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Molecular analysis of genetic predispositions to Crohn´s disease and ulcerative colitis
ROUBÍKOVÁ, Natálie
This bachelor thesis focuses on idiopathic inflammatory bowel diseases, which include Crohn's disease and ulcerative colitis. These are chronic inflammatory diseases of the intestines caused by several causes. Studies have shown that the development of these diseases is most commonly due to genetic predisposition, external factors such as stress and smoking, infection, and in some cases, an abnormal immune system reactivity to components of the gut microbiome. The genetics of these diseases have not yet been fully elucidated, but it is clear that genetic factors are more apparent in Crohn's disease than in ulcerative colitis. The most common genes associated with the development of idiopathic inflammatory bowel diseases are NOD2/CARD15, NOD1/CARD4, and ICAM-1. It is known that the NOD2/CARD15 gene is more involved in the development of Crohn's disease, while the ICAM-1 gene is more involved in the development of ulcerative colitis. In the methodological part, this thesis aims to demonstrate at least one relevant gene mutation in 12 probands, of whom six suffered from Crohn's disease, three from ulcerative colitis, and three were healthy individuals who were related to the affected individuals. The relevant genomic DNA was isolated from the samples and used in the PCR method to amplify a specific gene segment. The PCR products were visualized using gel electrophoresis, then enzymatically purified and sequenced by the GenSeq company. Finally, the relevant sequences were verified and evaluated using the BLAST internet tool, the GenBank internet database, and the Geneious Prime program. As a result, a gene mutation was demonstrated in two cases - NOD1/CARD4 (D372N) and ICAM-1 (K469E).
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Optimization of calcium chloride concentration for removal of polysaccharide contamination during plant DNA isolation
Frnčová, Ekaterina ; Šlosárová, Katarína (referee) ; Fialová, Lenka (advisor)
The greatest difficulty in isolating DNA is the presence of contaminants that cause side effects. Polysaccharides are the most common contaminants in fruits. They can distort the results in spectrophotometric determination of purity or act as inhibitors in PCR analysis together with other substances (for example, proteins or phenolic substances). The aim of this work was to investigate the influence of different concentrations of calcium chloride on the process of DNA isolation. In the experimental part, DNA from the apple was isolated using different concentrations of calcium chloride. The isolation was carried out four times, and each time the sample was adjusted in different ways. It was found that the isolation method used works only with a sample that has been lyophilized. Isolation of DNA from fresh fruit provided very low yields. Probably, this was due to the large water content in the sample, and the proportion of the solid component was smaller. Subsequently, PCR analysis and electrophoresis were performed to determine the amplifiability of the isolated DNA. Two sets of primers with different specificity were used for this analysis. Amplifiability was confirmed only when using primers specific to apple DNA when using 100 mM solution of CaCl2. Other samples have been amplifiable using both types of primers. Probably, samples isolated using a 100 mM solution of CaCl2 had a larger amount of inhibitors that do not affect all PCR reactions equally, which may also indicate a small effectiveness of this amount of CaCl2.
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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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Yeasts and wine
Palíková, Petra ; Vadkertiová, Renata (referee) ; Vránová, Dana (advisor)
This thesis deals with isolation and identification wine yeasts from grapes and must. For analysis was used white wine Sauvignon that was grown and producing after needs ecological agriculture. Remove samples were processed in laboratory and by the help of dilution method were obtained pure culture isolated yeasts. In the following step, by the application of commercial kit UltraCleanTM Microbial DNA Isolation Kit we were able to isolated individual DNA that it was used to the next analysis. Isolated DNA was amplification by PCR method with ITS1 and ITS4 primers. PCR products were detected on agarose gel. Amplification samples were chopped five restriction endonucleases: HaeIII, HinfI, TaqaI, AluI and MseI. Chopped DNA was detected by the same way as PCR products and it was compared with restriction patterns of collection yeasts. In the next step it was compared genetic similarity of isolated yeasts by using BioNumerics software. As a criterion it was used Pearson coefficients and UPGMA clastering analysis. The result is dedrogram of genetics similarity isolated yeasts.
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Preprocessing of 1D gel electrophoresis image
Svozilová, Veronika ; Škutková, Helena (referee) ; Vítek, Martin (advisor)
The aim of this work is to propose an algorithm in MATLAB, allowing image processing and evaluation of one-dimensional gel electrophoresis. At first describes the detection of samples based on the horizontal segmentation of electrophoresis gel image, followed by detection of the minimum intensity values of the image, a vertical segmentation and determination of the sample boundaries in the image. The final phase involves testing of the method on publicly available images and evaluation of the results.
