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Nucleic acids isolation for diagnostic purposes using polymeric carriers
Syslová, Ivona ; Horák, Daniel (referee) ; Rittich, Bohuslav (advisor)
The isolation of deoxyribonucleic acid (DNA) was studied in the diploma thesis by using three different methods: phenol extraction, salting with sodium chloride and magnetic separation with reversible adsorption of nucleic acids on different magnetic carriers. There were used five different properly functionalized carriers for the isolation of DNA: magnetic silicagel, P(HEMA-co-GMA) ox. I, P(HEMA-co-GMA) ox. II, Dynal DNA Direct and Perovskit 439. The reversible imobilization of DNA on the magnetic carrier was proceeded under the conditions of high concentration of NaCl and poly(ethyleneglycol) (PEG). There was induced the condensation of DNA by 2 M NaCl and PEG with molecular mass 6000 for binding of the DNA to the magnetic carriers and the final concentration of PEG in the separation mixture was 8 and 16 %. The aim was to gain the DNA of quality suitable for polymerase chain reaction (PCR). The DNA was isolated from the bacterial cultures of three probiotic strains, L. amylovorus CCM 4380T, L. zeae CCM 7069 T, L. plantarum CCM 7039T, which were cultivated in MRS medium. The DNA was also isolated from the fermented dairy products: Jihočeský zákys s ovocem jahoda (the fermented dairy product with the probiotic culture of Lactobacillus acidophilus, Bifidobacterium lactis and Streptococcus thermophilus), Revital active (the yogurt with inulin and the probiotic culture of Lactobacillus rhamnosus, Lactobacillus acidophilus and Bifidobacterium sp.) and Actimel višeň (the dairy product with the probiotic culture of Lactobacillus casei). When the PCR with the isolated DNA was passed off, the PCR products were detected by the gel electrophoresis with agarose. The success of the DNA isolation of the probiotic bacteria by phenol extraction, salting with NaCl and by magnetic separation, was verified by the PCR method. The method of magnetic separation using magnetic carriers was also verified for the isolation of DNA of quality suitable for PCR from the probiotic fermented dairy products.
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Study of aggregation process and physical stability of polymeric micelle by fluorescence probe method
Chovancová, Romana ; Pekař, Miloslav (referee) ; Mravec, Filip (advisor)
Amphiphilic block copolymer poly(ethylene glycol)-b-poly(–caprolactone) (PEG–PCL) was synthesized from poly(ethylene glycol) (PEG) and poly(–caprolactone) (PCL) by using ring-opening polymerization. The structure and composition of this copolymer was determined by IR spectroscopy. Polymeric micelles were prepared by membrane dialysis method and direct dissolution of copolymer in water. The process of aggregation and physical stability in water solution was studied by fluorescence spectroscopy using pyrene and perylene as fluorescent probes.The results of steady-state and time resolved fluorescence measurements indicate that system of PEG–PCL forms both unimolecular and multimolecular micelles, which depends on copolymer concentration. The critical micelle concentration (CMC) was around 0,002 g/L. Measurement of micelles size by dynamic light scattering method suggested that systems with higher concentration form bigger aggregates. In addition, copolymer behavior was explored under physiological conditions. The results show that CMC of copolymer increased in 0,15 mol/L sal solution at temperature 37°C to 0,02 g/L when compared to copolymer in water.
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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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Nucleic acids isolation for diagnostic purposes using polymeric carriers
Syslová, Ivona ; Horák, Daniel (referee) ; Rittich, Bohuslav (advisor)
The isolation of deoxyribonucleic acid (DNA) was studied in the diploma thesis by using three different methods: phenol extraction, salting with sodium chloride and magnetic separation with reversible adsorption of nucleic acids on different magnetic carriers. There were used five different properly functionalized carriers for the isolation of DNA: magnetic silicagel, P(HEMA-co-GMA) ox. I, P(HEMA-co-GMA) ox. II, Dynal DNA Direct and Perovskit 439. The reversible imobilization of DNA on the magnetic carrier was proceeded under the conditions of high concentration of NaCl and poly(ethyleneglycol) (PEG). There was induced the condensation of DNA by 2 M NaCl and PEG with molecular mass 6000 for binding of the DNA to the magnetic carriers and the final concentration of PEG in the separation mixture was 8 and 16 %. The aim was to gain the DNA of quality suitable for polymerase chain reaction (PCR). The DNA was isolated from the bacterial cultures of three probiotic strains, L. amylovorus CCM 4380T, L. zeae CCM 7069 T, L. plantarum CCM 7039T, which were cultivated in MRS medium. The DNA was also isolated from the fermented dairy products: Jihočeský zákys s ovocem jahoda (the fermented dairy product with the probiotic culture of Lactobacillus acidophilus, Bifidobacterium lactis and Streptococcus thermophilus), Revital active (the yogurt with inulin and the probiotic culture of Lactobacillus rhamnosus, Lactobacillus acidophilus and Bifidobacterium sp.) and Actimel višeň (the dairy product with the probiotic culture of Lactobacillus casei). When the PCR with the isolated DNA was passed off, the PCR products were detected by the gel electrophoresis with agarose. The success of the DNA isolation of the probiotic bacteria by phenol extraction, salting with NaCl and by magnetic separation, was verified by the PCR method. The method of magnetic separation using magnetic carriers was also verified for the isolation of DNA of quality suitable for PCR from the probiotic fermented dairy products.
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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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Study of aggregation process and physical stability of polymeric micelle by fluorescence probe method
Chovancová, Romana ; Pekař, Miloslav (referee) ; Mravec, Filip (advisor)
Amphiphilic block copolymer poly(ethylene glycol)-b-poly(–caprolactone) (PEG–PCL) was synthesized from poly(ethylene glycol) (PEG) and poly(–caprolactone) (PCL) by using ring-opening polymerization. The structure and composition of this copolymer was determined by IR spectroscopy. Polymeric micelles were prepared by membrane dialysis method and direct dissolution of copolymer in water. The process of aggregation and physical stability in water solution was studied by fluorescence spectroscopy using pyrene and perylene as fluorescent probes.The results of steady-state and time resolved fluorescence measurements indicate that system of PEG–PCL forms both unimolecular and multimolecular micelles, which depends on copolymer concentration. The critical micelle concentration (CMC) was around 0,002 g/L. Measurement of micelles size by dynamic light scattering method suggested that systems with higher concentration form bigger aggregates. In addition, copolymer behavior was explored under physiological conditions. The results show that CMC of copolymer increased in 0,15 mol/L sal solution at temperature 37°C to 0,02 g/L when compared to copolymer in water.
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