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Development of a new DNA mutation detection technology for application in cancer diagnostics
Janoušková, Kristýna ; Minárik, Marek (advisor) ; Klepárník, Karel (referee)
The aim of this diploma thesis was to test a new technology for DNA mutation detection and to optimize the parameters related to the introduction of a new method, that uses this technology, into oncological diagnostic practice. The new method consists of an experimental modification of an already used method for the detection of somatic mutations in tumor DNA, which is based on heteroduplex analysis and denaturing capillary electrophoresis. The new aspect of the technology is the use of short oligonucleotide probe, which significantly simplifies the interpretation of data and also enables the simultaneous examination of multiple mutations in one reaction (multiplexes). Key words: DNA, denaturating capillary electrophoresis, heteroduplex, mutation, PCR, probe
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Development and optimization of oligonucleotide separation using a novel recycle HPLC approach
Holoubková, Barbora ; Minárik, Marek (advisor) ; Tesařová, Eva (referee)
1 Abstract Separation of biologically important substances such as proteins, peptides and nucleic acids, has become an important area of application for various modes of high-performance liquid chromatography in recent years. One of those applications is preparative chromatography for isolation of pure oligonucleotide synthesis products. Oligonucleotides are of fundamental importance in the field of contemporary scientific and commercial spheres. They play and irreplaceable role in the form of short sequences (primers or probes) for a variety of molecular methods including polymerase chain reaction (PCR), DNA sequencing, detection of pathogens by specific hybridization etc. The preparation of perfectly defined and pure oligonucleotides is often crucial for the success of these methods, so the efficiency of isolating the target sequence from incomplete synthesis products or other unwanted impurities is very important. Current procedures do not have sufficient enough resolution. The concept of recycling chromatography, where the analytes are returned to the column for repeated passage after leaving the column and thus their resolution is improved, has been known since the 1960s. Recently, however, a completely new concept was introduced, the so-called Repetto system, where segments of the separated mixture...
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Brain Tumor Cells Genome Instability. Correlation of clinial, morphological and molecular-cytogenetic data
Kramář, Filip ; Michalová, Kyra (advisor) ; Minárik, Marek (referee) ; Novák, Zdeněk (referee)
Gliomas are brain tumors arising from neuroglia. In most cases astrocytic or oligodendroglial component is the main element of the tumor. Non-random chromosomal abberations are found in tumor cells as was revealed previously. The aim of this study was a fluorescence in-situ hybridisation analysis (FISH) of tissue samples obtained during neurosurgical procedures, determine the frequence of selected chromosomal abberations, further correlation with morphological and clinical data and statistical analysis of the results. During six years 264 tissue samples were gained in which FISH with defined probes was performed. The acquired results were compared with histological analysis and selected clinical data (age, Karnofsky score, extent of resection, overall survival). The whole series was divided into 7 groups by tumor type for further statistical analysis. In every group median and mean survival time was calculated, Kaplan-Meier analysis was focused on influence of selected parameters to overall survival. In some categories Cox regression model was created to achieve a hazard ratio of selected parameters. In WHO Grade II and III tumors the risk of malignant progression and tumor upgrading is significantly higher in comparison with samples where specific abberations were not found (EGFR amplification, CDKN2A and...
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Using modern molecular-genetic methods in prevention, early diagnosis, prediction of therapy response and prognosis
Benešová, Lucie ; Minárik, Marek (advisor) ; Brdička, Radim (referee) ; Klepárník, Karel (referee)
55 5 Závěr V prezentovaných pracích jsme se zabývali vývojem a praktickou aplikací metod pro rychlou, citlivou, vysokokapacitní detekci molekulárních markerů u různých typů nádorových onemocnění. Vyvinuli jsme v této souvislosti dvě nové metodiky. První je založená na cyklujícím teplotním gradientu, který poskytuje efektivnější separaci DNA molekul a možnost opakovaného dávkování v jedné analýze a tedy výrazné zvýšení celkového počtu analyzovaných vzorků 1 . Druhá metodika založená na dynamickém značení DNA molekul interkalačním činidlem během elektroforetické analýzy odstraňuje potřebu fluorescenčně značených primerů a dělá tak analýzy finančně i prakticky dostupnějšími 3 . Obě tyto metodiky jsme následně použili při analýzách klinických vzorků pacientů s nádorovým onemocněním. Výsledky uvedených publikací zaměřených na klinické aplikace ukazují, že vyšetření molekulárních markerů zahrnující mutace, alelické ztráty a jednonukleotidové polymorfismy má široké uplatnění. Charakterizace vzorků kolorekta na molekulární úrovni, jehož optimalizaci jsme provedli, může mít význam při popisu stádia transformace adenomu v karcinom nebo při odhadu prognózy onemocnění na základě detekovaných typů mutací 2 . U pacientů s podezřením na karcinom pankreatu může molekulárně-biologické vyšetření v kombinaci s cytologickým...
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Development and validation of a new method for enrichment and detection of circulating tumor DNA in cancer patients
Pláničková, Lenka ; Minárik, Marek (advisor) ; Kuthan, Martin (referee)
Tumors are one of the leading causes of death worldwide. Generally, the prognosis is better if the treatment begins at an early stage. Nowadays, the conventional chemotherapy treatment of cancer, known for its limited efficacy and side effects, is being gradually replaced by targeted biological treatment, which is used when specific genetic mutations are found. A part of the treatment is a detection of a potential progression, which is mainly based on the tumor biomarkers monitoring. Currently, further investigation of a so-called liquid biopsy method are ongoing, on which this thesis is focused. The main aim of this work was the experimental development and validation of the method for detection of the ctDNA in the plasma samples based on the somatic mutations presence. For the development and optimization of the system on the principle of denaturation capillary electrophoresis, the samples of cancer patients with KRAS mutation were used. Subsequently, a clinical part of the research was performed on a pilot set of 21 plasma samples. Finally, the method was optimized for the detection of BRAF and EGFR markers. A partial objective was to improve the detection sensitivity and increase the capture of the ctDNA in patients with advanced stage of the disease. The results of this work suggest the...
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Brain Tumor Cells Genome Instability. Correlation of clinial, morphological and molecular-cytogenetic data
Kramář, Filip ; Michalová, Kyra (advisor) ; Minárik, Marek (referee) ; Novák, Zdeněk (referee)
Gliomas are brain tumors arising from neuroglia. In most cases astrocytic or oligodendroglial component is the main element of the tumor. Non-random chromosomal abberations are found in tumor cells as was revealed previously. The aim of this study was a fluorescence in-situ hybridisation analysis (FISH) of tissue samples obtained during neurosurgical procedures, determine the frequence of selected chromosomal abberations, further correlation with morphological and clinical data and statistical analysis of the results. During six years 264 tissue samples were gained in which FISH with defined probes was performed. The acquired results were compared with histological analysis and selected clinical data (age, Karnofsky score, extent of resection, overall survival). The whole series was divided into 7 groups by tumor type for further statistical analysis. In every group median and mean survival time was calculated, Kaplan-Meier analysis was focused on influence of selected parameters to overall survival. In some categories Cox regression model was created to achieve a hazard ratio of selected parameters. In WHO Grade II and III tumors the risk of malignant progression and tumor upgrading is significantly higher in comparison with samples where specific abberations were not found (EGFR amplification, CDKN2A and...
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