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DNA extraction from plant tissues for polymerase chain reaction analysis
Trojánek, Zdeněk ; RNDr.Roman Matyášek, CSc. (referee) ; Rittich, Bohuslav (advisor)
Extraction of nucleic acids is an important step for all molecular biological studies. The process of isolation of plant DNA is complicated due to the presence of polyphenols, polysaccharides and other metabolites. They can be co-isolated with DNA and act as PCR inhibitors. The aim of this study was to compare CTAB extraction procedure, Qiagen DNA easy kit, direct homogenization, carboxyl-functionalised magnetic non-porous HEMA based microspheres and combination of the above mentioned methods for DNA isolation from different plants. The DNA was evaluated regarding concentration, purity and amplification in PCR. All methods provided DNA that could be used in downstream PCR applications. However, there were differences regarding yield, purity, labour intensiveness and cost. Combination of direct homogenization and magnetic microspheres coated by carboxyl groups was isolated DNA from various plants and plant foods in a quality suitable for convectional PCR, real time PCR and restriction analysis. This method is fast, simple and does not require work with harmful substances.
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DNA isolation from probiotic lactic acid bacteria in food additives
Tvrdíková, Jana ; Vojtíšková, Marie (referee) ; Španová, Alena (advisor)
In this work the functionalised magnetic particles were tested with streptavidin to selective DNA isolation. The method of selective DNA isolation was tested by using DNA probiotic strain Lactobacillus paracasei subsp. paracasei CCDM 211/06. A test was done on the biotinyl oligonucleotic particles, which was immobilised by containing streptavidin and it was used like a DNA probe for isolation complementary DNA chain by means of DNA/DNA hybridization. The primer R 5´ bio and the biotinyl denatured specific PCR product were tested for species Lb. paracasei as a DNA probe. These following experimental conditions were optimized for selective DNA isolation: temperature and time of hybridization, amount of DNA and the release of DNA from microspheres. Isolation of DNA was verified by PCR with specific generic primers. The specific generic PCR product was amplified in extent 250 bp, which was detected by using electrophoresis in agarose gel. This optimized method was successfully used in selective isolation of DNA Lactobacillus from a complementary sample of supplementary food (BIFI pangamin).
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Identification of lactic acid bacteria in fermented dairy products using amplification methods
Tycová, Martina ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Polymerase chain reaction (PCR) is molecular diagnostic method which allows the identification of lactic acid bacteria used in food industry. In this work species-specific PCR primers (targeted on highly conserved 16S rDNA region) were used for identification of bacteria of species Streptoccocus thermophilus in 10 randomly commercially accessible fermented milk products and for identification of species Streptococcus thermophilus in 25 lyophilisates collected in Culture Collection of Dairy Microorganisms Laktoflora (CCDM, Tábor, Czech Republic). The PCR products (968 bp) were detected using electrophoresis in 1,2 % agarose gel. Bacterial DNA was isolated from crude cell lysates by magnetic carriers P(HEMA co GMA) containing carboxyl groups. DNA was reversibly bind on their surface in the presence of high concentrations of poly(ethylene glycol) (PEG 6000) and sodium chloride. Phenol extraction of DNA was used as control. Streptococcus thermophilus strains were identificated using PCR in all analysed samples.
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The use of magnetic microparticles for DNA isolation
Jelínek, Zdeněk ; Horák, Daniel (referee) ; Rittich, Bohuslav (advisor)
The effectiveness of magnetic microparticles in isolation of DNA from Lactobacillus rhamnosus CCM 1825T and DNA from chicken erythrocytes were studied in diploma thesis. Magnetic HEMA based microparticles coated by carboxylic groups and hyperbranched styrene-divinylbenzene particles (IMC AS ČR, Prague, Czech Republic) were used for DNA isolation. Magnetic microparticles Dynabeads® DNA DIRECT™ Universal (Dynal, Norway) based on polystyrene and MPG® Uncoated (PureBiotech, USA) based on magnetic glass were used as a control. The dependence of amount of eluted DNA on concentration of DNA in the base solution and the dependence of amount of eluted DNA on concentration of magnetic microparticles were studied. The affinity of magnetic microparticles to RNA for various concentrations of RNA solution was studied, too. The ability of tested particles to isolate DNA from real samples was validated using milk product Actimel. The quality of isolated DNA of Lactobacillus genus was proved using genus specific PCR.
