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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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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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