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Enzymatic synthesis and study of properties of base-modified DNA
Viktorinová, Klára ; Hocek, Michal (advisor) ; Vrábel, Milan (referee)
The influence and specific interactions of chemical modifications in the DNA major groove have been systematically investigated for a long time. This work focuses on the enzymatic preparation of DNA bearing phenyl substituents on bases in milligram scale. For this synthesis, four 2'-deoxyribonucleoside triphosphates bearing an electron-rich phenyl group at position 7 of 7-deazapurines and at position 5 of pyrimidines (dAPhTP, dGPhTP, dCPhTP, dUPhTP) were prepared in prof. Hocek's group. The DNA polymerase KOD(exo-), which was expressed for the purpose of this work, was used to carry out the enzymatic synthesis. All four phenyl-modified 2'-deoxyribonucleoside triphosphates were successfully incorporated into the oligonucleotide chain using the primer extension and the DNA was synthesized in a milligram scale. The individual products were purified by dialysis and HPLC and their denaturation temperature was further monitored on UV-Vis spectrophotometer and their structure by spectroscopic method CD. The main objective of this and further extensive studies is mainly to understand the various biological processes and bioorthogonal reactions of macromolecules. [IN CZECH] Key words DNA, modifications, oligonucleotides, polymerases, PEX, PCR, structural analysis, CD
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