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Optimization of parameters of the polymerase chain reaction.
VOJTOVÁ, Magda
Abstract The polymerase chain reaction (PCR), is a molecular biological method, which is capable to amplify a particular DNA sequence. Above all, there are recently appeared modifications working on the principle of PCR, which became indispensable not only for the diagnosis of some viral, bacterial and tumoral, but also for the diagnosis of genetically conditioned diseases. The duty of my bachelor thesis is to understand the whole system of this technology, to describe its individual parts, process and principle. The main task of my work was to optimize PCR, in order to achieve its highest specifity, accuracy and yields of PCR products, which would be sufficient for next research and analysis. All optimization steps of amplification were done using cDNA (Spinacia oleracea) of proteins PsbR and PsbQ from photosystem II of higher plants. Instead of PsbR, which amplification was optimized with physical impacts, PsbQ protein was optimized using chemical factors. In the case of these chemical factors used to improve PCR product yields that were different concentrations of individual elemental components as: DNA template, Taq DNA polymerase, dNTPs, primers and magnesium ions. For physical impacts the increase of amplification yields was concentrated primarily on optimization of number of cycles, the right temperature of annealing and the time of extesion In the practical part of this bachelor thesis I present the whole set of resulting gels, with the clear conclusion, which conditions for amplification of PCR products were the most suitable, and which on the contrary were problematical. The well adjusted method was analyzed using the electrophoretic division. Namely it was observation of sharpness of corresponding band and the formation of negative or falsely positive results. For the minimisation of undesirable products were experimentally determined for amplification of PsbQ gene as the most appropriate the following parameters: the lowest concentration of DNA (0.001 ?g/?l), for Taq DNA polymerase the resulting activity 0.05 U/?l. The higher values of both components bring during the amplification formation of secondary products, which restrict the yields of reaction. The finding of the right concentration of dNTPs confirms the need of optimization for amplification of each gene. As the most suitable concentration was choosen 0.2 mM, only twice higher levels of this concentration inhibit the reaction. The concentrations of used primers were corresponding to direct proportion, it means that the higher the levels 0,1-2 ?M were used, the higher the yields of PCR products were obtained in observed 446 bp band. Some publications focused on the evaluation of the right concentration of magnesium ions warn us about the fact that the levels lower than 1.5 mM inhibit the reaction. All higher levels guarantee high-quality yields of amplification. The same conclusion was reached in the case of amplification of the PsbQ gene. Also the principles of optimization of amplification of PsbR gene were based on findings obtained from scientific literature focused on this problematic. The ideal temperature of annealing was 63 °C, indisputed intensification of amplification was accomplished owing to the increase of number of cycles. At least 30 cycles of PCR reaction were sufficient enough to ensure the formation of enough intense 290 bp band. The high-quality amplification was also ensured by 2 minutes long extension, shorter time resulted in none or low yield. All optimized parameters were used for analysis of several unknown samples, where the presence of above mentioned genes ? PsbR, PsbQ had to be ascertained and thus confirmed the optimization of PCR method for investigated genes. The results of optimization will be used in the laboratory like starting protocol.
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