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Optimization of DNA amplification isolated from buccal swabs for sequencing purposes
ROŠTÍKOVÁ, Kristýna
The topic of my bachelor thesis was the issue of taking primary DNA samples from the buccal mucosa, determination of the ideal length and optimal conditions and subsequent analysis of these samples. In the theoretical part, I focused on the preanalytical phase of the laboratory examination. It was about, the methods of sampling and the type of material that is most often used used for DNA isolation in the molecular biological laboratory. Subsequently, I focused on the methods themselves, which are used for DNA analysis. These included DNA isolation, DNA amplification methods, nucleic acid electrophoresis and DNA sequencing. In the practical part, I processed 42 samples. First, I isolated DNA from the samples and measured its concentration and purity. Then I performed electrophoresis in these samples and based on its results I decided which samples I would process further by PCR. Subsequently, I determined by electrophoresis whether DNA was amplified in the samples. Finally, I selected samples that I sent for sequencing. The aim of my bachelor thesis was to determine the optimal length and storage conditions of samples from the buccal mucosa for further processing. Then I evaluated how these parameters affect the quality, purity and quantity of isolated DNA as well as the course of the polymerase chain reaction. According to the obtained results, the best option is to store the samples in a refrigerator temperature and perform their analysis in the shortest possible time, or to store samples in a refrigerator temperature and process them within weeks to three months.
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