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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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