POLYMERASE CHAIN REACTION

POLYMERASE CHAIN REACTION

16.      POLYMERASE CHAIN REACTION
PCR is the method of in vitro gene cloning. This technique results in exponential amplification of the selected region of DNA molecule or gene of interest with the help of DNA polymerase enzyme. It facilitates the million-fold copies of the selected DNA region. It discovered by “Karry Mulis” in 1984, who worked in Cetus, a California Biotech Company. In 1993 Mulis with Michael Smith jointly received Nobel prize in chemistry. 
16.1.    The constituent of PCR Reaction 
One or more molecules of target DNA.
Two oligonucleotide primer, forward primer and Reverse primer.
All four Deoxyribonucleoside triphosphate (dNTP), where N=A/C/G/T. 
Thermostable DNA polymerase, buffer,  Mg2+.    
16.2.    Steps in the PCR cycle


Denaturation at 95°C, 0.5minute, at this stage dsDNA is converted into two single strands.  
This formula should be used if primer base length is less then ~20 nucleotide.  
Tm =4(G+C) +2(A+T).
If primer length is 17-30 nucleotide, then this formula should be used.              
Tm =22+1.46 [2*(G+C) + (A+T) ].
If the primer length is 14-17nucleotide, then this formula should be used.             
Tm = 81.5+16.6 [log10 (J+)]+0.41 (%GC) – (600/l). 
(J+) = monovalent cation’s molar concentration, l= oligonucleotide’s length.
Annealing: It is a second step of the PCR cycle. Annealing steps allow the hybridization of primers with its target DNA. Hybridization can occur only at low temperature. The annealing temperature is normally 69°C. The duration of the annealing step is 1.5 minute. The annealing temperature can be calculated by Ta = 0.3*Tm (primer’s Tm) + 0.7Tm (product’s Tm) – 14.9.
Extension: The extension step is a 3rd step of the PCR cycle. The extension allows the binding of Taq polymerase with 3' OH of primer. The temperature of the extension step is 72° which is higher than the annealing step of PCR. The higher temperature causes dissociation of non-specific primers from target DNA. The high stringency condition (high temperature) reduce the non-specific primer extension and thus reduce the noise.
Enzymes use in PCR 
Taq polymerase is isolated from Thermus aquaticus, which is found in hot spring. It used to carry out the DNA synthesis in PCR because it is stable at high temperature like 90°C which is required for denaturation of DNA. Taq polymerase works optimally at 75°-80°C. 
Taq polymerase is identical to the Klenow domain of E.coli DNA polymerase. It lacks the 3’-5’ exonuclease proofreading activity, thus show the error rate of 1nucleotide per 1000 nucleotide. It possesses 5’-3’ exonuclease activity as well as 5’-3’ polymerization activity. 
Amplitaq DNA polymerase: It is a thermostable recombinant DNA polymerase. The mutation in 5'-3' exonuclease activity enhance the processivity is Taq polymerase. The Ampli Taq DNA polymerase is created in the lab by the mutation in 46th Glycine of 5'-3' exonuclease pocket. In that mutation, the glycine is replaced by Aspartic amino acid. 
Taq polymerase requires Mg2+ for their activity because it acts as a cofactor for the enzyme. A standard PCR efficiently amplifier target DNA sequence up to 3 kb, more than get amplify non specifically, Target sequence up to 1.5 kb define as ideal for PCR reaction. Tris-Hcl buffer use in PCR reaction (pH - 6.8-7.8).
Pfu polymerase. It is a DNA polymerase isolated from (Pyrococcus furiosus)  having optimum activity at 75°C. At 70°-80°C. It has an error rate of 1.5 x 10-6 (lowest one). It has 3’-5’ exonuclease activity. It is used in high fidelity PCR and primer extension.
Vent polymerase, it is natural polymerase isolated from (Thermococcus clitoris) having optimum activity at 70°-80°C along with error rate intermediate between Taq and Pfu polymerase, it has 3’-5’ proofreading activity and works with the difficult template like GC rich and looped.
Tth DNA polymerase, it is a recombinant polymerase isolated from Thermus thermophilus having optimum activity at 75°C-80°C along with low fidelity, it has 5’-3’ exonuclease activity. It is used in RT-PCR, PCR and primer extension with the requirement of Mn2+.
16.3.    The result of PCR: 
PCR reaction is performed within thermal cycler, After completion of PCR reaction, the agarose gel electrophoresis is performed with the final outcome of PCR reaction if the desired sample gets amplify it get visualize in the form of a band on the gel.
In PCR the cycle repeat is up to 30 times (standard 25-35 cycle), after each cycle the number of template doubles and after 30 cycles the number reaches 1 x 109 but the exact no of the product after completion of reaction calculate by 
N = Ni  x  2n, where Mf = final product number, Mi = initial number of molecule and n = number of cycle. 
The decrease in the exponential rate of PCR beyond a certain cycle of PCR. Plateau effect occurs due to reactant’s degradation (dNTPs, DNA polymerase), depletion of reactant like the exhaust of primer, pyrophosphate or end product inhibition, thus the PCR efficiency less than 100 %. 
PCR product yield can be calculated by PCR product yield = input target amount*(1+ % efficiency) x number of cycle.
The desire product yield should be calculated by 2n – (n+1) because the desired product starts to form from the third cycle.
Primer designing
In PCR, primer designing is the most important aspect of selective amplification. For primer design, some prior DNA sequence information from the target DNA is required. The information is used to design two primers (amplimers), which are specific to sequences flanking the target DNA sequence. So, for most PCR reactions, it is very important to reduce the chance of the primers binding to other locations in the DNA than the desired one.
Primer length
Length of the primers should not be very short or long. If the primers are too short, they might hybridize to non-target sites and give undesired amplification products.
Length of primer should be 17-30 nucleotide, less than that it does not show specific binding, in turn, form non-specific product and increase in length of primer results in inhibition of PCR reaction because they pair themselves and form diner of primer.
40-60 % GC content should be present in the primer, up to three G or C within the last 5 bases at 3’ end show strong pairing but not more than three G or C.
Primer should not possess repeating motif or nucleotide (3 bp) because of this result in the wrong hybridization of primer at the template.
The primer can be designed if we know the DNA sequence which we need to amplify. If DNA sequence not known but protein is sequence known then also we can design primer by protein, but due to the degeneracy of codon we choose the segment of protein which includes the amino acid which possesses the least degeneracy and this type of primer known as the degenerate primer.
PCR product : (Initial amount of DNA)  X  (1 + % efficiency)No. of cycle
16.4.    Variant of PCR
16.4.1    Real-time PCR or qPCR or qRT-PCR or RT-qPCR or kinetic PCR
The detection of amplification of DNA in PCR is posted gel electrophoresis. l.e. Amplification can be detected only after gel electrophoresis. However, in real time PCR the detection of amplification is in real time life. during the PCR cycle, itself.


