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Monitoring the success of transfection of cell line 293 HEK
Dvořák, Tomáš ; Španová, Alena (referee) ; Ševčík, Mojmír (advisor)
Diploma thesis is based on monitoring the succes of transfection of cell linie HEK293. In theoretical part are described principles of transfection methods, cell lines, vectors and reporter genes. HEK293 cells EBNA1 were used for practical part. It was studied the difference between GFP and EGFP plasmids. As well as using various transfection reagents under different culture conditions.
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Probiotic bacteria and authenticity of milk products
Storozhko, Viktoriya ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Probiotics are living microorganisms that have positive effects on human health after consumption. The theoretical part of the bachelor thesis describes properties and health effects of probiotics and their use in food industry. The experimental part was focused on the isolation of PCR-ready DNA from two dairy probiotic products. The presence of target bacterial DNA was confirmed using PCR methods.
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Development of a fast method for site-directed mutagenesis in Streptococcus zooepidemicus
Černý, Zbyněk ; Španová, Alena (referee) ; Pepeliaev,, Stanislav (advisor)
This diploma thesis is focused on development of a fast method for site-directed gene mutagenesis in Streptococcus zooepidemicus based on the mechanism of natural competence. Several genes were selected based on experimental data which highly probably influence hyaluronic acid synthesis. The deletion of the selected genes from genomic DNA was performed as proof of concept, and the resulting recombinant strains were characterized regarding changes of hyaluronic acid precursor concentrations (glucuronic acid and N-acetylglucosamin) in time of cultivation and the end production of hyaluronic acid.
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Nucleic acids as terapeutic agent
Ráčková, Lucie ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
With the development of molecular biology development of oncotherapy proceeds. The major progress of modern medicine is gene therapy. In the gene therapy are two categeories, namely, viral vectors and nonviral vectors which are used mainly. Nonviral vectors include plasmids. Plasmid DNA used in medicine must be perfectly purified. Chromatographic methods are mainly used at present. Research and development deals with other methods for example two-phase aqueous systems and magnetic carriers. In experimental part of this thesis, isolation of pUC 19 plasmid DNA from Escherichia coli JM 109 (pUC 19) cell culture was performed via method of alkaline lysis. Quality of isolated plasmid DNA was verified spectrophotometrically and by agarose gel electrophoresis. Isolated plasmid DNA was purified using three methods: RNA in plasmid DNA was precipitated by lithium chloride, RNA was degraded by immobilized RNase A and plasmid DNA was purified using two-phase aqueous system.
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Reversible immobilisation of DNA on newly designed magnetic carriers
Kubisz, Petr ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
The aim of work was an optimization of separation deoxyribonucleic acid (DNA) with the use of nucleic acid reversible adsorption to the surface of magnetic particles coated by functional groups. Six carriers were verificated for DNA isolation: P (HEMA-co-GMA) ox, F-kol B 30 ox, F-kol 77 ox, F-kol B100 ox, F-kol 135 ox, coated with carboxyl groups and Perovskit 439 (coated by silicone). Bacterial DNA was isolated by phenol extraction procedure, first. DNA was reversibly bond to magnetis carrier in the presence of high concentration of NaCl ( 5 M) and poly (ethylene glycol) (PEG 6000). The final PEG and NaCl concentrations of 16.0 % (w/v) and 2.0 M, respectively, were used.DNA was eluted into TE buffer. The quality of extracted DNA was checked by PCR amplification. It was found out that although different quantities of DNA were isolated, the quality of isolated DNA was always compatible with PCR. Nanoparticles Perovskit 439 had the best separative characteristics in comparison to the other magnetic carriers because highest amounts of DNA was isolated. However, next optimisation of DNA separation procedure is required for the use of studied microspheres in real samples.
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Optimalisation of a new micromethod of DNA isolation from foods
Surá, Tereza ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
The thesis were focused on the optimalization of micromethod for isolation of DNA in quality for polymerase chain reaction (PCR) using magnetic microparticles from plant food products. There were chosen a red beetroot (fresh, frozen, dried and sterilized) for the analysis and food products containing red beetroot. Different approaches of processing of homogenates were compared and optimized. The homogenates were prepared in lysis buffer with cetyltrimethylammonium bromide (CTAB) with different amounts of NaCl with or without addition of organic extraction agents chloroform-octanol and isopropanol. Microisolation of DNA was performed using magnetic particles P(GMA). The concentration of NaCl and polyethylene glycol (PEG) 6000 in separation mixtures was tested. The influence on quantity and purity of isolated DNA was compared and the optimum amounts of NaCl in CTAB buffer and optimal concentration of PEG 6000 in separation mixtures were compared. The optimized separation mixture for the DNA isolation from red beetroot was applied to food products containing red beetroot. Amplifiability of DNA was tested in conventional PCR using specific primers for plant DNA. PCR products of length 700 bp were detected by agarose gel electrophoresis.
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Isolation of PCR-ready DNA from dairy products for baby nutrition
Mantlová, Jana ; Eva, Kvasničková (referee) ; Španová, Alena (advisor)
The work was focused on isolation of PCR-ready DNA and the identification of probiotic lactic acid bacteria that were isolated from five milk product for infant nutrition. DNA was isolated from crude cell-lysates of the products by magnetic P(HEMA-co-GMA) microspheres. DNAs isolated from crude cell lysates of control strains using phenol extraction method were used as positive controls. Using PCRs DNA of genera Bifidobacterium and species B. animalis, B. bifidum, B. breve, B. infantis, B. longum and Streptococcus thermophilus species were identified in products. The results obtained are consistent with the data declared by the manufacturers.
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Testing of food authenticity using DNA analysis
Kocianová, Vanda ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Authenticity verification of food products is an important step before their release to the market. This testing ensures that the product contains what the label states and also confirms the medical harmlessness. One of methods possible to use for authenticity verification is polymerase chain reaction (PCR). The experimental part was focused on the DNA isolation using magnetic particles on PCR – ready quality from food product – ketchup. DNA isolated from seeds and stalk of tomato were used as a controls. Amplificalibity of DNA isolated with magnetic particles using magnetic separator and needle was compared. The method requires further optimization.
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DNA extraction from cheeses for polymerase chain reaction analysis
Mohelský, Tomáš ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
This work was focused on DNA isolation from cheeses for the use in polymerase chain reaction. First, there was optimised the procedure of homogenisation of different types of cheeses from commercial sources, cell lysis and DNA isolation. DNA was isolated using magnetic microspheres and phenol extraction. It was shown that the DNA was amplified in PCR for domain Bacteria after dilution. Next, there was optimised the procedure of DNA isolation from fresh cheeses and from contaminated fresh cheeses and their pickles. DNA from all samples was amplified in PCR. The presence of DNA of domain Bacteria and yeast DNA was demonstrated. In the last part of the work, there were optimised the preparation of PCR mixtures and bacterial DNA amplification in PCR with primers with clamp (F357-GC and R518). Synthetized PCR products were analysed using DGGE. It was shown that amplicons of DNA isolated from cheeses and pickles differ in positions and numbers. Larger number of bands of different intensities was detected after amplification of DNA isolated from contaminated pickles.
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