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Optimization of isolation and PCR methods for the identification of CMS in onion
PETERKA, Václav
This bachelor thesis deals with the optimization of different methods of DNA isolation from different types of onion (Allium cepa L.) samples and the subsequent detection of the Ms locus and the detection of cytoplasmic male sterility (CMS). The different types of DNA extractions performed and their results were compared and evaluated. The best quality DNA was isolated from leaves using the CTAB-PVP. These test samples were also used for PCR for the detection of CMS and Ms locus using several different markers, which were compared with each other. The most suitable markers were then used in the PCR for the main samples. DNA isolation by CTAB-PVP was also used for further DNA isolation from the main samples of onion, which were two sterile lines and four fertile lines. The extracted DNA from these samples was first used to detect the Ms locus. Detection was performed using the marker AcSKP1. The samples were collected over several weeks. The Ms locus was detected in all samples (C1C6), regardless of the age of the plant. However, the Ms locus was detected in various forms of zygosity in samples from lines designated C2, C4 and C5. Furthermore, the DNA samples were used to detect CMS in plants and the type of CMS was determined. The markers cob and orfA501were used for this detection. Using these markers, it was found that the lines that were declared sterile (lines designated C1 and C4) were indeed sterile and had the CMS-T sterility type. The lines C2, C3 and C6 that were labelled as fertile were also detected as sterile samples. This means that these lines are not 100% fertile. In the line designated as C5, no sterility was detected in any of the samples.
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Metagenomic analysis of animal gut microbioma based on diet
Spanakis, Martin Nikolaos ; Čejková, Darina (referee) ; Bartoň, Vojtěch (advisor)
This bachelor’s thesis deals with the sequencing of the microbiome and its composi- tion. It focuses on the gut microbiome of animals, which differs among animals with different diets. The work describes theoretical knowledge about metagenomic analysis of the microbiome, such as sampling procedures, various sequencing methods and data processing. The practical part of the work includes the preparation of the dataset, which includes the collection of data and their preparation for the following metagenomic anal- ysis. The result of the work is the taxonomic classification of bacterial species in the samples and the analysis of their diversity according to the type of diet of individual animals.
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Unravelling molecular mechanisms of the abasic crosslink DNA repair
Hušková, Andrea ; Šilhán, Jan (advisor) ; Bařinka, Cyril (referee) ; Pavlíček, Jiří (referee)
4 Abstract DNA as the primary carrier of genetic information guarantees organisms to live, grow, develop and reproduce. However, this most vital molecule in the cell is subject to various damages every moment. If it is not repaired, the cell and the organism will eventually succumb to inevitable destruction. One of the most serious damages is abasic site interstrand crosslink (Ap-ICL). Ap-ICL is formed spontaneously when an abasic site covalently pairs with an adenine on the opposite strand. The lack of information on repair mechanisms, the influence of the local sequence and its stability leads to questions about the fate, toxicity and occurrence of these lesions in cells. During evolution, several mechanisms have evolved to repair these and other damages to ensure the organism's survival. A recently discovered pathway known to repair Ap-ICL is named after the DNA glycosylase responsible for removing Ap-ICL. NEIL3 DNA glycosylase is recruited to Ap-ICL by ubiquitylation of DNA helicase, which is part of the DNA replication complex. NEIL3 glycosylase contains several zinc-finger domains, that bind to the damaged DNA and ensure its catalytic function. The molecular mechanism of the NEIL3 glycosylase repair process is currently not known. In order to answer the aforementioned unknowns, the rate of formation,...
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Influence of makroelements from food on DNA and epigenetic profile
Veselý, Zdeněk ; Kouřilová, Xenie (referee) ; Brázda, Václav (advisor)
The macroelements contained in food have an important function for the human body. They are involved in several of biochemical reactions in the body and their abundance can prevent serious diseases. The theoretical part of the bachelor thesis describes the function of minerals in the human body, the function of DNA and epigenetic mechanisms such as DNA methylation or histone modification. The influence of nutrition and function of selected macroelements – sodium, magnesium, calcium, potassium on epigenetic modifications and on the stability of G-quadruplexes was described. The aim of the experimental part of this work was to study the effect of these substances on DNA structures in vitro and to prepare them experimentally for in vivo studies.
