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Isolation and purification of nucleic acids using magnetised micro- and nanoparticles
Dostálová, Simona ; Adam, Vojtěch (referee) ; Kizek, René (advisor)
Nucleic acids are a completely unique component of every organism. They determine the appearance, behaviour or disposition to a certain disease. Despite their importance, there is still a lot of information unknown. In order to study the nucleic acids, they need to be purified from other components of the organism. The routine methods of isolation and purification are often laborious and time consuming; therefore experiments with magnetic separation are carried out. This thesis deals with magnetic micro- and nanoparticles and their application in this field. General conditions of this isolation are summarized in the thesis. Detection methods of nucleic acids are listed, focusing on electrochemical methods. A procedure for isolation and purification of nucleic acids using magnetic particles and square-wave voltammetry is described. This procedure was modified in experimental part of the thesis to achieve the maximum yield. Partial steps including washing, immobilization and elution were optimized. Results showed that the phosphate buffer was optimal for washing of beads before immobilization of nucleic acids on their surface. The optimal immobilization was carried out for 5 minutes at 20 °C and microtube needed to be firmly shaken during the process. Immobilization solution composed of 0.1M Na2HPO4 + 0.1M NaH2PO4, 0.6M guanidinium thiocyanate, 0.15M Trizma base adjusted by HCl on pH 7.5 and 2.5M CsCl. 5M NaCl was optimal for washing of the complex “beads-nucleic acid” after the immobilization. Elution was carried out for 15 minutes at 99 °C and the highest yield was obtained using elution solution composed of Tris-EDTA pH at 9.1
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Ancient-DNA analysis of teeth and skeletal remains with utilization of miniSTR loci.
Kvítková, Dana ; Brouček, Jaroslav (advisor) ; Dobisíková, Miluše (referee)
During the last twenty years, immense progress occurred in the area of analysis of DNA extracted from historical material. Considering the common level of preservation of tissue material, this analysis is usually executed on samples procured from bones and teeth. The analysis of soft mummified tissue is possible only in rare cases. Limiting factor of these analyses is a high degree of degradation and small amount of DNA extractable from this kind of material. First researches concentrated only on short sections of mainly mitochondrial DNA. Today, the analysis of the complete mitochondrial genome of both contemporary and extinct organisms was made possible. In case of analyses conducted on human remains, sections of nuclear DNA are far more valuable, because they can reveal information including not only subject's sex, but also possible kinship between subjects found e.g. in the same grave. Fundamental component of the whole analysis is the process of extracting DNA from cells. Probably every laboratory working with historical DNA uses a differently modified extract protocol. The main requirement for methods of extraction is to secure enough DNA with such a level of purity that would allow its use for following steps of the analysis. Taking in consideration high fragmentation of DNA, it is necessary...
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Ancient-DNA analysis of teeth and skeletal remains with utilization of miniSTR loci.
Kvítková, Dana ; Brouček, Jaroslav (advisor) ; Dobisíková, Miluše (referee)
During the last twenty years, immense progress occurred in the area of analysis of DNA extracted from historical material. Considering the common level of preservation of tissue material, this analysis is usually executed on samples procured from bones and teeth. The analysis of soft mummified tissue is possible only in rare cases. Limiting factor of these analyses is a high degree of degradation and small amount of DNA extractable from this kind of material. First researches concentrated only on short sections of mainly mitochondrial DNA. Today, the analysis of the complete mitochondrial genome of both contemporary and extinct organisms was made possible. In case of analyses conducted on human remains, sections of nuclear DNA are far more valuable, because they can reveal information including not only subject's sex, but also possible kinship between subjects found e.g. in the same grave. Fundamental component of the whole analysis is the process of extracting DNA from cells. Probably every laboratory working with historical DNA uses a differently modified extract protocol. The main requirement for methods of extraction is to secure enough DNA with such a level of purity that would allow its use for following steps of the analysis. Taking in consideration high fragmentation of DNA, it is necessary...
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Isolation and purification of nucleic acids using magnetised micro- and nanoparticles
Dostálová, Simona ; Adam, Vojtěch (referee) ; Kizek, René (advisor)
Nucleic acids are a completely unique component of every organism. They determine the appearance, behaviour or disposition to a certain disease. Despite their importance, there is still a lot of information unknown. In order to study the nucleic acids, they need to be purified from other components of the organism. The routine methods of isolation and purification are often laborious and time consuming; therefore experiments with magnetic separation are carried out. This thesis deals with magnetic micro- and nanoparticles and their application in this field. General conditions of this isolation are summarized in the thesis. Detection methods of nucleic acids are listed, focusing on electrochemical methods. A procedure for isolation and purification of nucleic acids using magnetic particles and square-wave voltammetry is described. This procedure was modified in experimental part of the thesis to achieve the maximum yield. Partial steps including washing, immobilization and elution were optimized. Results showed that the phosphate buffer was optimal for washing of beads before immobilization of nucleic acids on their surface. The optimal immobilization was carried out for 5 minutes at 20 °C and microtube needed to be firmly shaken during the process. Immobilization solution composed of 0.1M Na2HPO4 + 0.1M NaH2PO4, 0.6M guanidinium thiocyanate, 0.15M Trizma base adjusted by HCl on pH 7.5 and 2.5M CsCl. 5M NaCl was optimal for washing of the complex “beads-nucleic acid” after the immobilization. Elution was carried out for 15 minutes at 99 °C and the highest yield was obtained using elution solution composed of Tris-EDTA pH at 9.1
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