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Applications of real-time PCR for characterization particles suitable for DNA isolation
Ondrejková, Martina ; Šálek, Petr (referee) ; Trachtová, Štěpánka (advisor)
The theoretical part of the diploma thesis was focused on core-shell type magnetic carriers, used mainly in medical, molecular-biological and biochemical applications. Encapsulation of the core is essential for these applications due to the decrease od non-specific protein adsorbtion, increase of biocompatibility and the possible functionalization of magnetic carriers. In the experimental part, the DNA (E. coli) was amplified by real-time PCR in the presence of poly(hydroxymethacrylate-co-glycidylmethacrylate) (P(HEMA-co-GMA)) magnetic carriers with/without carboxyl groups. The inhibitory effect of different concentrations of magnetic carriers in the PCR mixture was evaluated from the calibration curve parameter values obtained by regression analysis. The presence of a specific PCR product was verified by agarose gel electrophoresis. Most of magnetic carriers without carboxyl groups extinguished the fluorescence in the concentration range of 2,0 – 4,0 g.l-1 in the PCR mixture, without inhibition of DNA amplification - the carriers were biocompatible. Magnetic carriers with carboxyl groups extinguished the fluorescence in the lower concentration range (0,4 – 4,0 g.l-1 in the PCR mixture). Their inhibition of amplification was in the concentration range of 2,0 – 4,0 g.l-1 in the PCR mixture, from the concentration 0,8 g.l-1 in the PCR mixture, the inhibition did not occur and the carriers were biocompatible. The results do not depend on the characteristic properties of the magnetic carriers but on the presence of the carboxyl groups on the surface of the carrier and the degree of coverage of the magnetic core by the polymer. Real-time PCR has become an effective tool for studying magnetic core encapsulation and the influence of functional groups on the surface of the polymeric layer.
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Preparation and Characterization of Magnetic Carriers from Hypercrosslinked Polystyrene Microspheres and their Application in a Biosensor.
Šálek, Petr ; Šňupárek, Jaromír (referee) ; Šafařík,, Ivo (referee) ; Horák, Daniel (advisor)
With the aim to develop and characterize a functionalized highly magnetic polymer carrier of micrometer size and of a narrow particle size distribution that will be suitable for biological application, hypercrosslinked microspheres were prepared. Simultaneously, the relation between structure and properties of product was observed. Condition of dispersion polymerization were optimized to obtain starting monodisperse poly(styrene-co-divinylbenzene) [P(St-DVB)] microspheres. The P(St-DVB) microspheres of different degree of crosslinking were prepared and effect of some polymerization parameters such as type of solvent, initiator, concentration and mode of DVB addition on morphology, size and particle size distribution were investigated. The starting microspheres were hypercrosslinked to obtain microporous inner structure. Hyperosslinked particles had very large specific surface area (> 1000 m2/g) and a high content of micropores (ca. 0.6 ml/g). First, P(St-DVB) microspheres were chloromethylated using three different chloromethylation agents to regulate their porous properties. Hypercrosslinking was achieved by the addition of stannic chloride as a catalyst and by increasing a temperature. The hypercrosslinked microspheres were then functionalized with sulfo- or aminogroups. The functional groups captured precipitated iron oxide inside the porous structure of the microspheres and also served as a reactive site for intended immobilization of the protein. A solution of ferrous and ferric chloride was imbibed under vacuum into the porous structure and the iron oxide was precipitated by an aqueous ammonia solution. Finally, the magnetic functionalized hypercrosslinked micropsheres were integrated into a biosensor for qualitative detection of ovalbumin.
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Study of reversible adsorption of nucleid acids on magnetic carriers
Šálek, Petr ; Ing.Daniel Horák, CSc. (referee) ; Rittich, Bohuslav (advisor)
Reversible adsorption of nucleid acids on magnetic carriers was studied in this diploma thesis. Magnetic P(HEMA-co-GMA) microspheres and magnetic glass particles were used. The aim of the study was to isolate DNA in suitable quality for polymerase chain reaction (PCR). Adsorption of DNA on magnetic carriers was achieved after DNA condensation by PEG and NaCl in separation mixture. PEGs of various molecular weight (600 and 6000 g/mol) and different concentrations of PEG in separation mixture (4, 8, 12, 16%) were used. Quantity of eluted DNA incerased with molecular weight and concentration of PEG in separation mixtures. Optimized experimental conditions were applied for the separation of DNA from chicken erythrocytes, purified DNA, DNA in crude lysates of bacterial cells of Lactobacillus paracasei ssp. paracasei CCDM 211/06 and from real samples (liquid dairy products, hard cheese). The presence of target DNA in eluates was tested using genus specific PCR (genus Lactobacillus) or species specific PCR (species Bifidobacterium longum) Aqueous two-phase system (liquid-liquid) was used for separation of DNA from real symplex, too. At first the condiotions aqueous two-phase systém creation were studied. It was created by 16% PEG of various molecular weight (600, 6000 g/mol) and by various concentration of ammonium sulphate. Reversible DNA adsorption on carboxyl group-containing magnetic nonporous P(HEMA-co-EDMA) microspheres for the isolation PCR-ready DNA from liquid dairy products containing PCR inhibitors was studied, too. The quality of isolated DNA was checked by PCR amplification.The presumption on the elimination of PCR inhibitors from DNA samples was confirmed.
