|
| |
|
|
Preparation of conjugates oligonucleotide probes with functional molecules.
Grossová, Petra ; Miletín, Miroslav (advisor) ; Demuth, Jiří (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Supervisor: doc. PharmDr. Miroslav Miletín, Ph.D. Consultant: Mgr. Michaela Beranová Student: Petra Grossová Title of Thesis: Preparation of conjugates oligonucleotide probes with functional molecules The aim of this thesis was to find the optimal conditions for conjugation of an oligonucleotide to a fluorescent dye by solid-phase click chemistry. The traditional fluorescent dye Cyanine5 was chosen as the labelling molecule. The effect of the concentration of fluorescent dye, the position of the labelling, the type of solid phase and the reaction time were examined. The deprotection conditions were optimized. Two series of reactions followed by deprotection and purification were performed. The efficiency of the reaction was evaluated by comparing the peak areas under the curve of conjugated and unconjugated molecules in the chromatograms of the samples after analysis by HCPL with UV detection. Peak integration was performed in LabSolutions software. The reactions running on the polystyrene solid phase gave the highest yields, with labelling at any position of the chain. For labelling at the 5'-end of the chain, more than 90% efficiency was achieved using all tested solid...
|
|
|
Preparation of the transcription factor FOXK1 DNA binding domain
Procházková, Valérie ; Novák, Petr (advisor) ; Košek, Dalibor (referee)
Transcription factors are proteins that regulate gene expression in different cell types. They play an important role in many cellular processes including regulation of cell cycle and cell differentiation. They possess DNA binding domains to recognize and bind specific DNA sequences. One type of DNA binding domain is the forkhead domain, which contains a region of 100-110 amino acid residues. This sequence is referred to DBD FOX and its spatial arrangement resembles a "winged helix". Proteins of the FOX family interact with double- stranded DNA via the α-helix H3, which represents highly conserved region within the proteins of this family. Other regions of the DBD further contribute in DNA binding, but as not significantly conserved, and their different properties are responsible for variable affinities of individual FOX proteins against binding motifs. Differences in three-dimensional structure may also alter biological functions of FOX proteins in the organism. FOX proteins are divided into 19 subfamilies, including the FOXK subfamily, consisting of two members, FOXK1 and FOXK2. FOXK proteins regulate aerobic glycolysis, cell proliferation and carcinogenesis. Their increased expression has been reported in cancer cells of skeletal tissue, stomach, colon, breast, lung, ovary, etc. However, the...
|
|
|
The importance and role of reverse transcriptases in gene expression analysis
Žucha, Daniel ; Valihrach, Lukáš (advisor) ; Španielová, Hana (referee)
The continuously advancing field of gene expression analysis enables the evaluation of even the slightest changes that occur in the cell transcriptome. In order to ensure accuracy of the observed biological variances, it is fundamentally important to be aware of the possible biases introduced during sample processing. In gene expression research, the methods of reverse transcription−quantitative PCR (RT−qPCR) and RNA- Sequencing (RNA-Seq) are often the primary choice, mostly because of their high precision and reproducibility. Since these both methods require DNA template, they are coupled with the same initial step - reverse transcription (RT), a reaction producing DNA complementary to its RNA template. It is well known that RT introduces bias. As a result, it is therefore of importance to thoroughly evaluate the effects of these biases. One such annotated source of artifacts is the reverse transcriptase (RTase) itself. However, it has been shown that the enzyme does not account for most of the variance alone. Surprisingly, choice of primers or RNA template may influence the reaction outcome even more than the bias introduced from the enzyme. This is especially the case with recent advances in protein engineering. Production of highly efficient RTases may pronounce the variation originating from...
|
|
| |
|
|
Study of DNA oligonucleotides interactions with ethidium bromide by partial-filling affinity capillary electrophoresis
Růžička, Martin ; Koval, Dušan ; Kašička, Václav
A partial filling affinity capillary electrophoresis was developed and applied to investigation of non-covalent molecular interactions between double stranded DNA oligonucleotide (Dickerson dodecamer) and classical DNA intercalator ligand –ethidium bromide (EtBr). Binding constants of DNA EtBr complexes were determined from the dependence of migration time changes of DNA oligomer (applied as analyte) on the length of ligand zones introduced beforehand as plugs of various lengths in hydroxypropylcellulose coated fused silica capillary. Binding constants of DNA-EtBr complex were found to be in the range 4.2 x 103 – 1.5 104 L/mol.
|
guest ::
