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The employment of HPLC for separation of bioconjugates of azaphthalocyanines II.
Sčensná, Anna ; Kučera, Radim (advisor) ; Lochman, Lukáš (referee)
7 ABSTRACT Charles University, Faculty of Pharmacy in Hradec Králové Department Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate Anna Sčensná Supervisor doc. PharmDr. Radim Kučera, Ph.D. Title of Thesis The employment of HPLC for separation of bioconjugates of azaphthalocyanines II. Bioconjugates of azaphthalocyanines are studied as dark quenchers in molecular probes to monitor real-time PCR. The detection is based on energy transfer between a fluorophore and a quencher, both labelled to the oligonucleotide chain. The fluorophore emits a radiation and the dark quencher switches it off. The efficiency of fluorescence quenching depends on the distance between fluorophore and quencher. Probes can be mono-labelled, in which two oligonucleotides are used, one attached to a fluorophore and the other one to a quencher. The second type is called double-labelled probes, which contain both (fluorophore and quencher) on the same oligonucleotide chain. An unbound probe is randomly coiled, thus fluorophore and quencher approach each other. The fluorescence is not emitted. After hybridisation with a complementary chain, the quencher and fluorophore move away from each other. The terminal nukleosid at 5'end of Taq-man probe is cleaved after hybridisation, where the fluorophore is bonded. After...
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