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Paraprobiotics, postbiotics and other -biotics
Crha, Richard ; Šálek, Petr (referee) ; Trachtová, Štěpánka (advisor)
The theoretical part of the thesis deals with a wide range of microorganisms and substances derived from probiotics. Based on recent publications, probiotics, prebiotics, synbiotics, paraprobiotics, postbiotics, pharmabiotics, psychobiotics and oncobiotics are defined. Furthermore, the purpose and use of the mentioned -biotics is specified for the current understanding of their function. In the experimental part, DNA was isolated from a sample of probiotic capsules using a commercially available bacterial kit and magnetic particles. Its purity and concentration were then determined spectrophotometrically. Using the isolated DNA as a matrix for conventional PCR and detection on agarose gel, the composition of the probiotic product as declared by the manufacturer was demonstrated. Finally, the PMA-PCR method was optimized.
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Nanoparticle-Mediated Delivery System for Gene Therapy
Dvořáková, Nikola ; Ellederová, Zdeňka (advisor) ; Šálek, Petr (referee)
Gene editing with the CRISPR/Cas9 system is one of the options that sets a new trend in the development of gene therapy. The most commonly used delivery of DNA into the cells are via viruses. Nevertheless, they are often unable to take CRISPR/Cas9 system, which can be bigger than several kb. Nanoparticles (NPs), as non-viral transporters, seem to be a good alternative delivery system. For this work magnetic Fe3O4 NPs (MNPs) were selected, because of their excellent properties such as multifunctionality, biocompatibility, easy degradation and simple synthesis. The aim of this work was to synthesise MNPs and a complex of MNPs coated with PEI/CRISPR-Cas9 plasmid and to characterize them by physicochemical methods. The created complex MNPs/PEI/CRISPR-Cas9 was defined by exact parameters that are suitable for possible cell uptake. The hypothesis of stabilization of the MNPs/CRISPR-Cas9 plasmid complex by polyethylenimine (PEI), which can also protect plasmid DNA against restriction endonucleases, was verified. Next a stable modified cell line HEK293-TLR3, designed to evaluate the efficacy of double strand break (DSB) repair by nonhomologous end joining (NHEJ) or homologous recombination (HR) was, transfected with the synthesised MNPs/PEI/CRISPR-Cas9 complex. The results indicate a 25% transfection...
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Nanoparticle-Mediated Delivery System for Gene Therapy
Dvořáková, Nikola ; Ellederová, Zdeňka (advisor) ; Šálek, Petr (referee)
Gene editing with the CRISPR/Cas9 system is one of the options that sets a new trend in the development of gene therapy. The most commonly used delivery of DNA into the cells are via viruses. Nevertheless, they are often unable to take CRISPR/Cas9 system, which can be bigger than several kb. Nanoparticles (NPs), as non-viral transporters, seem to be a good alternative delivery system. For this work magnetic Fe3O4 NPs (MNPs) were selected, because of their excellent properties such as multifunctionality, biocompatibility, easy degradation and simple synthesis. The aim of this work was to synthesise MNPs and a complex of MNPs coated with PEI/CRISPR-Cas9 plasmid and to characterize them by physicochemical methods. The created complex MNPs/PEI/CRISPR-Cas9 was defined by exact parameters that are suitable for possible cell uptake. The hypothesis of stabilization of the MNPs/CRISPR-Cas9 plasmid complex by polyethylenimine (PEI), which can also protect plasmid DNA against restriction endonucleases, was verified. Next a stable modified cell line HEK293-TLR3, designed to evaluate the efficacy of double strand break (DSB) repair by nonhomologous end joining (NHEJ) or homologous recombination (HR) was, transfected with the synthesised MNPs/PEI/CRISPR-Cas9 complex. The results indicate a 25% transfection...
