Polymerase chain reaction (PCR) is a technique used to exponentially amplify a specific target

DNA sequence, allowing for the isolation, sequencing, or cloning of a single sequence among
many. PCR was developed in 1983 by Kary Mullis, who received a Nobel Prize in chemistry in
1993 for his invention. The polymerase chain reaction has been elaborated in many ways since its
introduction and is now commonly used for a wide variety of applications including genotyping,
cloning, mutation detection, sequencing, microarrays, forensics, and paternity testing.

PCR is based on three simple steps required for any DNA synthesis reaction: (1) denaturation of the
template into single strands; (2) annealing of primers to each original strand for new strand
synthesis; and (3) extension of the new DNA strands from the primers.

The sample containing a dilute concentration of template DNA is mixed with a heat-stable DNA
polymerase, such as Taq polymerase, primers, deoxynucleoside triphosphates (dNTPs), and
magnesium. In the first step of PCR, the sample is heated to 95–98°C, which denatures the double-
stranded DNA, splitting it into two single strands. In the second step, the temperature is decreased
to approximately 55–65°C, allowing the primers to bind, or anneal, to specific sequences of DNA at
each end of the target sequence, also known as the template. In the third step, the temperature is
typically increased to 72°C, allowing the DNA polymerase to extend the primers by the addition of
dNTPs to create a new strand of DNA, thus doubling the quantity of DNA in the reaction. This
sequence of denaturation, annealing, and extension is repeated for many cycles, resulting in the
exponential amplification of the template DNA.

The polymerase chain reaction (PCR): is essentially DNA replication in vitro. The PCR can
copy segments of DNA by up to a billion fold in the test tube, a process called amplification.

(In vitro A test performed in vitro ("in the glass") means that it is done outside of a living organism
and it usually involves isolated tissues, organs or cells)

(In vivo refers to when research or work is done with or within an entire, living organism.
Examples can include studies in animal models or human clinical trials).

A template is defined as a single-stranded nucleic acid a DNA or RNA polymer made of

A nucleotide is the basic building block of nucleic acids (RNA and DNA). A nucleotide consists of
a sugar molecule (either ribose in RNA or deoxyribose in DNA) attached to a phosphate group and
a nitrogen-containing base. The bases used in DNA are adenine (A), cytosine (C), guanine (G) and
thymine (T). The bases used in RNA adenine (A), cytosine (C), uracil (U), and guanine (G). Uracil
is a pyrimidine that is structurally similar to the thymine, another pyrimidine that is found in DNA.

A nucleoside consists of a nitrogenous base covalently attached to a sugar (ribose or deoxyribose)

The original PCR technique employed the DNA polymerase Escherichia coli Pol III, but because of
the high temperatures needed to denature the double-stranded copies of DNA, the enzyme was also
denatured and had to be replenished every cycle. This problem was solved by employing a
thermostable DNA polymerase isolated from the thermophilic hot spring bacterium Thermus
aquaticus. DNA polymerase from T. aquaticus, called Taq polymerase, is stable to 95 °C and thus
is unaffected by the denaturation step employed in the PCR. The use of Taq DNA polymerase also
increased the specificity of the PCR because the DNA is copied at 72 °C rather than 37 °C.

Applications and Uses of PCR Technique:

PCR is a powerful tool. It is easy to perform, extremely sensitive and specific, and highly efficient.
During each round of amplification the amount of product doubles, leading to an exponential
increase in the DNA. This means not only that a large amount of amplified DNA can be produced
in just a few hours, but that only a few molecules of target DNA need be present in the sample to
start the reaction.

1. PCR is extremely valuable for obtaining DNA for cloning genes or for sequencing purposes
because the gene or genes of interest can easily be amplified if flanking sequences are
known.
2. PCR is used routinely in comparative or phylogenetic studies to amplify genes from various
sources. It also has been used in forensics to identify human individuals from very small
samples of their DNA.
 3. PCR is a highly accurate and rapid method for duplicating genetic material. The
discovery of thermostable polymerase enzymes has permitted the automation of PCR,
thus reducing the manpower required to conduct these experiments.
4. 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.
5. Polymerase chain reaction may also help to identify a potential role for viruses or
other microbial pathogens in neurologic and psychiatric diseases of uncertain causes,
including schizophrenia, multiple sclerosis, Alzheimer disease, and other
neurodegenerative diseases.
6. The PCR technique is in widespread use in microbial ecology and has revealed the
enormous diversity of the microbial world, much of it not yet cultured.
7. PCR can be used to amplify very small quantities of DNA. For example, PCR has been
used to amplify and clone DNA from sources as varied as mummified human remains and
fossilized plants and animals.
8. The ability of PCR to amplify and analyze DNA from cell mixtures has also made it a
common tool of diagnostic microbiology. For example, if a clinical sample shows evidence
of a gene specific to a particular pathogen, then it can be assumed that the pathogen was
present in the sample. Treatment of the patient can then begin without the need to culture
the organism, a time-consuming and often fruitless process.

What is the difference between nucleotide and nucleoside in DNA and RNA?

The main difference lies in their molecular composition as Nucleosides contain only sugar and a
base whereas Nucleotides contain sugar, base and a phosphate group as well. A nucleotide is what
occurs before RNA and DNA, while the nucleoside occurs before the nucleotide itself.