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Současné metody sekvenování nukleových kyselin
Navrátil, Jiří
The Bachelor´s thesis deals with the description of the techniques of nucleic acids sequencing. Based on an analysis and synthesis of findings in scientific resources, the characteristics of the methods, a brief history, advantages and disadvantages, possible modifications and future applications are described in the theoretical section, whereas special reference is placed on the principle on which these methods work. A special cha-pter is devoted to the Human Genome Project, which is considered the greatest milestone in the history of sequencing. In the practical section, animal genome of order Lepidoptera was sequenced. The entire process included DNA extraction, its purification, amplification, evaluation of quality on an agarose gel, sequencing and, last but not least, an analysis of the output data using specialized software.
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Štúdium možností elektrochemickej detekcie špecifických sekvencií nukleových kyselín
Vaňová, Veronika
This work deals with the study of the possibility of creating an electrochemical biosensor for the detection of specific nucleic acid sequences. The theoretical part describes various types of RNA, with a special focus on miRNAs and possibilities of its detection. It also describes various electrochemical detection approaches, both nucleic acids, and more specifically miRNAs. miRNA is a potential biomarker that can be used for early and non-invasive diagnosis of cancer. The experimental part is aimed at designing, optimizing and preparing a sensitive biosensor for the detection of miRNA-21. A biosensor was prepared to detect the lowest concentration of miRNA-21 in the sample. A linear concentration range for the calibration curve from 1 nM to 1 µM concentration was measured. We measured LOD of 1 fM and by calculation from the regression equation to 3.2 zM and LOQ was 10.8 zM. Subsequently, samples were measured in artificial urine to verify the functioning of the sensor in real conditions. The results showed a minimal effect of the matrix on the determination of the target miRNA.
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Computational Study of the TiO2-Catalyzed Synthesis of Acyclonucleosides from Formamide: Implications for the Origin of Life
Mládek, Arnošt ; Burda, Jaroslav (advisor) ; Futera, Zdeněk (referee)
The TiO2-catalyzed synthesis of nucleosides in non-aqueous formamide environ- ment via so-called acyclonucleoside intermediates represents an alternative way for the emergence of nucleic acids monomeric units, which could address the the fundamental problem associated with the formation of a --glycosidic bond between a nucleobase and a sugar moiety. In this computational contribution we present a plausible reaction route for the prebiotic TiO2-catalyzed synthesis of purine C2- and C3-acyclonucleosides in formamide, which does not require photocatalytic or radical chain mechanisms. The maximum computed activation energy along the proposed reaction channel is ≥ 32 kcal·mol≠1 , which is clearly feasible under the experimental conditions of the Saladino synthesis. We show that the rate determining step of the entire reaction path is the deprotonation of the formaldehyde hydrate methylene carbon occurring likely on defective binding sites of an anatase surface. Our calculations thus support the view of Saladino et al. about the catalytic role of the TiO2 surface in the one-pot synthesis of purine acyclonucleosides in heat formamide solution.
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Education of Molecular Biology at Secondary School and Analysis of Preconception of Nucleic Acids Term
Hanušová, Eva ; Ehler, Edvard (advisor) ; Pavlasová, Lenka (referee)
The theoretical part assembles methodological starting points for the research - the content and meaning of curricula documents for science education; the contemporary scientific knowledge about selected molecularly biological topics taught at secondary schools; the characteristics of student preconceptions and misconceptions, their diagnosis and importance. The practical part contains the setting of research problems and aims related to their object, presented as the content analysis of curriculum and the teaching methods concerning molecularly biological topics at a selected secondary school; the diagnosis of preconceptions regarding the term "nucleic acids" with pupils (first, third and fourth grade) studying at a selected secondary school (ninety- five respondents) that served to the determination of overall character of stated preconception (genesis, modification and conceptual change during the study at a secondary school). The incorporation of new laboratory exercises with the topic of nucleic acids to the education in the fourth grade and the assessment of their impact on the shape of the preconception has been also the part of the project. The evaluation was done by comparing of success in solving the tasks involved in the didactic test for pupils in the first and third grade and pupils...
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Study of Biomolecular Interactions with Surface Plasmon Resonance Biosensors
Šípová, Hana ; Homola, Jiří (advisor) ; Houska, Milan (referee) ; Skládal, Petr (referee)
Surface plasmon resonance (SPR) biosensors represent one of the most advanced sensing technologies for real-time studies of biomolecular interactions. In this thesis, methods for functionalization of SPR substrates were optimized and studied via spectroscopic methods. Effects related to the SPR sensor microfluidic interface on the measured biomolecular interactions were analyzed, and furthermore, means to decrease mass-transport limitations were proposed. Several SPR-based assays regarding the detection of nucleic acids were developed, which allow for the detection of physiologically relevant concentrations of nucleic acids as well as point mutations in a nucleic acid sequence. Assays for the determination of the enzymatic activity of HIV integrase and ribonuclease H were developed. These assays can be employed for the design and synthesis of molecules that function either as antiviral drugs or as gene-regulating agents.
