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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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Simple Electrochemical DNA Biosensor for Detection of DNA Damage Caused by UV Radiation
Arustamian, Daria ; Vyskočil, Vlastimil (advisor) ; Dejmková, Hana (referee)
Ultraviolet (UV) radiation is a common DNA damaging agent. Major DNA lesions, such as cyclobutane pyrimidine dimers (CPDs) and pyrimidine-(6-4)-pyrimidone (6-4PPs) photoproducts, are carcinogenic and mutagenic. UV induced DNA damage was investigated using a simple electrochemical DNA biosensor based on an ultra-trace graphite electrode (UTGE) and low molecular weight doble-stranded DNA (dsDNA) from salmon sperm. Biosensor was prepared using adsorption of dsDNA on a surface of the UTGE and then used to detect UV-induced DNA damage. Effects of UV radiation were investigated using a combination of several electrochemical technics: square-wave voltammetry (SWV) for direct monitoring of DNA base oxidation and cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), as non-direct methods, using redox-active indicator [Fe(CN)6]4-/3- . CV and EIS, which allow characterization of electrode surface, were used to optimize preparation of the dsDNA/UTGE biosensor. Prepared dsDNA/UTGE biosensor was exposed to UV radiation using UV lamp with two set wavelengths: UVC of 254 nm and UVA of 365 nm. UVC radiation was used to damage DNA. Relative signal decrease was 50% after 20 minutes of exposure to UVC radiation. UVA radiation was used to compare effects of different types of UV radiation. Obtained...
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Simple Electrochemical DNA Biosensor for Detection of DNA Damage Caused by UV Radiation
Arustamian, Daria ; Vyskočil, Vlastimil (advisor) ; Dejmková, Hana (referee)
Ultraviolet (UV) radiation is a common DNA damaging agent. Major DNA lesions, such as cyclobutane pyrimidine dimers (CPDs) and pyrimidine-(6-4)-pyrimidone (6-4PPs) photoproducts, are carcinogenic and mutagenic. UV induced DNA damage was investigated using a simple electrochemical DNA biosensor based on an ultra-trace graphite electrode (UTGE) and low molecular weight doble-stranded DNA (dsDNA) from salmon sperm. Biosensor was prepared using adsorption of dsDNA on a surface of the UTGE and then used to detect UV-induced DNA damage. Effects of UV radiation were investigated using a combination of several electrochemical technics: square-wave voltammetry (SWV) for direct monitoring of DNA base oxidation and cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), as non-direct methods, using redox-active indicator [Fe(CN)6]4-/3- . CV and EIS, which allow characterization of electrode surface, were used to optimize preparation of the dsDNA/UTGE biosensor. Prepared dsDNA/UTGE biosensor was exposed to UV radiation using UV lamp with two set wavelengths: UVC of 254 nm and UVA of 365 nm. UVC radiation was used to damage DNA. Relative signal decrease was 50% after 20 minutes of exposure to UVC radiation. UVA radiation was used to compare effects of different types of UV radiation. Obtained...
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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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Electrochemical Study of Triazaborine Chromophores
Mikysek, T. ; Josefík, F. ; Ludvík, Jiří
This contribution describes a basic electrochemical behaviour of a series of newly synthesized chromophores based on triazaborine core. The main attention has been paid to the investigation of the first oxidation and first reduction process of such compounds using cyclic voltammetry, rotating disk voltammetry and polarography. The oxidation is mostly a two-electron irreversible process, whereas the first reduction is in most cases revesible, involving one-electron. For better understanding of the relationship between the structure and redox properties, the Linear Free Energy relationship (LFER) approach using sigma (para) constants of Hammett type was applied, the oxidation and reduction centers were localized, the difference between E(ox) and E(red) was correlated with the HOMO-LUMO gap and the anomalous cases were elucidated.
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