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National Repository of Grey Literature 2 records found  Search took 0.01 seconds. 
Non-template activities of DNA/RNA polymerases and their significance
 Pokorná, Kristýna ; Vopálenský, Václav (advisor) ; Pospíšil, Jiří (referee)
Non-templated addition of adenosine on the 3' end of the product of several DNA polymerases commonly used in molecular biology has been a problematic yet unexplained phenomenon for a few decades now. The only other instance where adenosine is added to the 3' end of an information molecule in a cell is polyadenylation. At the same time, several RNA viruses appear to perform similar action to either edit or polyadenylate their mRNAs. This work focuses on the details of these three highlighted processes and relationship between them and has found out that all of them appear to stem from a common conserved property of the ancestral polymerase. Key words: DNA polymerase, RNA polymerase, non-templated addition, Taq polymerase, polyadenylation
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Construction of modified DNAs with selected reactive or protective groups
 Vaníková, Zuzana ; Hocek, Michal (advisor) ; Křen, Vladimír (referee) ; Zimčík, Petr (referee)
This PhD thesis is focused on the synthesis of DNA modified with photocleavable 2- nitrobenzyl protecting groups in major groove and its applications in the regulation of gene expression in the level of transcription. In the first part of my thesis, the synthesis of photocaged 2'-deoxyribonucleosides triphosphates and their photolysis to unprotected 5-hydroxymethylated nucleotides is described. All prepared nucleoside triphosphates were good substrates for their enzymatic incorporation into DNA. Synthesized 5-(2-nitrobenzyloxy)methyl-2'-deoxyuridine-5'- monophosphate (dUNBMP) and DNA with one 5-(2-nitrobenzyloxy)methyl- modification in the sequence were used for the detailed kinetic studies of photocleavage reactions. In the second part of the thesis, the series of modified DNAs with specific sequences were prepared by primer extension (PEX) and/or polymerase chain reaction (PCR). A cleavage of prepared modified DNAs was studied by selected restriction endonucleases (REs). In all cases, the nitrobenzylated DNA fully resist the cleavage by REs. The deprotection/ photocleavage conditions for nitrobenzylated DNA were studied in the case of DNAs with positive restriction endonuclease digestion of hydroxymethylated DNA. The resulting photocleaved DNA was fully digested by REs, therefore 2-nitrobenzyl...
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