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Comparison of different types of magnetic carriers for DNA microisolation from foods
Koplík, Jerguš ; Lunerová,, Jana (referee) ; Kovařík, Aleš (advisor)
Micro-isolation of PCR-ready from fresh and dried legumes seeds and food products containing legumes (hummus) was tested. For optimization process magnetic microparticles PGMAox was used. Optimum weight plants and food material was 200 mg. For isolation of DNA from fresh legumes, mixture containing 500 L of CTAB extraction buffer, 1 L -mercaptoethanol and 500 L chloroform-octanol was used. For isolation of DNA from dried legumes seeds and food products volume of CTAB extraction buffer was increased to 1 000 L. To achieve higher purity of DNA some prepared homogenates was precipitate by isopropylalcohol. The optimized process of DNA isolation was used to prepare a homogenate from food products which DNA was isolated by different types of magnetic carriers. For comparison, non-porous magnetic carriers poly(glycidyl methacrylate) PGMAox, poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) P(HEMA-co-GMA)ox covered by carboxyl groups and porous fully crosslinked microparticles poly(styrene-co-divinylbenzene) HPS-B-M-22-NH2, magnetic porous glass MPG and nanoparticles of iron oxides covered by poly(L-lysine) PLL were used. In average the highest concentration and the best purity of DNA was isolated by magnetic carriers P(HEMA-co-GMA)ox a PGMAox.
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Magnetic carriers and their practical use
Chlopková, Barbora ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
The theoretical part summarizes the current knowledge for practical use of magnetic carriers in molecular diagnostics. It includes both already used methods and methods with potential for the future. In the experimental part was tested by use of magnetic media for isolation of DNA from a dairy product and a bacterial culture. It was confirmed that the magnetic carrier DNA was isolated in quantity and quality suitable for carrying out polymerase chain reaction.
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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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Isolation of bacterial DNA from foods using magnetic carriers
Bubeníková, Lucia ; Horák, Daniel (referee) ; Španová, Alena (advisor)
The aim of the work was the selective isolation of bacterial DNA with help of magnetic carriers covered by streptavidine (PGMA-NH2-STV, MPG® Streptavidin). Conditions of functionalisation of carriers using two biotinylated probes were optimized: the amount of carrier, the amount of probe, binding of biotinyled probe to streptavidine. Purified DNA Lactobacillus was used for hybridization. DNA binding to the probe (DNA/DNA hybridization) and nospecific adsorption of DNA to the carrier were tested. Target DNA eluted from the carrier was identified using PCR with primers R16-1 and LbLMA1-rev and with primers P_eub and F_eub. The amount of probe bound to the carrier was estimated using UV spectrophotometry. It was estimated that biotinyled probe can be used for functionalisation in concentration 5 pmol/µl added to the carrier in the ration carrier : probe 1:1. It was shown that nonspecific DNA adsorption to the MPG® Streptavidin is significantly lower than to the carrier PGMA-NH2-STV.Using DNA/DNA hybridization and the MPG® Streptavidin, DNA from pure culture Lactobacillus was isolated. Procedure was applicated for DNA isolation from milk products.
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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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Identification of lactic acid bacteria in hard cheeses using amplification methods
Herzogová, Jitka ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Diploma thesis was focused on identification of lactic acid bacteria of species Lactococcus lactis and subspecies Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris using species and subspecies specific polymerace chain reaction (PCR). PCR method was used for identification of bacteria of species Lactococcus lactis in 10 samples of hard cheeses. The method of sample preparation was evaluated for hard cheeses with the aim to receive sufficient amount of cells for the preparation of crude cell lysates. Whole DNA in quality suitable for PCR was separated using magnetic microspheres P(HEMA-co-GMA) in the presence of polyethylenglycol (PEG 6000) and sodium chloride. DNA isolated by phenol extraction was used as control of DNA isolation. PCR was used to the analysis of 7 strains of Lactococcus lactis from Collection of dairy microorganisms Laktofora (CCDM). Altogether 5 or 2 strains were identified into subspecies Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris, respectively.
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