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Polymerase Chain

Reaction
(PCR)
PCR
The polymerase chain reaction (PCR) is one of
the most important and powerful technique, that
is being applied in virtually all fields of modern
molecular biology.
PCR is a method for thein vitroexponential
amplification of a specific DNA region
(calledtarget region), that lies between two
regions (calledprimers) of known DNA
sequence, resulting in a large quantity of DNA
(this means micrograms of DNA).
Most organisms copy their DNA in the same
way: DNA polymerases carry out the synthesis of
a complementary strand of DNA in the 5' to 3'
The PCR mimics this process, only it does it in a
test tube and employs two primers, each
complementary to opposite strands of the region
of DNA.
The PCR amplification occurs by repeated cycles
of three temperature dependent steps:-
(1)Denaturation
(2)Annealing(1)Denaturation
(3)Elongation.
(2)Annealing
(3)Elongation
Reagents
All components of a polymerase chain reaction
are readily available from commercial suppliers. A
PCR test tube should contain:-
- DNA polymerase.
- Reaction buffer with magnesium chloride.
- Template (a piece of DNA).
- Large quantities of the four nucleotides (dNTPs).
- Large quantities of the primers.
Detection and analysis of
PCR products
The product of a PCR should be a fragment or
fragments of DNA of defined length.
Many techniques can be used to detect
amplified sequences.
Detection Visualization
-EtBr staining (UV
transilluminator, image
analyzer)-Southern blotting
Agarose gel and/or
(hybridization with labeled
polyacrylamidegel
probe)
electrophoresis
-Incorporation of label into
amplifying sequence
-Silver staining
Restriction -Agarose or
endonucleasedigestion polyacrylamide gel-HPLC
Hybridization with labeled
Dot blots
probe
High-pressure liquid
UV detection
chromatography
Voltage-initiated chemical
Electrochemiluminescence
reaction/photon detection
Radioactive or fluoescent-
Direct sequencing
based DNA sequencing
The simplest and commonly used technique is
electrophoresis of the PCR product on an agarose
gel with EtBr (ethidium bromide).
EtBr is a fluorescent dye that intercalates into
the DNA. Size markers can be electrophoresed on
the gel to allow size determination of the PCR
An example of detection of PCR products on the
agarose gel with EtBr is shown in Figure below.
Applications
The PCR can be applied in virtually all fields of
modern molecular biology:-
(1)In the introducing of restriction site
sequencesin.
(2)The construction of cDNA libraries.
(3)In the isolation and construction of DNA
clonesin. (4)Modifications of DNA sequences.
(5)In mutagenesis and analysis of known
mutationsin sequencingin.
(6)Detecting of pathogens (viruses, bacteria).
The most important applications, in structural
molecular biology, are:-
(1)The sequencing of DNA, which allows the
determining of the primary structure of proteins.
(2)The introducing of mutations, which allows
substitution of amino acids and searching for
changes of the structure and activity of the
macromolecules.