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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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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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Isolation and detection of DNA from plant species important for food prodution
Orel, Matúš ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
In the food industry, it is very important to take care of the quality, safety and organoleptic properties of the products supplied. For this reason, food must be checked. However, not all information can be found using conventional techniques such as immunoassays, chromatographic techniques, etc. DNA-based techniques can be used for these cases where traditional procedures are insufficient. Among them, the best known technique is PCR. The aim of the thesis was to isolate DNA from vegetable samples (broccoli, beetroot, carrot and pepper). DNA was isolated using the magnetic particle method and the traditional CTAB method. Both methods were able to isolate the DNA from the vegetable samples in quality and at a concentration suitable for PCR, where the 35S rDNA gene region was amplified (more precisely about 700 bp of the 18S-ITS1-5,8S region). After amplification, the PCR products were subjected to restriction reactions and the results compared to bioinformatic analysis. These steps have succeeded in finding suitable enzymes for diferentiation of PCR products from the tested vegetable species.
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Identification of DNA from plant foods using molecular techniques
Papala, František ; Langová, Denisa (referee) ; Němcová, Andrea (advisor)
The aim of this bachelor thesis is the identification of plant DNA in a complex matrix using molecular techniques. The work first focuses on a search of literature sources concerned with molecular techniques of DNA identification. Next, it deals theoretically with methods of isolation and characterization of plant DNA. Specifically, we shall work with blueberry DNA. In the experimental part, DNA from four commercial products was isolated and subsequently subjected to PCR and HRM analysis.
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Identification of DNA from plant foods using molecular techniques
Papala, František ; Langová, Denisa (referee) ; Němcová, Andrea (advisor)
The aim of this bachelor thesis is the identification of plant DNA in a complex matrix using molecular techniques. The work first focuses on a search of literature sources concerned with molecular techniques of DNA identification. Next, it deals theoretically with methods of isolation and characterization of plant DNA. Specifically, we shall work with blueberry DNA. In the experimental part, DNA from four commercial products was isolated and subsequently subjected to PCR and HRM analysis.
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|
|
Isolation and detection of DNA from plant species important for food prodution
Orel, Matúš ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
In the food industry, it is very important to take care of the quality, safety and organoleptic properties of the products supplied. For this reason, food must be checked. However, not all information can be found using conventional techniques such as immunoassays, chromatographic techniques, etc. DNA-based techniques can be used for these cases where traditional procedures are insufficient. Among them, the best known technique is PCR. The aim of the thesis was to isolate DNA from vegetable samples (broccoli, beetroot, carrot and pepper). DNA was isolated using the magnetic particle method and the traditional CTAB method. Both methods were able to isolate the DNA from the vegetable samples in quality and at a concentration suitable for PCR, where the 35S rDNA gene region was amplified (more precisely about 700 bp of the 18S-ITS1-5,8S region). After amplification, the PCR products were subjected to restriction reactions and the results compared to bioinformatic analysis. These steps have succeeded in finding suitable enzymes for diferentiation of PCR products from the tested vegetable species.
|
|
|
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.
|
|
|
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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