POLYMERASE CHAIN REACTION: PRINCIPLES, METHODS AND APPLICATION

INTRODUCTION
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. Polymerase Chain Reaction was developed in
1984 by the American biochemist, Kary Mullis. Mullis received the Nobel Prize and the Japan
Prize for developing PCR in 1993. PCR is now a common and often indispensable technique used
in medical and biological research labs for a variety of applications. The polymerase chain reaction
is a powerful technique that has rapidly become one of the most widely used techniques in
molecular biology because it is quick, inexpensive and simple. The technique amplifies specific
DNA fragments from minute quantities of source DNA material, even when that source DNA is of
relatively poor quality. PCR; the quick, easy method for generating unlimited copies of any
fragment of DNA, is one of those scientific developments that actually deserve timeworn
superlatives like "revolutionary" and "breakthrough. From the daily practicalities of medical
diagnosis to the theoretical framework of systematics, from courts of law to field studies of animal
behavior, PCR takes analysis of tiny amounts of genetic material-even damaged genetic material
to a new level of precision and reliability. Furthermore, many important contributions to the
development and application of PCR technology have been made.
Basic concept of PCR
The basic PCR principle is simple. As the name implies, it is a chain reaction: One DNA molecule
is used to produce two copies, then four, then eight and so forth. This continuous doubling is
accomplished by specific proteins known as polymerases, enzymes that are able to string together
individual DNA building blocks to form long molecular strands.
To do their job polymerases require a supply of DNA building blocks, i.e. the nucleotides
consisting of the four bases adenine (A), thymine (T), cytosine (C) and guanine (G). They also
need a small fragment of DNA, known as the primer, to which they attach the building blocks
as well as a longer DNA molecule to serve as a template for constructing the new strand. If
these three ingredients are supplied, the enzymes will construct exact copies of the templates.
PCR is a method used to acquire many copies of any particular strand of nucleic acids. It’s a means
of selectively amplifying a particular segment of DNA. The segment may represent a small part of
a large and complex mixture of DNAs e.g. a specific exon of a human gene. It can be thought of
as a molecular photocopier. PCR can amplify a usable amount of DNA (visible by gel
electrophoresis) in ~2 hours. The template DNA need not be highly purified — a boiled bacterial
colony. The PCR product can be digested with restriction enzymes, sequenced or cloned.
The polymerase chain reaction relies on the ability of DNA copying enzymes to remain stable at
high temperatures. PCR has transformed the way that almost all studies requiring the manipulation
of DNA fragments may be performed as a result of its simplicity and usefulness.

Steps in PCR

There are three major steps involved in the PCR technique: denaturation, annealing, and
extension.

In step one; the DNA is denatured at high temperatures (from 90 - 97 degrees Celsius).

In step two, primers anneal to the DNA template strands to prime extension.

In step three, extension occurs at the end of the annealed primers to create a complementary copy
strand of DNA. This effectively doubles the DNA quantity through the third steps in the PCR cycle.

Step 1: To amplify a segment of DNA using PCR, the sample is first heated so the DNA denatures,
or separates into two pieces of single-stranded DNA. Next, an enzyme called "Taq polymerase"
synthesizes - builds - two new strands of DNA, using the original strands as templates. This process
results in the duplication of the original DNA, with each of the new molecules containing one old
and one new strand of DNA. Then each of these strands can be used to create two new copies, and
so on, and so on.

Step 2: The annealing phase happens at a lower temperature, 50-60°C. This allows the primers to
hybridize to their respective complementary template strands, a very useful tool to forensic
chemistry. The newly-formed DNA strand of primer attached to template is then used to create
identical copies off the original template strands desired. Taq polymerase adds available
nucleotides to the end of the annealed primers.
Step 3: The extension of the primers by Taq polymerase occurs at approx 72°C for 2-5 minutes.
DNA polymerase I cannot be used to elongate the primers as one would expect because it is not
stable at the high temperatures required for PCR.

The beauty of the PCR cycle and process is that it is very fast compared to other techniques and
each cycle doubles the number of copies of the desired DNA strand. After 25-30 cycles, whoever
is performing the PCR process on a sample of DNA will have plenty of copies of the original DNA
sample to conduct experimentation. Assuming the maximum amount of time for each step, 30
cycles would only take 6 hours to complete.

N.B: The entire cycling process of PCR is automated and can be completed in just a few hours. It
is directed by a machine called a thermocycler, which is programmed to alter the temperature of
the reaction every few minutes to allow DNA denaturing and synthesis.

Fig 1: A thermocycler
As the process of denaturation, annealing, and polymerase extension is continued the primers
repeatedly bind to both the original DNA template and complementary sites in the newly
synthesized strands and are extended to produce new copies of DNA. The end result is an
exponential increase in the total number of DNA fragments that include the sequences between the
PCR primers, which are finally represented at a theoretical abundance of 2n, where n, is the number
of cycles.

The Taq DNA polymerase is thermostable, at the beginning of the PCR reaction. The thermostable
properties of the DNA polymerase activity were isolated from Thermus aquaticus (Taq) that grow
in geysers of over 110°C, and have contributed greatly to the yield, specificity, automation, and
utility of the polymerase chain reaction. The Taq enzyme can withstand repeated heating to 94°C
and so each time the mixture is cooled to allow the oligonucleotide primers to bind the catalyst for
the extension is already present. After the last cycle, samples are usually incubated at 72°C 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
sequence

METHODS

In molecular biology, real-time polymerase chain reaction, also called quantitative real time
polymerase chain reaction is a laboratory technique based on the PCR, which is used to amplify
and simultaneously quantify a targeted DNA molecule. Traditionally, PCR is performed in a tube
and when the reaction is complete the products of the reaction (the amplified DNA fragments) are
analyzed and visualized by gel electrophoresis.