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Use of molecular techniques to characterize yeasts of the genus Metschnikowia
Schneiderwindová, Nicole ; Brázda, Václav (referee) ; Němcová, Andrea (advisor)
This diploma thesis deals with the possibilities of implementation and use of molecular methods for the characterization of yeasts of the genus Metschnikowia and the application of methods in biotechnology or the food industry. The theoretical part focuses on a brief description of yeast, specially selected species that were used during the practical part of the work, the possibilities of their use, and especially on a detailed description of all molecular techniques used. The practical part focuses on the optimization of the molecular methods, namely the method of pulsed gel electrophoresis and the method of denaturing gradient gel electrophoresis. Initially, yeast was cultured under optimal conditions that are specific to this genus. Furthermore, their DNA was isolated using isolation techniques, which were subsequently processed using PFGE and PCR–DGGE methods. The DNA isolation procedure needed to be optimized the most. Several optimizations of the concentration of lysis enzymes, especially the lyticase enzyme, were performed. It was also necessary to determine the correct ratio of low-melting agarose and isolated DNA, which was essential for the correct consistency of the isolated DNA blocks and their further application in PFGE analysis. Finally, the PFGE method was optimized, which brought the correct distribution of chromosomes, and it was possible to describe the individual chromosomes according to their size according to the standard used CHEF of the yeast Hansenula wingei. To properly optimize the DGGE analysis process itself, it was first necessary to isolate the yeast DNA using a kit, then it was used as a template for the PCR reaction. The annealing temperature was also optimized for the individual groups of primers. The amplicons obtained by this reaction were separated by the DGGE method. This technique mainly required the optimization of basic parameters such as the range of the denaturation gradient or the total separation time. According to the measurement results, it can be determined that the process of yeast DNA isolation and their subsequent analysis using molecular methods of pulsed gel electrophoresis and denaturing gradient gel electrophoresis was successful. We were able to describe the genome and determine the number of chromosomes in all used yeast species of the genus Metschnikowia at least partially.
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Biochemical analyses of the DNA interaction partners
Valchová, Michaela ; Coufal,, Jan (referee) ; Brázda, Václav (advisor)
This thesis was focused on DNA analysis. The fluorescently labelled oligonucleotides were at first hybridised and subsequently analysed by HRM analysis to determine the melting temperatures of the oligonucleotides depending on the environment. This thesis describes the change of melting temperature of oligonucleotides in environments containing mono and bivalent ions and the influence of protein binding on the stability of these DNA structures. From determined melting points, it was specified whether the ion/protein stabilised or destabilised the oligonucleotide. Furthermore, plasmids were isolated and analyzed by atomic force microscopy.
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Fourier transformation for exon prediction
Rusina, Michal ; Škutková, Helena (referee) ; Maděránková, Denisa (advisor)
In this bachelor thesis there are described the methods of the prediction of exon. The first part is aimed at the difference between the prokaryotic and eukaryotic organisms, the description of the DNA structure and the explanation of the terms exon and intron. The second section of the theoretic part includes four methods of the prediction of exons namely dynamic programming, neural networks, hidden Markov models and discrete Fourier transform. In the practical part there was created the program called Predikce_exonu that searches exons in nucleotide sequences and works on the principle of the Fourier transform. This algorithm together with 3 freely available programs was tested on 25 sequences and the success of their prediction was described by sensitivity and specificity.
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Analysis of Genomic Data with the Assistance of Algorithm BLAST
Křížová, Martina ; Burgetová, Ivana (referee) ; Jaša, Petr (advisor)
This Bachelor Thesis describes analysis of genomic data with assistance of algorithm BLAST. In theoretic part, it describes genomes, their importance and structure. In this thesis, you can find a description of algorithm BLAST, Smith-Waterman and Needleman-Wunsch and with algorithm BLAST connected Karlin-Altschul statistics. This thesis is discussing types of databases such as spatial, deductive and relational which store genomic data. Second part is concerned with design and implementation of application which use mentioned algorithm and database Oracle 10g R2. Testing and confrontation output of algorithm is also a part of this work.
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DNA Sequence Classification
Heczková, Petra ; Burgetová, Ivana (referee) ; Martínek, Tomáš (advisor)
The work deals with DNA sequence classification. The first part summarizes information about existing methods a their characteristics. In the second part there are description of implementation and experiments. Average sensitivity of method was 65% and specificity 92%.
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Analysis of local DNA structures
Kratochvíl, Jaromír ; Trenz, Oldřich (referee) ; Šťastný, Jiří (advisor)
This diploma thesis is focused on the description and implementation of the user interface for the analysis of local DNA structures as a web application in the Vue.js framework. The local structure can be described as an alternative DNA structure which arises in a specific DNA sequence that occurs in important regions of the genome. The first part of the thesis is theoretical and examines the field of DNA research and a description of selected technologies. The second part deals with the implementation and testing of the user interface on specific DNA sequences.
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