POLYMERASE CHAIN REACTION
Introduction
The polymerase chain reaction (PCR) is a laboratory technique for DNA replication
that allows a “target” DNA sequence to be selectively amplified. PCR can use the smallest
sample of the DNA to be cloned and amplify it to millions of copies in just a few hours.
Discovered in 1985 by Kerry Mullis, PCR has become both and essential and routine tool
in most biological laboratories.
Principle
The PCR involves the primer mediated enzymatic amplification of DNA. PCR is
based on using the ability of DNA polymerase to synthesize new strand of DNA
complementary to the offered template strand. Primer is needed because DNA polymerase
can add a nucleotide only onto a preexisting 3′-OH group to add the first nucleotide. DNA
polymerase then elongate its 3 end by adding more nucleotides to generate an extended
region of double stranded DNA.
Components of PCR
1. DNA Template: The double stranded DNA (dsDNA) of interest, separated from the
sample.
2. DNA Polymerase: Usually a thermo stable Taq polymerase that does not rapidly
denature at high temperatures (98°), and can function at a temperature optimum of
about 70°C.
3. Oligonucleotide primers: Short pieces of single stranded DNA (often 20-30 base
pairs) which are complementary to the 3’ ends of the sense and anti-sense strands of
the target sequence.
4. Deoxynucleotide triphosphates: Single units of the bases A, T, G, and C (dATP,
dTTP, dGTP, dCTP) provide the energy for polymerization and the building blocks
for DNA synthesis.
5. Buffer system: Includes magnesium and potassium to provide the optimal conditions
for DNA denaturation and renaturation; also important for polymerase activity,
stability and fidelity.
Working Procedure
All the PCR components are mixed together and are taken through series of 3 major
cyclic reactions conducted in an automated, self-contained thermocycler machine.
1. Denaturation: This step involves heating the reaction mixture to 94°C for 15-30
seconds. During this, the double stranded DNA is denatured to single strands due to
breakage in weak hydrogen bonds.
2. Annealing: The reaction temperature is rapidly lowered to 54-60°C for 20-40
seconds. This allows the primers to bind (anneal) to their complementary sequence in the
template DNA.
3. Elongation: Also known at extension, this step usually occurs at 72-80°C (most
commonly 72°C). In this step, the polymerase enzyme sequentially adds bases to the 3′
each primer, extending the DNA sequence in the 5′ to 3′ direction. Under optimal
conditions, DNA polymerase will add about 1,000bp/minute
Applications
Some common applications of PCR in various fields can be explained in following
categories. Medical Applications:
1. Genetic testing for presence of genetic disease mutations. Eg: hemoglobinopathies,
cystic fibrosis, other inborn errors of metabolism
2. Detection of disease causing genes in suspected parents who act as carriers.
3. Study of alteration to oncogenes may help in customization of therapy
4. Can also be used as part of a sensitive test for tissue typing, vital to organ transplantation
genotyping of embryo
5. Helps to monitor the gene in gene therapy
Infectious disease Applications
1. Analyzing clinical specimens for the presence of infectious agents, including HIV,
hepatitis, malaria, tuberculosis etc.
2. Detection of new virulent subtypes of organism that is responsible for epidemics.
Forensic Applications
1. Can be used as a tool in genetic fingerprinting
2. This technology can identify any one person from millions of others in case of crime
scene, rule out suspects during police investigation, paternity testing even in case of
availability of very small amount of specimens (stains of blood, semen, hair etc)
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