·
PCR is a very simple process.
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All that happens in PCR is a short region of DNA
molecule, let’s take for example a single gene, and is copied many times by DNA
polymerase enzyme.
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The PCR has a variety of applications in
genetics, research and in broader areas of biology.
An outline of polymerase chain reaction:
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It results in selective amplification of chosen
reaction of the DNA molecule.
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For amplification, a region of DNA is chosen
(whose sequences in border regions are known)
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The border sequences of the DNA fragment to be
amplified must be known, because in order to carry PCR, two short
oligonucleotides must hybridize to the DNA molecule, one to each strand of
double helix.
·
These oligonucleotides are used as DNA primers
for DNA synthesis reaction.
·
Amplification is usually carried out by DNA polymerase
I enzymes derived from thermus aquaticus (this bacteria
inhabits the hot streams)
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The polymerase is named taq polymerase
after the name of bacteria from which it is derived.
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Taq polymerase is
thermostable and can withstand temperature up to 96 ⁰ C.
·
The PCR is a very sensitive technique; it can
even start from a single target molecule.
·
The size of DNA that can be amplified by this
technique is 10 to 40 Kb.
Components required to carry out polymerase chain reaction (PCR):
·
DNA template (with known end sequence)
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Primers (
the primers complementary to the known sequence of target DNA molecule,
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taq
DNA polymerase (isolated from bacteria thermus aquaticus) living
in hot springs. It can withstand temperature up to 96 ⁰ C),
·
fixed
buffer( to maintain favourable environment during the PCR),
·
Divalent
cations (Mg2+ is used in general. Mn2+ can be also used,
but at higher concentration it causes mutation),
·
monovalent cations (K+ is used in
general)
·
Large number of DNA nucleotides
Procedures of PCR:
·
The PCR consists of 20 to 40 thermal cycles.
·
Each cycles has discrete steps
1.
Hold: the cycle starts with a temperature of 96 ⁰
C. the hold lasts for a brief period.
2.
Initializing step: the temperature is raised
further to 94 to 96 ⁰ C. (if the DNA polymerase to be used is highly
thermostable then the temperature can be raised up to 98 ⁰
C.)
This step lasts up to 1 to 6 minutes.
3.
Denaturation step: at the 94⁰ C
to 96 ⁰
C the tubes containing the target DNA is placed in the machine.
At this temperature the DNA melts—the strands get separated by breading
of hydrogen bonds. At the end of the denaturation step the result is the 2 separated
ssDNA molecules.
4.
Annealing step: in this step the temperature is
decreased down to 50 to 60 ⁰ C and primers are added and are
carried out for 20 to 40 seconds.
Also the DNA polymerase is added too. The primers pair with the
complementary sequences on target DNA molecules.
Note: annealing temperature should always be 2 to 3 ⁰ C
less than melting temperature of primers, to prevent the primers from melting
down.
At this temperature H-bonds are formed and DNA polymerase binds to end of
primers.
5.
Elongation: at this step the temperature is
raised to 70 to 75 ⁰ C.
At this temperature taq DNA polymerase acts at its best (best
temperature is 72 ⁰ C)
6.
Final elongation: at this step the temperature
is maintained from 70 to 74 ⁰ C for 5 to 15 minutes after last cycle
of PCR, to ensure that all last DNA are fully extended.
