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Examination of the polymorphism of the gene ApoE
NOVOTNÁ, Tereza
Alzheimer´s disease (AD) belongs to the most frequent cause of dementia in the world. It is determined that it is about 65-75 % of all the dementias (Preiss, Přikryková Kučerová, 2006). The term Alzheimer Dementia, as well as Pre-senile Form (AD) is used with the patients at the age up to 65 years (Seidl, 2015). After the age of 65 years we use the term Senile Dementia of the Alzheimer Type. (SDAT). Up to the age of 45 this desease is rare (Seidl, Obenberger, 2004). Causal treatment of this disease does not exist, because etiopathogenesis of the disease is not entirely clarified (Seidl, 2015). The aim of this bachelor´s work was to write a professional search to the given topic. In this theoretical part there is summed up the information about neurodegenerative disease, about dementia and (AD). Most importantly there are described the symptoms, the causes, phase, treatment, diagnostics and genetic disposition of AD. The description of polymerase chain reaction (PCR) and reverse hybridization on strips is also an important part of this theoretical part. In the practical part, the main goal was to master molecular biological methods. To achieve the goals and to get the best results, I chose two methods. In addition to optimizing the PCR ARMS method, reverse hybridization was used on strips. The above-mentioned second method was performed using the commercially available ApoE StripAssay? kit from ViennaLab. In this part of the paper are described all the procedures by which I achieved the results. Molecular biological methods were performed in a laboratory at the Health and Social Faculty of the University of South Bohemia in České Budějovice.
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Significance of detection of the ApoE gene and MTHFR gene polymorphisms for osteoporosis.
ŠILHOVÁ, Markéta
Osteoporosis is the sceletal metabolic disease characterized by changes of bone tissue and disfunction of mechanic bone resistance which can lead to numerous bone fractures. Osteoporosis is frequently diagnosed in patients older than 60 years, also advanced age patients are not an exception for this disease. The most frequently affected group of patients is postmenopausal women. Prevalence of osteoporosis still rises. Nowadays more than 200 millions of people suffer from osteoporosis and one million of patients from Czech republic. Osteoporosis have became a serious complication affecting not only medical but also an economic field. Based on these findings, osteoporosis should be considered as an important topic even from the perspective of genetic risk factors. The aim of my bachelor thesis is to write a specialized research focused on introduced topic, summarize accessible information about osteoporosis, including diagnosis and genetic predisposition. I have focused especially on genes ApoE and MTHFR677. ApoE plays an important role in sceletal homeostasis. The highest risk for osteoporosis development is the presence of alel 4 gene for alipoprotein E. Gen MTHFR677 is mostly associated with bone mineral density located in femoral neck. An experimental part of bachelor thesis was based on laboratory practice in molecular biology field inluding isolation of DNA from peripheral blood, PCR technique, detection of PCR products by gel agarose electrophoresis, analysis of ApoE gel by MyTaq Red DNA Polymerase kit and 2x PCRBIO HS Taq Mix, analysis of MTHFR677 gene by MyTaq Red DNA Polymerase kit. Analysis of ApoE gene is based on ARMS PCR method, on the other hand analysis of MTHFR677 is based on RFLP PCR method. I have performed the laboratory practice within the Laboratory of genetics GENLABS in České Budějovice.
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Application of the method RFLP PCR , PCR ARMS and reverse hybridization for detecting the most common thrombophilic mutations in the Czech population
MATĚJKOVÁ, Markéta
Recently the term thrombophilia is becoming a current topic. It is an increased precondition for the creation of tromboses and the following complications as the result of increased blood coagulation which could be very serious or even fatal. A timely diagnosis of thrombophilia mutations helps patients to avoid many critical situations (e.g. in case of operations, pregnancy etc.). In these days there are at least 30 genetic laboratories in the Czech Republic which research thrombophilia mutations. Unfortunately this medical examination is covered by insurance companies just in case of indicated symptoms which are under strict criteria. If someone is interested in a medical examination and a doctor does not indicate him with the symptoms, they are forced to pay the examination on their own. There are a few insurance companies which contribute with some amount for these examinations as a part of their preventive programmes (e.g. Česká průmyslová zdravotní pojišťovna, Zdravotní pojišťovna ministerstva vnitra ČR or Zdravotní pojišťovnu Revírní bratrská pokladna). The aim of my bachelor thesis was a practical mastery RFLP-PCR method, PCR ARMS method and a reverse hybridization on strips to detect the most common mutations of thrombophilia in the population of the Czech Republic and deviloving on the issue. In the theoretical part, I focus on a description of the most important thrombophilic mutations in the Czech population and their risks. I also describe particular genetic methods by which each of thrombophilia mutations is being examined in genetic laboratories. My own results of the examination of four major thrombophilia mutations and polymorphs are shown in the practical part. These are the Leiden mutation, prothrombin mutation, MTHFR 1298 and MTHFR 677 polymorphs. For my examinations I used these genetic methods: RFLP-PCR method, PCR ARMS method, reverse hybridization on strips. I made all these methods in the genetic laboratory Genlabs in České Budějovice. For the examinations I used DNA samples of my family.
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Detection of mutations associated with increased incidence of breast and ovarian tumors using modern molecular biology methods
RÁJOVÁ, Kristýna
Breast cancer and ovarian cancer is one of the most common malignancies what women have in the Czech Republic and the incidence of new diagnoses has a tendency to increase. It is estimated that 5-10 per cent of these tumours is cause by pathogenic mutations in highly penetrance tumour suppressor genes BRCA1 or BRCA2. The main function of these genes are repair of double strand DNA breaks and another function is control of cell division and participate on regulation of transcription and chromatin remodeling. The aim of this bachelors thesis is to summarize current knowledge of the "Gene mutations" topic, the function of the genes BRCA1 and BRCA 2 and their link with hereditary breast and ovarian cancer, an overview of possible detection methods and particularly also the summary of the Czech studies of detection mutations in the Czech Republic. It pointed out to the strong effect of the founder mutation. Among the most frequently found mutations are mutations as c.5266dupC, c.181T> G and c.3700_3704del5 in the gene BRCA1 and c.7913_7917del5 and c.8537_8538del2 in the BRCA2 gene. In the experimental part of the work I have tried to optimize ARMS PCR method for point mutation c.5266dupC. This mutation has proved to be one of the most common mutations not only in the Czech Republic but also in Europe. The objective was to optimize the PCR ARMS in the conditions of the GENLABS Ltd. laboratory. The main goal of the optimization was the selection of appropriate PCR mix and optimal temperature of PCR profile at which the quality and quantity of the yield was visualized on gel electrophoresis as specific and highest as it is possible. The aim of this part is also practically to do the DNA isolation from peripheral blood and buccal swab, preparation and execution of the PCR reaction and detection of PCR products by gel electrophoresis. Eventually, we managed to optimize a method for the two different matrices for the DNA isolates, and peripheral blood. Unfortunately the method for the buccal swab was not successful. During gradually temperature increasing, we started the process of annealing at 55°C, the best results were achieved at a temperature of 65.5 ° C using gb Basic PCR Master Mix from the Generi Biotech firm. In the future this optimization might allow the using of method for mutation in the gene BRCA1 c.5266dupC for the routine practise in the laboratory.
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