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Optimization of plasmid DNA isolation by magnetic particles
Chlopková, Barbora ; Zemanová, Jana (referee) ; Brázda, Václav (advisor)
The theoretical part summarizes information on the isolation and purification of plasmid DNA and nucleic acids as. Plasmid DNA is often used in gene engineering as a vector for the transfer of a particular gene. Its insulation and transportation in sufficient quality is crucial for other processes associated with it. Isolation and survival of pDNA using magnetic carriers of different concentrations of PEG 8000 in combination with 1M NaCl was investigated in experimental parts. Furthermore, the isolation of pDNA using commercial kits was examined.
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Use of high resolution melting analysis for the study of lactic acid bacteria
Knápková, Monika ; Němcová, Andrea (referee) ; Brázda, Václav (advisor)
Currently, there is a growing interest in the use of probiotic products, and there are many of them in the market. With the growing interest, greater emphasis is placed on the identification of declared probiotic microorganisms. Precise identification of microbial composition is often a difficult task and it requires more advanced methods especially in the field of molecular diagnostics. The diploma thesis was focused on the verification of the presence od declared probiotic microorganisms in probiotic food supplements GS Laktobacily Forte 21, Biopron 9 Premium and Linex® Forte. DNA was isolated from the complex matrices by phenol extraction, commercial kit and magnetic carriers F79/L3-PLL in the quality suitable for PCR. Subsequently, the isolated DNA was amplified by real-time polymerase chain reaction using genus- and species-specific primers. The specific PCR product was subjected to agarose gel electrophoresis, whereas species identification was not always in compliance with the data declared by producers. The next part of the thesis was focused on polymerase chain reaction with high-resolution melting analysis to distinguish bacterial strains belonging to the Lactobacillus group and to identify probiotic microorganisms present in the complex matrices of the probiotic food supplements. Eight primer sets were tested (V1F HRM a V1R-HRM, CHAU-V3F a CHAU-V3R, CHAU-V6F a CHAU-V6R, LAC2 a LAC4, LAC1 a LAC2, P1V1 a P2V1, poxcDNAFw a poxPromRVC, poxcDNAFw a poxPromRVT). Three primer pairs (V1F HRM a V1R-HRM, poxcDNAFw a poxPromRVC, poxcDNAFw a poxPromRVT) were evaluated as the most suitable for distinguishing Lactobacillus bacterial strains.
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Using different methods of DNA isolation of lactic acid bacteria in molecular biological methods
Chvalkovská, Eva ; Skoumalová, Petra (referee) ; Brázda, Václav (advisor)
This thesis focused on the probiotic bacteria, DNA isolated from these bacteria by three different methods and the effect of isolation on DNA identification using molecular biological methods. Probiotic bacteria are an important part of human intestinal tract. They have an important role in the function of the immune system due to adhesion to the mucosa of the intestinal flora. They create a inhostile environment for pathogens. Probiotic bacteria are commonly taken in the food like dairy products or food supplements. However, overuse of antibiotics is at risk of passing on the intrinsic resistance that probiotic bacteria have to the pathogenic bacteria. The intrinsic resistence they have to maintain the natural homeostasis of the intestinal tract. It is important to effectively identify risky probiotic bacteria that have the ability to transmit resistance to eliminate their presence in food and dietary supplements. Three methods of DNA isolation like phenol extraction method, magnetic particle isolation and commercial kit isolation were used in the experimental part. DNA was isolated from three dietary supplements, namely Biopron 9 premium, Linex forte and GS Lactobacily forte 21. The purity and concentration of the isolated DNA was detected spectrophotometrically. The presence of individual DNA strains in dietary supplements was confirmed by real-time polymerase chain reaction. The best method of isolation in terms of purity and concentration of isolated DNA was evaluated by RT-PCR and spectrophotometry using a commercial kit isolation method.
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Fluorescence detection in pathogen analysis
Tomečková, Kristýna ; Kristýna, Zemánková (referee) ; Bezděková, Jaroslava (advisor)
This bachelor thesis deals with isolation and detection of bacteria. The used method is called molecular imprinting and is connected with fluorescence microscopy. The theoretical part concentrates on comparison of the developed method with methods that have been used till now. The practical part describes preparation and optimization molecularly imprinted polymers. These polymers were prepared on two different carriers – multititration wellplate and magnetic particles. The bacteria used as imprinted template was Enterococcus faecalis. Staphylococcus aureus was used as its competitor.
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Tau protein, a biomarker of Alzheimer's disease: in vitro phosphorylation and tau-reactive antibodies characterization
Hromádková, Lenka ; Bílková, Zuzana (advisor) ; Fialová, Lenka (referee) ; Krejsek, Jan (referee)
Tau protein, a microtubule-associated protein localized in axonal projections of neurons, is a key molecule in the pathology of Alzheimer's disease (AD), the most common cause of dementia in the elderly population. Tau belongs to the group of natively unfolded proteins without globular structure and is prone to numerous posttranslational modifications (PTMs). Under pathological conditions, abnormal PTMs and misfolding of tau protein occurs and leads to oligomerization and aggregation into paired helical filaments forming neurofibrillary tangles, the histopathological hallmark of AD. Currently available drugs applied in AD treatment can only slow the disease progression and those, which halt the AD-specific neurodegenerative processes, are still missing. Very promising and evolving therapeutic approach is immunotherapy, and even immunomodulation by administration of intravenous immunoglobulin (IVIG) products, a reservoir of natural antibodies from the plasma of healthy donors, has been already tested. The discovery of naturally occurring antibodies directed to tau (nTau-Abs) in body fluids of both AD and healthy subjects and their presence in IVIG begin the investigation of their therapeutic potential. Considering a wide range of possible modifications of tau and of various tau species (oligomers,...
