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Nucleic acids as terapeutic agent
Ráčková, Lucie ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
With the development of molecular biology development of oncotherapy proceeds. The major progress of modern medicine is gene therapy. In the gene therapy are two categeories, namely, viral vectors and nonviral vectors which are used mainly. Nonviral vectors include plasmids. Plasmid DNA used in medicine must be perfectly purified. Chromatographic methods are mainly used at present. Research and development deals with other methods for example two-phase aqueous systems and magnetic carriers. In experimental part of this thesis, isolation of pUC 19 plasmid DNA from Escherichia coli JM 109 (pUC 19) cell culture was performed via method of alkaline lysis. Quality of isolated plasmid DNA was verified spectrophotometrically and by agarose gel electrophoresis. Isolated plasmid DNA was purified using three methods: RNA in plasmid DNA was precipitated by lithium chloride, RNA was degraded by immobilized RNase A and plasmid DNA was purified using two-phase aqueous system.
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Plasmid DNA vaccines
Machan, Radoslav ; Chroboková, Maria (referee) ; Rittich, Bohuslav (advisor)
Plasmid DNA vaccines are the new generation of vaccines with a great potential in prevention of many diseases. Recent studies and clinical test are aimed at prevention against cancer, hepatitis, malaria, HIV, influenza and other diseases. Recent main challenges covering plasmid DNA vaccines are associated with optimalization of each step of production and mainly purification steps allowing production of pDNA at kilogram levels. Main purification techniques used are based upon chromatographic methods, but research and development also shows other potential methods, like two-phase aqueous systems or magnetic microparticles as carriers. In experimental part of this thesis isolation of pUC 19 plasmid from Escherichia coli JM 109 (pUC 19) cell culture was performed via method of alkaline lysis. Isolation was verified by agarose gel electrophoresis. Isolated samples were purified in four repetitions with lithium chloride and magnetic microparticle carriers and the extent of purification was verified spectrophotometrically. Purified samples were visualised via agarose gel electrophoresis and results were compared.
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Nucleic acids as terapeutic agent
Ráčková, Lucie ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
With the development of molecular biology development of oncotherapy proceeds. The major progress of modern medicine is gene therapy. In the gene therapy are two categeories, namely, viral vectors and nonviral vectors which are used mainly. Nonviral vectors include plasmids. Plasmid DNA used in medicine must be perfectly purified. Chromatographic methods are mainly used at present. Research and development deals with other methods for example two-phase aqueous systems and magnetic carriers. In experimental part of this thesis, isolation of pUC 19 plasmid DNA from Escherichia coli JM 109 (pUC 19) cell culture was performed via method of alkaline lysis. Quality of isolated plasmid DNA was verified spectrophotometrically and by agarose gel electrophoresis. Isolated plasmid DNA was purified using three methods: RNA in plasmid DNA was precipitated by lithium chloride, RNA was degraded by immobilized RNase A and plasmid DNA was purified using two-phase aqueous system.
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