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PCR-PRINCIPLE, PROCEDURE,
APPLICATIONS, NOTES
Home / Fundamentals / PCR-Principle, Procedure, Applications, Notes
In this article we will discuss about the basic fundamentals, such as principle,
procedure etc., of the Polymerase Chain Reaction, widely known as PCR.
The enzyme chain reaction (PCR) technique, developed by Kary Mullis in 1985, is
extremely powerful. It generates metric weight unit (Mg) quantities of
deoxyribonucleic acid copies (up to billion copies) of the required
deoxyribonucleic acid (or DNA) section, gift when one copy within the initial
preparation, during a matter of few hours. The PCR method has been utterly
automatic and compact thermal cyclers square measure obtainable within the
market.
The machine utilizes the following:
(1) a deoxyribonucleic acid preparation containing the required section to be
amplified,
(2) 2 oligonucleotide primers (about twenty bases long) specific, i.e.,
complementary, to the 2 3′-borders (the sequences gift at the 3′-ends of the 2
strands) of the required section,
:
(3) the four deoxynucleoside triphosphates, viz., TTP (thymidine triphosphate),
dCTP (deoxycyctidine triphosphate), dAP (deoxyadenosine triphosphate) and
dGTP (deoxyguanosine triphosphate), and (a) a heat stable DNA polymerase, e.g.
 2
Taq (isolated from the bacteria Thermus acquaticus), Pfu (from Pyrococcus
furious) and Vent (from Thermococcus litoralis) polymerases. Pfiu and Vent
polymerases square measure a lot of economical than the Tag enzyme. Taq
enzyme, lacks proof-reading ability (3′ -› 5′ nuclease activity); as a result, it
commits errors at a high rate (2 × 10-*). however Vent and Pfu polymerases have
proof-reading ability, and that they commit errors at a lot of lower rates of 4 x 10S and 7 x 10, correspondingly.
Procedure of PCR
At the beginning , the deoxyribonucleic acid from that a section is to be amplified,
an way over the 2 primer molecules, the four deoxyriboside triphosphates and
also the DNA polymerase enzyme square measure mixed together within the
reaction mixture that has acceptable quantities of Mg+2. the subsequent
operations square measure currently performed consecutive. (Fig 1)
1. Denaturation
The reaction mixture is initial heated to a temperature between 90-98°C
(commonly 94°C) that ensures deoxyribonucleic acid denaturation. this is often
the denaturation step. The length of this step within the initial cycle of PCR is
sometimes a pair of min at 94°C, however it’s just one min within the future
cycles.
2. Annealing
:
The mixture is currently cooled to a temperature (generally, between 40-60°C)
that allows annealing of the primer to the complementary sequences within the
deoxyribonucleic acid. In fact, the tempering temperature depends on the length
and also the base sequence of the primers being employed for amplification. As a
rule, the sequences to that the primers toughen square measure settled at the 3′ends of the 2 strands of the section to be amplified. This step is termed
tempering. The length of tempering step is sometimes I min throughout the
primary likewise because the future cycles of PCR.
Since the primer concentration is unbroken terribly high relative to it of the
templet deoxyribonucleic acid, primer template hybrid formation is greatly
favored over reannealing of the templet strands.
3. Primer Extension
The temperature is currently therefore adjusted that the deoxyribonucleic acid
enzyme synthesizes the complementary strands by utilizing the 3′-OH of the
primers; this reaction is that the same as that happens in vivo during replication
of the leading strand of a deoxyribonucleic acid duplex. The primers square
measure extended towards each other in order that the deoxyribonucleic acid
section lying between the 2 primers is copied; this is often ensured by using
primers complementary to the 3′-ends of the section to be amplified. The
duration of primer extension is sometimes a pair of min at 72°C. Tag enzyme
typically amplifies DNA fragments of up to a pair of kb; special reaction
conditions square measure necessary for the amplification of longer segments.
:
The completion of the extension step completes the primary cycle of
amplification; every cycle might take few (ordinarily 4-5) minutes. It ought to be
noted that extension of the primer continues until the strands square measure
separated throughout the denaturation step of successive laptop cycle.
Therefore, the product of this extension step, and so of each cycle supported the
first template deoxyribonucleic acid. is of indefinite length; this PCR product is
sometimes known as the long product.
( Image.1 )
Image Source: B.D Singh book
:
(Fig. 1). At the second cycle of PCR, primers can toughen to the long product’ a
lot of before its 3′-end, wherever sequences complementary to them square
measure settled. Extension step during this cycle can turn out a product that may
be a lot of shorter than the long product’; this is often the ‘correct’ PCR product
and represents the target sequence (Fig. 2). the first templet sequence also will
be traced throughout the second and every one future cycles to get the long
product’. Therefore, the long product continues to extend linearly, whereas the
‘correct PC’ product can multiply exponentially.
The next cycle of amplification is initiated by denaturation (Step 1), that separates
the newly synthesized deoxyribonucleic acid strands from the recent
deoxyribonucleic acid strands. This step is sometimes of 1 minute as against a
pair of min within the initial cycle. tempering permits the primers to base-pair with
each the new and recent strands, the entire range of strands being double their
original range. Synthesis of latest strands takes place, that doubles the quantity
of copies of the required deoxyribonucleic acid section present at the tip of step
one. This completes the second cycle.
Thus at every cycle, each new and recent strands toughen to the primers and
function templates for deoxyribonucleic acid synthesis. As a result, at the tip of
every cycle, the quantity of copies of the required segment becomes double the
quantity gift at the tip of the previous cycle. therefore at the tip of n cycles 2′
copies of the section square measure expected; the important values square
measure quite on the brink of however lower than this expectation. Usually, 3045 cycles square measure disbursed in most PCR experiments.
In case of automatic laptop machines, known as thermal cyclers, the research
worker must solely specify the number and length of cycles, etc. once inserting
the whole reaction mixture for incubation, and also the machine performs the
whole program of operations exactly. After PCR cycles, the amplified
deoxyribonucleic acid section is pure by gel electrophoresis and might be used
for biological research. deoxyribonucleic acid sequencing, etc. However, Tag
enzyme doesn’t have proof-reading operate.
:
Therefore, PCR product have a lot of higher mutations (base substitutions) than
deoxyribonucleic acid obtained by in vivo replication. This facet must be guarded
against whereas mistreatment PCR products.
(Image.2)
Image source: B.D. Singh
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#Application #DNA polymerase #Fundamentals #Notes #PCR #Procedure
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