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Sample preparation for DNA analysis from foods of plant origin
Silná, Renata ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
The isolation of high quality DNA is nessecary for many molecular biology applications. However, plant DNA contains high amonts of polysaccharides, polyphenols and various secondary metabolites, which decrease yield and quality of isolated DNA. The aim of this study was preparation of samples and different food matrices for DNA isolation DNA by magnetic particles. It was about 5 species of vegetable and 10 species of processed plant food. Homogenization of samples was performed in CTAB buffer. Isolation of plant DNA was performed by magnetic particles covered with carboxyl groups. All DNAs were isolated in conventional PCR qualities using primers for 700 bp amplicons, in the case of heat processed products for 220 bp ampilicons and for real time PCR. The efficiancy of separation of magnetic particles with DNA by magnetic separator and magnetic needle was compared. It was find out that DNA of higher purity was isolated using magnetic needle. The micromethod of isolation of plant DNA from homogenates with CTAB with magnetic particles is suitable for different processed food.
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DNA isolation using newly designed magnetic carriers
Machan, Radoslav ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
Theoretical part of the master thesis was aimed on giving an overview of basic characteristics of magnetic particles, their morphology, basic methods of their synthesis, interaction with DNA and recent applications in biotechnology and biomedicine. The experimental part of thesis was aimed on application of new designed magnetic particles for isolation of both lactobacilli DNA and calf thymus DNA. Two types of magnetic beads were used: hyperbranched poly(glycidyl methacrylate-co-[2-(methacryloyloxy) ethoxy]acetic acid-co-ethylene dimethylacrylate) microbeads covered with amino groups (P(GMA-MOEAA-EDMA)-NH2) and magnetic non-porous poly(2-hydroxyethyl methacrylate) microbeads covered with carboxyl groups (P(HEMA-co-GMA)-COOH). For both types of microbeads two different protocols for preparation of separation mixtures with two different concentrations od poly(ethyleneglycol) 6000 (PEG 6000) as condensation agent were tested. Differences among both types of magnetic microbeads and DNAs used were found. It was shown that both types of microbeads are suitable for DNA isolation in the presence of 8% PEG 6000.
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DNA isolation from selected vegetable products (paprika)
Gőghová, Sabína ; Kuderová,, Alena (referee) ; Kovařík, Aleš (advisor)
The diploma thesis deals with micromethod of DNA isolation from ten differently processing food products containing pepper (Capsicum annum). PCR ready DNA was isolated by magnetic particles PGMA functionalized by carboxyl groups from homogenates prepared in lysis buffer with CTAB. Quantity and quality of DNA was estimated using spectrophotometric measurements and verified using PCR methods with primers specific for plant rDNA. Quality of isolated DNA varied depending on processing technology. DNA isolated from smoked grinded peppers and from heat treated food products was degraded and amplified with primers F_26S and R_26S (PCR product 220 bp) in contrary to the primers F_18S and R_5.8S (PCR product 700 bp). DNA isolated from the other food products was amplified with primers F_18S and R_5.8S (PCR product 700 bp). PCR product from one grinded pepper (Žitavská paprika) was cloned and sequenced.
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The application of magnetic particles for DNA isolation from cereal products
Starenkova, Anastasiia ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
The thesis has been focused on micro method for isolation of PCR- ready DNA iusing magnetic particles from cereal products. Cereal biscuits and cereal products for babies were selected for the analysis. These were homogenized using plastic copist in lysis buffer with cetyltrimethylammonium bromide (CTAB). The homogenates were purified using chloroform- octanol mixture. The effect of isopropanol in the preparation of homogenates was tested, too. Homogenates were used for DNA isolation by magnetic particles. Two ways to isolate magnetic particles with bounded DNA (magnetic separator and magnetic needle have been tested. Isolated DNA was analyzed spectrophotometrically its concentration and purity were assessed. . After that, amplification of the DNA was tested in PCR. Two sets of primers specific for plant ribosomal DNA were used for their amplification. PCR products of expected length 700 bp and 220 bp were detected by agarose gel electrophoresis. It was shown that DNA isolated from seeds and cereal products using magnetic particles was in PCR-ready quality.
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DNA microextraction from plant vegetable matrix
Cesnak, Filip ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
The aim of the thesis was the comparison of two DNA microextraction methods with the use of magnetic beads from food of plant origin. Samples had disparate and complex matrices and were either raw (broccoli) or processed (strawberry jam). The first method uses a magnetic separator for the manipulation of magnetic beads and was used as a standart for the comparison. The second method uses a paramagnetic needle, the advantage of which should be the possibility to isolate DNA of higher quality without a significant contamination by polyphenolic compounds or proteins. The former method was validated by statistic analysis of results obtained from both methods. DNA quality was judged by testing the amplificability of isolated DNA via PCR. The amplified products were visualised on an agarose gel with electrophoresis.
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