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  • FUNDAMENTALS OF THE REACTION OF THE POLYMERASE CHAIN

    The Chain Reaction (PCR) is a laboratory technique that permits copying in vitro of specific DNA sequences and has revolutionized the field of molecular biology. Conceptually, the PCR is a simple technique is to heat the separation of the two strands of DNA to be amplified is, and a copy simultaneously from a point determined by an artificial DNA fragment called primer, through the action of an enzyme called DNA polymerase. The result is a doubling of the number of molecules of a particular sequence of DNA. This process is repeated a number of times or cycles, such as 30, and achieved an increase or exponential amplification of the number of copies of template DNA fragment. Therefore, if we start from a single DNA molecule in the initial sample, and each cycle doubles the number of molecules, theoretically, the end of 30 cycles, you get 2 identical 30 molecules, ie molecules 1073741824.

    The great advantage of PCR is that it amplifies only the DNA fragment we want even if in minimal amounts (high sensitivity) or in the presence of large amounts of other similar DNA (high specificity). The reaction is effective even if one sample very little purified DNA in the presence of other components.

    The PCR was initially applied as a research laboratory technique, but its high specificity and sensitivity has allowed the development of specific techniques based on PCR in many fields of research and analysis. Thus, PCR has become a tool essential in fields as diverse as research (cloning of genes, detection of recombinant clones, DNA study of fossils), forensics and criminalistics (establishment of paternity, identification of individuals, identifying suspect / evidence in cases of murder, rape, etc.), animal health and breeding (detection of genetic diseases and infectious diseases, racial purity, etc..) and medicine (disease diagnosis).

    The PCR was developed by Kary Mullis (Saiki et al, 1985; Mullis, 1990) in 1985, using DNA polymerase Klenow fragment of DNA polymerase I from E. coli and carried out by changing the reaction tubes in each cycle, two baths that were at 94 ° C (denaturation) and 37 ° C (primer annealing and extension). Because this enzyme is denatured at temperatures above 37 º C, it was necessary to add in each cycle after the denaturation step, which made the PCR technique in a tedious and expensive. PCR has improved dramatically as a laboratory tool since 1988, thanks to two events that made the technique could automate: the first was the commercialization of the enzyme called Taq polymerase (Saiki et al, 1988) is an enzyme thermostable, so that remains active after prolonged incubations at 94 ° C, and should not be added in each amplification cycle. The second event was the appearance on the market for thermal cyclers who performed the first temperature changes automatically, without individual bathrooms. The great revolution that has occurred in molecular biology the emergence of PCR has made to their discoverer the Nobel Prize in 1993.

    Throughout the class will review the critical issues affecting the reaction, its components and the optimization of a PCR protocol.

    Published on October 24, 2012 · Filed under: DNA; Tagged as: , , , , , , ,
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