Polymerase chain reaction (PCR)
Generalities
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. Replication involves three steps: (1) denaturation of double-stranded DNA into single-stranded template; (2) hybridization of the oligonucleotides …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
Generalities
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. Replication involves three steps: (1) denaturation of double-stranded DNA into single-stranded template; (2) hybridization of the oligonucleotides …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