Principles Of Polymerase Chain Reaction (PCR)
PCR is a method developed in 1985 by Kary Mullis and to obtain, by coupling a heat-resistant DNA polymerase and without cloning, amplification of a fragment of DNA known. Initially, DNA polymerase was isolated from a bacterium thermophilne (resistant to significant increases in temperature), as Thermus aquaticus (Taq polymerase). Currently, we use recombinant enzymes, whose; elaboration is easier and greater efficiency.
The general principle of PCR is the in vitro amplification of a specific area of a nucleic acid. By a series of replication reactions repeated loop, the double-stranded DNA template undergoes exponential amplification products obtained for each cycle using the following cycles matrix. …show more content…
The denaturation step (separation of two DNA strands) is conducted at a temperature of 94ºC. Hybridization temperatures vary (45-60ºC) delivery using the pairs of primers and determine the stability of the paired hybrids. The calculation of these temperatures is done by removing approximately 5ºC Tm of the primer. This Tm are calculated using the formula Tm = 2(A+T) + 4(G+C) or A, T, G and C respectively correspond to the amounts of bases present in the oligonucleotide. However, this formula is an estimate, and a test performed in a range of temperature is indispensable to determine the optimal hybridization conditions. Finally, the polymerization is performed at 68 or 72ºC according to heat-resistant DNA polymerase used (manufacturer's …show more content…
Electrophoresis will be carried out in basic medium, composed of a TBE IX buffer (0,89M Tris, 0.8M Boique acid, 0.02 mM EDTA; pH 8.4 disodium). The nucleic acids, polyanionic macromolecules uniformly negativemen loads, when placed in a constant electric field will then migrate through the gel towards the anode. The speed of movement of the fragments through the gel mesh will therefore vary depending on the concetration in agarose gel, but also the molecular weight (number of base pairs) of each fragment. The agarose gels allowing separation of framents whose size is between 0.5 and 20 Kb. Ten microliters of amplification product are added 5 microliters of hare buffer (0.25% bromophenol blue, 30% glycerol, 40% sucrose); the mixture is then deposited in the wells. A negative witness is treated in the same conditions as the different samples: the 5 microliters of extracts are then replaced by 5 microliters of water purified in order to control for contamination.
In the gel are also introduced few microliters of BET (ethidium bromide); it is an intercalating agent to the level of the DNA base pairs and emitting an orange fluorescence under ultraviolet (UV) is 200-300nm. The DNA detection threshold by this technique is only a few ng.
Each gel is also deposited a molecular weight marker (100bp DNA Ladder GIBCO) to calibrate the size of the amplified fragments.