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Applications of real-time PCR for characterization particles suitable for DNA isolation
Ondrejková, Martina ; Šálek, Petr (referee) ; Trachtová, Štěpánka (advisor)
The theoretical part of the diploma thesis was focused on core-shell type magnetic carriers, used mainly in medical, molecular-biological and biochemical applications. Encapsulation of the core is essential for these applications due to the decrease od non-specific protein adsorbtion, increase of biocompatibility and the possible functionalization of magnetic carriers. In the experimental part, the DNA (E. coli) was amplified by real-time PCR in the presence of poly(hydroxymethacrylate-co-glycidylmethacrylate) (P(HEMA-co-GMA)) magnetic carriers with/without carboxyl groups. The inhibitory effect of different concentrations of magnetic carriers in the PCR mixture was evaluated from the calibration curve parameter values obtained by regression analysis. The presence of a specific PCR product was verified by agarose gel electrophoresis. Most of magnetic carriers without carboxyl groups extinguished the fluorescence in the concentration range of 2,0 – 4,0 g.l-1 in the PCR mixture, without inhibition of DNA amplification - the carriers were biocompatible. Magnetic carriers with carboxyl groups extinguished the fluorescence in the lower concentration range (0,4 – 4,0 g.l-1 in the PCR mixture). Their inhibition of amplification was in the concentration range of 2,0 – 4,0 g.l-1 in the PCR mixture, from the concentration 0,8 g.l-1 in the PCR mixture, the inhibition did not occur and the carriers were biocompatible. The results do not depend on the characteristic properties of the magnetic carriers but on the presence of the carboxyl groups on the surface of the carrier and the degree of coverage of the magnetic core by the polymer. Real-time PCR has become an effective tool for studying magnetic core encapsulation and the influence of functional groups on the surface of the polymeric layer.
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Preparation and Characterization of Magnetic Carriers from Hypercrosslinked Polystyrene Microspheres and their Application in a Biosensor.
Šálek, Petr ; Šňupárek, Jaromír (referee) ; Šafařík,, Ivo (referee) ; Horák, Daniel (advisor)
With the aim to develop and characterize a functionalized highly magnetic polymer carrier of micrometer size and of a narrow particle size distribution that will be suitable for biological application, hypercrosslinked microspheres were prepared. Simultaneously, the relation between structure and properties of product was observed. Condition of dispersion polymerization were optimized to obtain starting monodisperse poly(styrene-co-divinylbenzene) [P(St-DVB)] microspheres. The P(St-DVB) microspheres of different degree of crosslinking were prepared and effect of some polymerization parameters such as type of solvent, initiator, concentration and mode of DVB addition on morphology, size and particle size distribution were investigated. The starting microspheres were hypercrosslinked to obtain microporous inner structure. Hyperosslinked particles had very large specific surface area (> 1000 m2/g) and a high content of micropores (ca. 0.6 ml/g). First, P(St-DVB) microspheres were chloromethylated using three different chloromethylation agents to regulate their porous properties. Hypercrosslinking was achieved by the addition of stannic chloride as a catalyst and by increasing a temperature. The hypercrosslinked microspheres were then functionalized with sulfo- or aminogroups. The functional groups captured precipitated iron oxide inside the porous structure of the microspheres and also served as a reactive site for intended immobilization of the protein. A solution of ferrous and ferric chloride was imbibed under vacuum into the porous structure and the iron oxide was precipitated by an aqueous ammonia solution. Finally, the magnetic functionalized hypercrosslinked micropsheres were integrated into a biosensor for qualitative detection of ovalbumin.
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Study of reversible adsorption of nucleid acids on magnetic carriers
Šálek, Petr ; Ing.Daniel Horák, CSc. (referee) ; Rittich, Bohuslav (advisor)
Reversible adsorption of nucleid acids on magnetic carriers was studied in this diploma thesis. Magnetic P(HEMA-co-GMA) microspheres and magnetic glass particles were used. The aim of the study was to isolate DNA in suitable quality for polymerase chain reaction (PCR). Adsorption of DNA on magnetic carriers was achieved after DNA condensation by PEG and NaCl in separation mixture. PEGs of various molecular weight (600 and 6000 g/mol) and different concentrations of PEG in separation mixture (4, 8, 12, 16%) were used. Quantity of eluted DNA incerased with molecular weight and concentration of PEG in separation mixtures. Optimized experimental conditions were applied for the separation of DNA from chicken erythrocytes, purified DNA, DNA in crude lysates of bacterial cells of Lactobacillus paracasei ssp. paracasei CCDM 211/06 and from real samples (liquid dairy products, hard cheese). The presence of target DNA in eluates was tested using genus specific PCR (genus Lactobacillus) or species specific PCR (species Bifidobacterium longum) Aqueous two-phase system (liquid-liquid) was used for separation of DNA from real symplex, too. At first the condiotions aqueous two-phase systém creation were studied. It was created by 16% PEG of various molecular weight (600, 6000 g/mol) and by various concentration of ammonium sulphate. Reversible DNA adsorption on carboxyl group-containing magnetic nonporous P(HEMA-co-EDMA) microspheres for the isolation PCR-ready DNA from liquid dairy products containing PCR inhibitors was studied, too. The quality of isolated DNA was checked by PCR amplification.The presumption on the elimination of PCR inhibitors from DNA samples was confirmed.
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