Thus is no need to waits to visualize the result. It examines fluorescence emitted throughout the reaction at each cycle as a sign of amplification at each PCR cycle. The principle of Real-time PCR depends on the fluorescent reporter’s detection and quantification, in turn, the initial amount of target template correlates with the first noteworthy increase in the amount of PCR product (CT -threshold cycle). Threshold cycle or CT define as the number of the cycle at which fluorescence crosses the preset threshold which is present above the baseline, the baseline is the point which defines the product accumulation. As CT -threshold cycle achieve, we can calculate the amplification by this formula- 
Tn=T0 (E)n
Where, Tn=amount of the target sequence at “n” cycle, T0= initial amount of template,  E= efficiency of amplification
The increment in fluorescence takes place in the positive sample with the cycle number but fluorescence remains at baseline in the negative sample, so the requirement of stable baseline or fluorescent intensity is necessary because from baseline graph for positive S-shaped curve begins and follow an exponential segment and at last come to an end with the plateau.
16.4.2.    Quantitative assay performed by three different method
1.    DNA binding agent:  PCR was initially quantified EtBr but now this method is not used. SYBR green, it binds to the minor groove of ds DNA and gives green colour, by SYBR green result is visualized in extension phase because in this phase more dsDNA are present if amplification of target DNA takes place.
2.    Hydrolysis Probe: A quencher fluorochrome system attached to the hydrolysis probe. The quencher absorbs the fluorescence of fluorochrome by fluorescence resonance energy transfer but when the quencher is cleaved with the 5’-3’exonuclease activity of polymerase then fluorescence emitted by free fluorochrome reporter is shown.


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