However, Real-Time PCR permits the analysis of the products while the reaction is actually in
progress. This is achieved by using various fluorescent dyes which react with the amplified product
and can be measured by an instrument.
This also facilitates the quantitation of the DNA.Quantitative PCR (Q-PCR), as this technique is
known, is used to measure the quantity of a PCR product (usually in a real-time PCR procedure).

It is the method of choice to quantitatively measure starting amounts of DNA, cDNA or RNA.
PCR is therefore often used to determine whether a DNA sequence is present in a sample and the
number of its copies in the sample. Another advantage of Real-Time PCR is rapidity of the assay,
since it is not necessary to perform electrophoresis or other procedure after the DNA amplification
reaction.

Digital PCR concept was conceived in 1992 by Sykes et al. It is a refinement of conventional
polymerase chain reaction methods that can be used to directly quantify and clonally amplify
nucleic acids including DNA, cDNA or RNA. The key difference between dPCR and traditional
PCR lies in the method of measuring nucleic acids amounts, with the former being a more precise
method than PCR. PCR carries out one reaction per single sample. dPCR also carries out a single
reaction within a sample, however the sample is separated into a large number of partitions and
the reaction is carried out in each partition individually. This separation allows a more reliable
collection and sensitive measurement of nucleic acid amounts.

Inverse PCR is a variant of the polymerase chain reaction that is used to amplify DNA with only
one known sequence. One limitation of conventional PCR is that it requires primers
complementary to both termini of the target DNA, but this method allows PCR to be carried out
even if only one sequence is available from which primers may be designed.

Nested polymerase chain reaction is a modification of polymerase chain reaction intended to

Touchdown polymerase chain reaction is a method of polymerase chain reaction by which

PCR is helping in the investigation and diagnosis of a growing number of diseases. It has also long
been a standard method in all laboratories that carry out research on or with nucleic acids. Even
competing techniques such as DNA chips often require amplification of DNA by means of PCR
as an essential preliminary step.

The polymerase chain reaction is used by a wide spectrum of scientists in an ever-increasing range
of scientific disciplines. The use of reverse transcriptase’s to evaluate RNA levels and the extension
of PCR technology to quantify DNA amplification in real time has brought major advances to the
application of PCR.

By allowing the determination and quantification of changes in gene expression, these techniques
have provided a greater understanding of disease processes and now serve as a foundation for
diagnostics and basic science research.

In microbiology and molecular biology, for example, PCR is used in research laboratories in DNA
cloning procedures, Southern blotting, DNA sequencing, recombinant DNA technology, to name
but a few. In clinical microbiology laboratories PCR is invaluable for the diagnosis of microbial
infections and epidemiological studies.

PCR is also used in forensics laboratories and is especially useful because only a tiny amount of
original DNA is required, for example, sufficient DNA can be obtained from a droplet of blood or
a single hair.Fortunately, the PCR method can detect the DNA of microorganisms in any sample,
whether of body fluids, foodstuffs or drinking water.

RT-PCR is commonly used in studying the genomes of viruses whose genomes are composed of
RNA, such as Influenza virus A and retroviruses like HIV.

Quantitative PCR provides additional information beyond mere detection of DNA. It indicates not
just whether a specific DNA segment is present in a sample, but also how much of it is there.
Another important application of quantitative PCR is in molecular diagnosis, i.e. the diagnosis of
diseases based on molecular findings rather than on physiological symptoms. In this connection
the diagnosis of viral diseases is an area that is gaining increasing importance.
PCR is the most sensitive test for herpes simplex virus, varicella-zoster virus, and human
papillomavirus infections. Other diagnostic uses, including tests for genetic diseases, cancers, and
other infectious diseases, are evolving.

Another important application in which quantitative PCR is used in the field of infectious diseases
is AIDS. It can detect the AIDS virus sooner during the first few weeks after infection than the
standard ELISA test.

Digital PCR has many potential applications, including the detection and quantification of low-
level pathogens, rare genetic sequences, copy number variations, and relative gene expression in
single cells. Clonal amplification enabled by single-step digital PCR is a key factor in reducing the
time and cost of many of the "next generation sequencing" methods and hence enabling personal
genomics.

Inverse PCR is especially useful for the determination of insert locations. For example, various
retroviruses and transposons randomly integrate into genomic DNA. To identify the sites where
they have entered, the known, "internal" viral or transposon sequences can be used to design
primers that will amplify a small portion of the flanking, "external" genomic DNA.

Nested PCR is one of these protocols for detection of only a few bacteria in clinical specimens.
Nested polymerase chain reaction is a key part of many genetics research laboratories, along with
uses in DNA fingerprinting for forensics and other human genetic cases.

PCR can identify genes that have been implicated in the development of cancer. There are
numerous applications for real-time polymerase chain reaction in the laboratory. It is commonly
used for both basic research and is deployed as a tool to detect newly emerging diseases, such as
flu, in diagnostic tests. PCR can be used for the diagnosis of Indian visceral leishmaniasis with
great accuracy. PCR can also be employed with significant precision to predict cure of the disease.
CONCLUSION

PCR and its applications hold scientific and medical promise. PCR has very quickly become an
essential tool for improving human health and human life. PCR has completely revolutionized the
detection of RNA and DNA viruses. PCR is valuable as a confirmatory test.

PCR is a rapid technique with high sensitivity and specificity. PCR has also been credited to have
been able to detect mixed infections with ease in many studies. PCR, a more sophisticated
technique, requires infrastructural support, is expensive but nevertheless, one cannot discount its
utilitarian advantages which are many compared to the existing conventional diagnostic methods.