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The application of magnetic nano- and microparticles for the isolation of DNA from selected foods
Ráčková, Lucie ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
In thesis was verified micromethod for isolation of plant DNA from different vegetable (onion and broccoli) and plant food products in quality for application in polymerase chain reaction (PCR). The micromethod allows isolation DNA using magnetic particles from crude lysates of cells obtained by direct homogenization of plant tissues. Various methods of processing homogenates were compared. Homogenization was performed by lysis buffer containing cetyltrimethylammonium bromide (CTAB). The effect of the organic extraction agents was tested (chloroform-octanol and isopropanol). DNA was purified from homogenates by reversible adsorption on magnetic particles (four different types of magnetic particles were tested). The quality of isolated DNA was verified by UV spectrophotometry. The amplificabilty of DNA was tested by polymerase chain reaction (PCR). Specific primers for plant ribosomal DNA (rDNA) were used. PCR products of lenght 700 and 220 bp were detected by agarose gel electrophoresis. Differences in yield and quality of DNA were depended on the homogenate processing and magnetic particles used. The proposed procedure with two magnetic particles was tested for the isolation DNA from plan food products (spreads). DNA was amplified in PCR. Micromethod is suitable for DNA analysis of foods.
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The application of magnetic particles for DNA isolation from thermally processed food products
Hronová, Aneta ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
The thesis has been focused on testing of micromethod of DNA isolation using magnetic particles from thermic-managed food products in a quality suitable for polymerase chain reaction (PCR). Currant jams were selected for the analysis. These were homogenized using plastic copist and stomacher in lysis buffer with cetyltrimethylammonium bromide (CTAB). The effect of chloroform-octanol and isopropanol in the preparation of homogenates was tested. Homogenates were used for DNA isolation by magnetic particles. Rough fraction of DNA was purified by binding on the magnetic particles after centrifugation of the CTAB complexes with proteins, polyphenols and polysaccharides. Two types of magnetic particles were tested: microparticles of poly(hydroxyethylmethacrylate-co-glycidylmethacrylate) - P(HEMA-co-GMA) and nanoparticles of iron oxides covered by poly(L-lysine) - PLL. Isolated DNA was analyzed spectrophotometrically - it was assessed its concentration and contamination by polyphenols and proteins. After that, amplification of the DNA was tested in PCR. Primers specific for plant ribosomal DNA were used. PCR products of expected length 700 bp were detected by agarose gel electrophoresis. It was shown that DNA isolated from currant jams using magnetic particles was in PCR-ready quality.
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The use of magnetic particles for DNA isolation from selected spices
Gaňová, Martina ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
The isolation of DNA from plant tissue of the required quality is very complicated, especially because of the presence of substances that can interfere during amplification of DNA. These substances are mainly polyphenols, polysaccharides, proteins and various dyes. The chemical diversity of such materials can have a significant effect on the yield and quality of DNA using one isolation procedure. The main aim of the work was to evaluate the use of microisolation protocol for related matrices to the quality of the isolated DNA as well as the evaluation of the effect of inhibitors isolated with the nucleic acid to the amplification in the PCR. DNA was isolated from dried paprika (Capsicum annuum). In the first step, the samples were homogenized using a lysis reagent with cetyltrimethylammonium bromide. Subsequently, the DNA was purified by reversible adsorption on magnetic particles. It was tested six different modified particles. The concentration and purity of the obtained DNA was determined by spectrophotometry measuring the absorbance of the DNA solution in TE buffer. The quality of the DNA was confirmed by amplification in PCR. For the PCR were used primers specific for plant ribosomal DNA (rDNA). The presence of PCR products was detected by agarose gel electrophoresis. It was found out that used microassay is suitable for isolating of the DNA of the corresponding purity that is suitable for the genetic analysis by PCR. The differences were found between the magnetic particles that were tested for DNA isolation.
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Modification of Speech Rate
Kovářík, Aleš ; Schwarz, Petr (referee) ; Szőke, Igor (advisor)
This diploma thesis discusses modification of a speech rate. The PSOLA (Pitch Synchronous OverLap Add) method was used for the rate modification. This algorithm works in time domain. Another method -- phase vocoder, which works in frequency domain is also presented in an overview. This thesis extends the PSOLA method with a phoneme recognition, which allows for better understandability of the speech output by considering characteristics of the phonemes beeing pronounced. To examine this proposed method, an application connecting PSOLA and a phoneme recognizer was developed.
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