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International Journal of Biomedical Research

POLYMERASE CHAIN REACTION: METHODS, PRINCIPLES AND


APPLICATION
Dr.Mohini Joshi1*, Dr.Deshpande J.D2.
1
Department of Anatomy, Rural Medical College, Pravara Institute of Medical Sciences,
Loni, Maharashtra, India
2
Department of Community Medicine,Rural Medical College, Pravara Institute of Medical
Sciences, Loni, Maharashtra, India

Corresponding author*: atharvamohini@gmail.com


This article is available online at www.ssjournals.com

ABSTRACT
The polymerase chain reaction (PCR) is a scientific technique in molecular biology to
amplify a single or a few copies of a piece of DNA across several orders of magnitude,
generating thousands to millions of copies of a particular DNA sequence. PCR is now a
common and often indispensable technique used in medical and biological research labs for
a variety of applications. There are three major steps involved in the PCR technique:
denaturation, annealing, and extension. PCR is useful in the investigation and diagnosis of a
growing number of diseases. Qualitative PCR can be used to detect not only human genes
but also genes of bacteria and viruses. PCR is also used in forensics laboratories and is
especially useful because only a tiny amount of original DNA is required. PCR can identify
genes that have been implicated in the development of cancer. Molecular cloning has
benefited from the emergence of PCR as a technique. The present paper is an attempt to
review basics of PCR.

KEY WORDS: PCR, Principles, Application

INTRODUCTION
The polymerase chain reaction (PCR) is a and polymerase purification issues.2 PCR
scientific technique in molecular biology is now a common and often indispensable
to amplify a single or a few copies of a technique used in medical and biological
piece of DNA across several orders of research labs for a variety of
magnitude, generating thousands to applications.3 The polymerase chain
millions of copies of a particular DNA reaction is a powerful technique that has
sequence. Polymerase Chain Reaction rapidly become one of the most widely
was developed in 1984 by the American used techniques in molecular biology
biochemist, Kary Mullis. Mullis received because it is quick, inexpensive and
the Nobel Prize and the Japan Prize for simple. The technique amplifies specific
developing PCR in 1993.1 However the DNA fragments from minute quantities of
basic principle of replicating a piece of source DNA material, even when that
DNA using two primers had already been source DNA is of relatively poor quality.4
described by Gobind Khorana in 1971. PCR; the quick, easy method for
Progress was limited by primer synthesis generating unlimited copies of any

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fragment of DNA, is one of those of as a molecular photocopier. PCR can


scientific developments that actually amplify a usable amount of DNA (visible
deserve timeworn superlatives like by gel electrophoresis) in ~2 hours. The
"revolutionary" and "breakthrough. From template DNA need not be highly purified
the daily practicalities of medical a boiled bacterial colony. The PCR
diagnosis to the theoretical framework of product can be digested with restriction
systematics, from courts of law to field enzymes, sequenced or cloned. PCR can
studies of animal behavior, PCR takes amplify a single DNA molecule, e.g. from
analysis of tiny amounts of genetic a single sperm. The polymerase chain
material-even damaged genetic material- reaction relies on the ability of DNA-
to a new level of precision and reliability. copying enzymes to remain stable at high
Furthermore, many important temperatures. PCR has transformed the
contributions to the development and way that almost all studies requiring the
application of PCR technology have been manipulation of DNA fragments may be
made; however the present paper is an performed as a result of its simplicity and
attempt to review basics of PCR. usefulness.5 In Mullis's original PCR
process, the enzyme was used in vitro.
Basic concept of PCR The double-stranded DNA was separated
into two single strands of DNA by heating
The basic PCR principle is simple. As the it to 96C. At this temperature, however,
name implies, it is a chain reaction: One the E.Coli DNA polymerase was
DNA molecule is used to produce two destroyed, so that the enzyme had to be
copies, then four, then eight and so forth. replenished with new fresh enzyme after
This continuous doubling is accomplished the heating stage of each cycle. Mullis's
by specific proteins known as original PCR process was very inefficient
polymerases, enzymes that are able to since it required a great deal of time, vast
string together individual DNA building amounts of DNA-Polymerase, and
blocks to form long molecular strands. To continual attention throughout the PCR
do their job polymerases require a supply process.6
of DNA building blocks, i.e. the
nucleotides consisting of the four bases Steps in PCR
adenine (A), thymine (T), cytosine (C)
and guanine (G). They also need a small There are three major steps involved in
fragment of DNA, known as the primer, the PCR technique: denaturation,
to which they attach the building blocks annealing, and extension. In step one; the
as well as a longer DNA molecule to DNA is denatured at high temperatures
serve as a template for constructing the (from 90 - 97 degrees Celsius). In step
new strand. If these three ingredients are two, primers anneal to the DNA template
supplied, the enzymes will construct exact strands to prime extension. In step three,
copies of the templates. PCR is a method extension occurs at the end of the
used to acquire many copies of any annealed primers to create a
particular strand of nucleic acids. Its a complementary copy strand of DNA. This
means of selectively amplifying a effectively doubles the DNA quantity
particular segment of DNA. The segment through the third steps in the PCR cycle.
may represent a small part of a large and To amplify a segment of DNA using PCR,
complex mixture of DNAs e.g. a specific the sample is first heated so the DNA
exon of a human gene. It can be thought denatures, or separates into two pieces of
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single-stranded DNA. Next, an enzyme conduct experimentation. Assuming the