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Identification of PHA producing bacteria employing molecular techniques
Gajdová, Barbora ; Obručová,, Hana (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with identification of bacteria which are capable of producing polyhydroxyalkanoates (PHAs). Work included testing variety of genera including Pseudomonas, Lactobacillus, Bifidobacterium, thermophilic cultures and samples gathered from natural sources. Bacteria were investigated by molecular technique polymerase chain reaction – PCR. An amplification of the PHA synthase gene (phaC) was analyzed. In the first reaction phaC and 16S rRNA genes were tested at the same time. 16S rRNA gene is used as control for bacterial DNA and as an identification tool for natural source samples. This multiplex PCR used multiple primers in PCR mix. Second reaction search for amplicon specific for catalysing biosynthesis mcl-PHA (phaC1). The presence of the PHA synthase gene was verified in 11 samples which were Bifidobacterium breve CCM 7825T, Lactobacillus rhamnosus CCM 1825T, Lactobacillus zeae CCM 7069T, Lactobacillus delbrueckii subsp. bulgaricus CCM 7190T, Lactobacillus plantarum CCM 7039T, Pseudomonas gessardii, Pseudomonas fulva, Arthrobacter protophormiae, Curtobacterium flaccumfaciens, Mycobacterium neoaurum and Staphylococcus lentus.
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Compensation of geometric distortion of electrophoretic gel image
Dvořáček, Tomáš ; Vítek, Martin (referee) ; Škutková, Helena (advisor)
This master thesis is engaged in problematics of creation and compensation of geometric distortions in 1D agarose electrophoresis. This master thesis analyze the problematics of cause of these distortions and summarize the theory needed for compensation of these distortions. Based on acquired theory and created electrophoretic phantoms, the master thesis contains several suggestions for compensation of incurred distortions. These suggestions are recreated into functions, which are connected into a functional user interface for gel image analysis and geometric distortions compensation.
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Evaluation of basic characteristics of thermal water with separation methods
Skálová, Lucie ; Mravcová, Ludmila (referee) ; Vávrová, Milada (advisor)
The presence of inorganic ions is one of the criteria for thermal water assessing. The most important anions that affect the water quality and allow their use for therapeutic purposes, include sulfates, chlorides, nitrates and bicarbonates. Ions (as sodium, potassium, calcium and magnesium) are the significant cations. The ion chromatography is used for separation of these substances and also some methods based on electrophoretic migration of ions in an electric field. The electromigration method of isotachophoresis was chosen for determination of selected ions in water samples collected from thermal boreholes in Pasohlávky.
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Analysis of low-molecular proteins in barley by the SDS-PAGE method during malting
Myslivcová, Pavla ; Svoboda, Zdeněk (referee) ; Mikulíková, Renata (advisor)
This diploma thesis deals with the analysis of low molecular weight proteins in barley during malting by SDS-PAGE method. Attention was paid to PR proteins, specifically LTP proteins and thionins, considered to be connected with gushing effect. Barley samples, malt intermediates and malt samples taken in 10 consecutive days to cover the whole malting process were used for the experiment. In total, 5 samplings were used for the analysis. Proteins extracted from the samples were separated by SDS-PAGE using a Tris-tricine buffer system. The protein lines of LTP proteins and thionins were identified in the resulting gels. The relative optical density values of the selected protein bands were obtained to assess the effect of malting on these proteins. A similar pattern of change in the content of mentioned low molecular weight proteins during the malting process was observed. This was confirmed by finding a statistically significant positive correlation between the relative optical density values of LTP proteins and thionins. Furthermore, the relationship between the low molecular weight protein content and the gushing potential and the microbiological contamination of the samples was investigated, but was not confirmed.
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Application for the Data Processing in the Area of Genome Engineering
Brychta, Jan ; Burgetová, Ivana (referee) ; Očenášek, Pavel (advisor)
This masters thesis has a few objectives. One of them is to acquaint with the problems of genome engineering, especially with fragmentation of DNA, the macromolecule DNA, the methods for purification and separation of the nucleic acids, the enzymes used for modification of these acids, amplification and get to know with cluster and gradient analysis as well. The next aim is to peruse the existed application and compare it to the layout of the proposed application, that is the third aim. The last one from the objectives is the implementation and the report how was the application tested by the real data. The results will be discussed as well as the possibilities of the further extension.
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