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Polymerase Chain Reaction: Methods, Principles and Application
Polymerase Chain Reaction: Methods, Principles and Application
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.
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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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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