called "Taq polymerase" synthesizes - maximum amount of time for each step,
builds - two new strands of DNA, using 30 cycles would only take 6 hours to
the original strands as templates. This complete.
process results in the duplication of the As the process of denaturation, annealing,
original DNA, with each of the new and polymerase extension is continued the
molecules containing one old and one new primers repeatedly bind to both the
strand of DNA. Then each of these strands original DNA template and
can be used to create two new copies, and complementary sites in the newly
so on, and so on.7 The annealing phase synthesized strands and are extended to
happens at a lower temperature, 50-60C. produce new copies of DNA. The end
This allows the primers to hybridize to result is an exponential increase in the
their respective complementary template total number of DNA fragments that
strands, a very useful tool to forensic include the sequences between the PCR
chemistry. The newly-formed DNA strand primers, which are finally represented at a
of primer attached to template is then used theoretical abundance of 2n, where n, is
to create identical copies off the original the number of cycles.5, 8
template strands desired. Taq polymerase Due to the introduction of a thermostable
adds available nucleotides to the end of DNA polymerase, the Taq DNA
the annealed primers. The extension of the polymerase once, at the beginning of the
primers by Taq polymerase occurs at PCR reaction.9 The thermostable
approx 72C for 2-5 minutes. DNA properties of the DNA polymerase
polymerase I cannot be used to elongate activity were isolated from Thermus
the primers as one would expect because aquaticus (Taq) that grow in geysers of
it is not stable at the high temperatures over 110C, and have contributed greatly
required for PCR. The beauty of the PCR to the yield, specificity, automation, and
cycle and process is that it is very fast utility of the polymerase chain reaction.
compared to other techniques and each The Taq enzyme can withstand repeated
cycle doubles the number of copies of the heating to 94C and so each time the
desired DNA strand. After 25-30 cycles, mixture is cooled to allow the
whoever is performing the PCR process oligonucleotide primers to bind the
on a sample of DNA will have plenty of catalyst for the extension is already
copies of the original DNA sample to present.10 After the last cycle, samples are
Y usually incubated at 72C for 5 minutes to
fill in the protruding ends of newly
synthesized PCR products. To ensure
success, care should be taken both in
preparing the reaction mixture and setting
up the cycling conditions. Increasing the
cycle number above ~35 has little positive
effect because the plateau occurs when the
reagents are depleted; accumulate. The
specificity of amplification depends on
the extent to which the primers can
recognize and bind to sequences other
than the intended target DNA sequences.
X

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METHODS sample, however the sample is separated