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Determination of structure and dynamics of biomolecules by theoretical calculations of NMR spectroscopic parameters
Benda, Ladislav ; Sychrovský, Vladimír (advisor) ; Munzarová, Markéta (referee) ; Hrabal, Richard (referee)
iv Abstract Subject: Determination of structure and dynamics of biomolecules by theoretical calcu- lations of NMR spectroscopic parameters Author: Ladislav Benda, ladislav.benda@gmail.com Department/Institute: Institute of Organic Chemistry and Biochemistry, AS CR Supervisor: Dr. Vladim'ır Sychrovsk'y, Institute of Organic Chemistry and Biochemistry, AS CR, vladimir.sychrovsky@uochb.cas.cz Abstract: This doctoral work was focused on theoretical modeling of nuclear magnetic resonance (NMR) parameters in peptides and nucleic acids. Dependences of NMR para- meters on molecular structure and solvation were primarily modeled. Great emphasis was put on the comparison of the calculated data with the NMR experiment. The molecular models studied included the l-alanyl-l-alanine di-peptide (AA) and the phosphate group of nucleic acid backbone. Conformations of all three charged forms of AA in solution were determined and the respective pH-induced changes of experimental NMR chemical shifts and nuclear spin-spin coupling constants were explained. Dependences of NMR cross-correlated relaxation rates on the AA backbone geometry were calibrated. The 31 P NMR parameters in nucleic acid phosphate were systematically calculated in dependence on the backbone conformation and the phosphate solvation pattern. Qualitative rules...
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Ribonucleic acids extraction in sweet cherry
Vojáčková, Karolína ; Sedlák, Petr (advisor) ; Korecký, Jiří (referee)
This bachelor thesis deals with the Isolaton RNA in sweet cherries. The turning point was the literature review on this topic. First chapters mentions the problemes of studying two main topics, which are RNA and a description of cherry (Prunus avium). Sweet cherry is a part of Noble hardwoods. Researches of sweet cherries can deal with an insufficient amount of quality planting material needed to breed new varieties. The biggest attention has given to the methods of RNA isolation, their comparison and analysis of already isolated RNA. The work describes five methods most commonly used for the isolation of RNA by phenol-chloroform, adsorption to silica, CTAB, TRIzol, magnetic separation and the using commercially marketed kits.
Phenol-chloroform method divides the lysate with a mixture of phenol, chloroform and isoamylalcohol in two phases, an upper aqueous, that includes the nucleic acids and lower organic.
Adsorption on silica using properties of nucleic acids bind to the surface of the silica in the presence of chaotropic salts.
CTAB method involves the use of extraction buffer with CTAB, which forms a precipitate with the nucleic acids. The pellet is then resuspended in DEPC-water.
TRIzol method uses a combination of guanidinium thiocyanate-phenol-chloroform. Like the phenol-chloroform method produces a precipitate, but treatment with guanidine thiocyanate, upper phase contained only RNA.
During the magnetic separation are the nucleic acids bound to the surface of the magnetized particles.
Subsequent genetic analysis that can detect and quantify the isolated RNA may be used in RT-PCR analysis, electrophoresis, and microarray hybridization methods northern blot and dot blot.
Bachelor thesis has written as a compilation literature review to inform the about mentioned topics. Any own research part is not here, because of the compilation character.
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Microarray Data Interpretation
Ludwig, Petr ; Šilhavá, Jana (referee) ; Smrž, Pavel (advisor)
This Bachelor thesis explains the basics of biochip or microarray data interpretation, starting with short introduction to genetics, especially genetic information significance evaluating. The focus was set mainly on the set of scripts transforming and analyzing the sample data. The data used in this thesis are a result of biochip analysis of the Colon Tumor tissues. The secondary result represents disclosing the main marker for this particular type of cancer, the primary result is evaluation of marker significance in the context of signaling pathways. The resulting pathways are sorted by relevance.
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Methodics of molecular detection of Barley yellow dwarf virus in vectors by RT-PCR
Jarošová, Jana ; Jaňourová, Blanka ; Kumar, Jiban
Barley yellow dwarf is one of the most important viral diseases of cereals in the world. It can cause significant yield losses in most cereal species, including wheat, barley, rice, corn, oat and rye. In this methodology we provide assays for reverse transcription - polymerase chain reaction (RT-PCR) for sensitive detection of barley yellow dwarf virus directly in the vectors, in aphids. This way it is possible to evaluate infectivity of vectors and from that conclude subsequent measures. The methodology is meant to serve the employees of State Phytosanitary Administration.
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Interactions of Zn2+ ions with proteins and nucleic acids
DVOŘÁKOVÁ, Kateřina
Interaction of Zn2+ ions with proteins and nucleic acids Zinc is an important metal in biological systems. It is a strong Lewis acid, forms a stable Zn2+ ion and can exist in several coordination geometries. Zinc is required for the activity of more than 300 enzymes. In proteins zinc can either participate directly in chemical catalysis or can be important for maintaining protein structure and stability. In this work a dataset of high quality (resolution better than 3 Ǻ) crystal structures deposited in the Protein Data Bank on internet have been examined to identify typical zinc binding sites and to establish their coordination geometries. Totally 135 zinc binding sites found in 74 structures have been analyzed. The most frequent coordinating atoms are sulphur in cystein, NE2 and ND1 nitrogens in histidine followed by oxygen atoms in carboxylates of aspartic and glutamic acids. 67% of zinc binding sites were found to be four-coordinated with tetrahedral coordination geometry. In contrast Mg2+ cations show a clear preference to bind to oxygen atoms and to form octahedral cavities. We have not found any structure with a direct zinc coordination to DNA except the 1ZQT structure which was however excluded due to its low resolution (> 3 {\AA}). Similarly only three zinc binding sites have been found in RNA structures which were however determined in the protein free environment. All other zinc binding sites have been located in proteins even in the presence of the nucleic acids.
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