. dehydrogenase, N-acetyltransferase, thiopurine
methyltransferase, glutathione S transferases
 Cloning and gene expression: (GSTM1, GSTT1, GSTP1), cytochromes P450
PCR is an essential component of cloning (CYP2C, CYP2D), and other enzymes contributing
techniques, enabling the amplification of DNA to human vulnerability have all been examined for
fragments for insertion into vectors. It is also used polymorphisms using Taqman-based allelic
in quantitative PCR (qPCR) or reverse transcription discrimination.
PCR (RT-PCR) to measure gene expression levels
and analyze gene function.  Exposure Monitoring:
The measurement of DNA-based viral
 Prenatal testing: loads in clinical pathology first made extensive use
PCR-based methods, such as of real-time PCR for quantitative detection of DNA
amniocentesis and chorionic villus sampling, are copy number. Studies evaluating the tissue
used for prenatal diagnosis of genetic disorders. distribution of DNA-based medications, such as
PCR amplification and analysis of fetal DNA DNA-based vaccinations, have more recently used
obtained from these procedures can identify genetic quantitative analysis of DNA. The monitoring of
abnormalities or chromosomal disorders in genetically modified organisms (GMOs) in food is
developing embryos. also applicable to this.
Figure 5: Exposure monitoring
 Molecular Toxicology: development than other expression analysis tools,

The majority of quantitative PCR real-time assays, particularly those using a specific
applications, or gene expression profiling, involve probe, have certain very specific applications: (1) A
checking the levels of gene expression. In addition quantitative examination of dose-response.
to helping with toxicologic disease, gene (2) Molecular epidemiological gene expression
expression profiling has been used to examine biomarker research, as an illustration.
changes in immune function and cytokine (3) Quantitative evaluations of tiny tissues or
expression caused by toxicants. Real-time methods samples, such as those acquired using laser capture
have occasionally been used as a stand-in for more microdissection (LCM).
traditional RNA detection techniques like Northern
blot analysis. Because they required more
 Toxicogenomic: by changes in certain gene sets is one of the goals

Genome-wide patterns of gene expression of a toxicogenomic approach.The toxicity of other
identified by microarray analysis are one of the chemicals with limited toxicological inception may
cutting-edge techniques that could revolutionize the therefore be predicted using these signals.
assessment of a compound's potential toxicity. According to recent bioinformatic methods, the
Microarray data on differential gene expression, best chance to distinguish between different groups
however, is frequently only semi-quantitative in of samples' gene expression profiles is found in the
nature. To expand initial findings to larger data sets examination of just 50 gene products. To
and account for interindividual biological supplement and extend initial microarray analyses
variability, a quantitative technology like RTAQ- for the identification of toxicological signatures,
RT-PCR can be utilised to confirm the results of the development of RTAQ-RT-PCR discrimination
microarray investigations in a high throughput and assay sets may thus be a suitable second-tier
quantitative manner. The ability to create molecular analysis.
signatures for classes of toxicants that are defined
Figure 6: Toxicogenomic
APPLICATIONS OF MULTIPLEX PCR : other mutation-amplifying multiplexes use a

 Gene deletion and mutation detection:By post-PCR modification of the reaction product.
using PCR or a subsequent study of PCR Breast cancer-related mutations in the human
products, multiplex assays can directly detect p53 tumor suppressor gene are found using
mutations and minor deletions in genes. For SSCP. Cycle sequencing of mice as oncogenes
RB1 gene exons amplified to reveal minor reveals activating mutations.Multiplex systems
deletions causing retinoblastoma and for have been developed to identify the numerous,
multiplex ARMS responses of frequent frequently population-specific mutations in the
population-specific beta-thalassemia and cystic cystic fibrosis transmembrane conductance
fibrosis mutations, results can be obtained right regulator gene. These multiplex systems
away by gel electrophoresis. As in a multiplex include allele-specific primers, restriction
reaction that recognizes a point mutation, a 4- enzyme digestion of naturally occurring
base deletion, and a full deletion of the alpha- restriction sites at amplified mutations, and
globin genes, numerous mutation types may be hybridization of exons to mutation-specific
investigated simultaneously. For diagnosis, oligonucleotides.
Figure 7: Gene deletion and mutation.
 Polymorphic Repetitive DNA: assessing any fungal load were effectively