into a large number of partitions and the
In molecular biology, real-time reaction is carried out in each partition
polymerase chain reaction, also called individually. This separation allows a
quantitative real time polymerase chain more reliable collection and sensitive
reaction is a laboratory technique based measurement of nucleic acid amounts.14
on the PCR, which is used to amplify and Inverse PCR is a variant of the
simultaneously quantify a targeted DNA polymerase chain reaction that is used to
molecule. Traditionally, PCR is amplify DNA with only one known
performed in a tube and when the reaction sequence. One limitation of conventional
is complete the products of the reaction PCR is that it requires primers
(the amplified DNA fragments) are complementary to both termini of the
analyzed and visualized by gel target DNA, but this method allows PCR
electrophoresis. However, Real-Time to be carried out even if only one
PCR permits the analysis of the products sequence is available from which primers
while the reaction is actually in progress. may be designed.15 Nested polymerase
This is achieved by using various chain reaction is a modification of
fluorescent dyes which react with the polymerase chain reaction intended to
amplified product and can be measured by reduce the contamination in products due
an instrument. This also facilitates the to the amplification of unexpected primer
quantitation of the DNA. Quantitative binding sites.16 Touchdown polymerase
PCR (Q-PCR), as this technique is known, chain reaction is a method of polymerase
is used to measure the quantity of a PCR chain reaction by which primers will
product (usually in a real-time PCR avoid amplifying nonspecific sequences.
procedure). It is the method of choice to The earliest steps of a touchdown
quantitatively measure starting amounts of polymerase chain reaction cycle have high
DNA, cDNA or RNA. PCR is therefore annealing temperatures. The annealing
often used to determine whether a DNA temperature is decreased in increments for
sequence is present in a sample and the every subsequent set of cycles.17
number of its copies in the sample.
Another advantage of Real-Time PCR is Applications of PCR
rapidity of the assay, since it is not
necessary to perform electrophoresis or PCR is helping in the investigation and
other procedure after the DNA diagnosis of a growing number of
amplification reaction. 11, 12 Digital PCR diseases. It has also long been a standard
concept was conceived in 1992 by Sykes method in all laboratories that carry out
et al.13 It is a refinement of conventional research on or with nucleic acids. Even
polymerase chain reaction methods that competing techniques such as DNA chips
can be used to directly quantify and often require amplification of DNA by
clonally amplify nucleic acids including means of PCR as an essential preliminary
DNA, cDNA or RNA. The key difference step. The polymerase chain reaction is
between dPCR and traditional PCR lies in used by a wide spectrum of scientists in
the method of measuring nucleic acids an ever-increasing range of scientific
amounts, with the former being a more disciplines. The use of reverse
precise method than PCR. PCR carries out transcriptases to evaluate RNA levels and
one reaction per single sample. dPCR also the extension of PCR technology to
carries out a single reaction within a quantify DNA amplification in real time
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has brought major advances to the any sample, whether of body fluids,
application of PCR. By allowing the foodstuffs or drinking water. Quantitative
determination and quantification of PCR provides additional information
changes in gene expression, these beyond mere detection of DNA. It
techniques have provided a greater indicates not just whether a specific DNA
understanding of disease processes and segment is present in a sample, but also
now serve as a foundation for diagnostics how much of it is there. This information
and basic science research.18 In is required in a number of applications
microbiology and molecular biology, for ranging from medical diagnostic testing
example, PCR is used in research through target searches to basic research.
laboratories in DNA cloning procedures, Another important application of
Southern blotting, DNA sequencing, quantitative PCR is in molecular
recombinant DNA technology, to name diagnosis, i.e. the diagnosis of diseases
but a few. In clinical microbiology based on molecular findings rather than
laboratories PCR is invaluable for the on physiological symptoms. In this
diagnosis of microbial infections and connection the diagnosis of viral diseases
epidemiological studies. PCR is also used is an area that is gaining increasing
in forensics laboratories and is especially importance. PCR is the most sensitive test
useful because only a tiny amount of for herpes simplex virus, varicella-zoster
original DNA is required, for example, virus, and human papillomavirus
sufficient DNA can be obtained from a infections. Other diagnostic uses,
droplet of blood or a single hair. In fact, a including tests for genetic diseases,
number of trials using PCR for detection cancers, and other infectious diseases, are
of a broad range of bacteria in CSF evolving.23Another important application
specimens have been reported.19-21 Since in which quantitative PCR is used in the
the culture of C. pneumoniae is difficult in field of infectious diseases is AIDS. It can
most clinical laboratories, determination detect the AIDS virus sooner during the
of this bacterium in clinical specimens has first few weeks after infection than the
been widely performed using the PCR standard ELISA test.
technique even though there is no Genetic factors are always involved in the
standardized PCR method for detection of development of cancer. Their contribution
this organism. Nested PCR is one of these varies greatly depending on the type of
protocols for detection of only a few cancer. Genes not only help to determine
bacteria in clinical specimens.22 progression of the disease but can also
Qualitative PCR can of course be used to have a substantial influence on the
detect not only human genes but also effectiveness of the available treatments.
genes of bacteria and viruses. One of the Identifying the genes that play a role in
most important medical applications of the development of cancer is therefore an
the classical PCR method is therefore the important step towards improving
detection of pathogens. Many viruses treatment. Both qualitative and
contain RNA rather than DNA. In such quantitative PCR play a crucial role in the
cases the viral genome has to be fight against cancer. PCR can identify
transcribed before PCR is performed, and genes that have been implicated in the
RTPCR is therefore used. Sometimes it is development of cancer. There are
also necessary to detect pathogens outside numerous applications for real-time
the body. Fortunately, the PCR method polymerase chain reaction in the
can detect the DNA of microorganisms in laboratory. It is commonly used for both
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basic research and is deployed as a tool to endonuclease recognition sites added to


detect newly emerging diseases, such as their 5 ends. 25
flu, in diagnostic tests. Digital PCR has
many potential applications, including the CONCLUSION
detection and quantification of low-level
pathogens, rare genetic sequences, copy The advancement of science has
number variations, and relative gene transformed our lives in ways that would
expression in single cells. Clonal have been unpredictable just a half-
amplification enabled by single-step century ago. Molecular methods have
digital PCR is a key factor in reducing the shown a promise in this aspect. PCR and
time and cost of many of the "next- its applications hold scientific and
generation sequencing" methods and medical promise. PCR has very quickly
hence enabling personal genomics.14 become an essential tool for improving
Inverse PCR is especially useful for the human health and human life. PCR has
determination of insert locations. For completely revolutionized the detection of
example, various retroviruses and RNA and DNA viruses. PCR is valuable
transposons randomly integrate into as a confirmatory test. PCR is a rapid
genomic DNA. To identify the sites where technique with high sensitivity and
they have entered, the known, "internal" specificity. PCR has also been credited to
viral or transposon sequences can be used have been able to detect mixed infections
to design primers that will amplify a small with ease in many studies. PCR, a more
portion of the flanking, "external" sophisticated technique, requires
genomic DNA.15 Nested polymerase infrastructural support, is expensive but
chain reaction is a key part of many nevertheless, one cannot discount its
genetics research laboratories, along with utilitarian advantages which are many
uses in DNA fingerprinting for forensics compared to the existing conventional
and other human genetic cases. diagnostic methods.
Conventional PCR requires primers
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