For mapping, illness linkage, gender separated from each other. Both have previously
determination, and DNA typing/identification, been found to be virulent to Anopheles mosquitoes
repetitive DNA polymorphisms are multiplexed. using three different qPCR assays. Quantification
Because they are plentiful, highly polymorphic, of co-infecting malarial parasites was made
and may be coamplified without overlapping size possible by a fourth assay to Plasmodium chabaudi.
ranges, short tandem repeats (STRs) of 1-6 bp are Every qPCR assay offers accurate, reliable
useful for multiplexing. While chromosomally quantification with a linear range higher than five
unlinked repetitions are used to identify orders of magnitude (seven orders of magnitude for
individuals, multiplexes of relatively near repeats the fungal assays). The B. bassiana-specific test
are used to link diseases. Multiplex tests for was used to track B. bassiana growth in mosquitoes
detecting gender incorporate repetitive DNA exposed to three distinct conidial challenge
regions. By co-amplifying a Y-specific repetitive dosages. This is the first description of
DNA locus with a gene sequence on both the X and entomopathogenic fungus replication in an insect
Y chromosomes, embryos from families with an X- host. This showed that, regardless of the challenge
linked disease can be sexed. Subsequent analyses dose, a sudden on-set of considerable nuclear
have verified the multiplex results. In a multiplex division occurs after many days of relatively
for forensic samples, the Y-specific STR is modest replication, followed by physical fungal
amplified with an X-specific STR. growth (hyphae) within the mosquito haemocoel
just before death. At each time point studied,
 Applications of PCR in malaria: increasing conidia exposure led to both a
A series of procedures for employing noticeably greater mosquito pickup and higher
quantitative real-time PCR to track the in vivo fungal loads. With increasing challenge dose, high
kinetics of entomopathogenic fungi in Anopheles fungal loads, comparable to those found in
in the presence or absence of malaria parasites. cadavers, were obtained more quickly and
Two well-known fungal entomopathogens, mortalities occurred earlier post-exposure. The
Beauveria bassiana isolate IMI391510 and qPCR assays discussed here will enable new study
Metarhizium anisopliae var. Acridumisolate areas that will help to improve fungal biopesticides
IMI330189 and a "generic" fungal test for against malaria and other vector-borne diseases.
Figure 8: PCR in Malaria
 The Applications of PCR to the polymorphisms within a species. The molecular

Diagnosis of Primary Cardiac lymphoma : phylogenies that presently dominate in
An aggravated stomach ulcer in a 75-year- categorization were first derived from comparisons
old man who had first complained of pollakiuria of the sequences of the same genes in various
and low abdominal pain caused significant species and from analyses of whole genomes.In the
bleeding before he passed away. Primary cardiac field of paleogenetics, where DNA sequences of
lymphoma was identified postmortem. Only the more or less ancient organisms are recovered and
epicardium and myocardium were affected by the analyzed, PCR has thus taken the lead. This is true
tumour, which was classified as a primary cardiac for remains preserved in museum collections as
lymphoma by the Armed Forces Institute of well as for historical sites where extinct organisms'
Pathology. Large cells made up the lymphoma, skeletal or mummified remains have been present
which also expressed CDZO, a B cell marker. for hundreds of thousands or even hundreds of
Polymerase chain reaction (PCR) was used to thousands of years. Thus, the applications of PCR
detect monoclonality at the DNA level in swiftly expanded to include other sectors or
lymphoma cells, which were demonstrated to specialties and were no longer restricted to the
comprise a monoclonal population. This was done study of biology.
despite the fact that chronic inflammation caused
by chronic renal failure was seen in the pericardium ACKNOWLEDGEMENT:
around the lymphoma. We would like to thank Dr. A. Meena, Principal,
and Dr. A. Shanthi, Vice Principal, K. K. College
IV. CONCLUSION: of Pharmacy for motivating us with our review
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International Journal of Pharmaceutical Research and Applications

Volume 8, Issue 3 May-June 2023, pp: 3502-3513 www.ijprajournal.com ISSN: 2249-7781

A Review of PCR, Principle, and Its Applications

biotechnology. Applications of qPCR in efficient whole genome amplification (WGA)

Figure 1: Step 1- Denaturation

Figure 2: Step 2- Annealing

Figure 3: Step 3- Extension

Figure 4- Steps in PCR

 Archaeology and paleontology:  Ancient DNA studies:

fossils and preserved remains, scientists can APPLICATIONS OF PCR IN

Figure 5: Exposure monitoring

 Molecular Toxicology: development than other expression analysis tools,

 Toxicogenomic: by changes in certain gene sets is one of the goals

APPLICATIONS OF MULTIPLEX PCR : other mutation-amplifying multiplexes use a

Figure 7: Gene deletion and mutation.

 Polymorphic Repetitive DNA: assessing any fungal load were effectively

Figure 8: PCR in Malaria

 The Applications of PCR to the polymorphisms within a species. The molecular

among suspected patients in Sudan. J Glob amplification of a nucleoprotein gene

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