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Utilization of Catalytic Hydrogen Evolution in Electrochemical Analysis of Nucleic Acids
Fojta, Miroslav ; Daňhel, Aleš ; Špaček, Jan ; Havran, Luděk ; Šebest, Peter ; Orság, Petr ; Pivoňková, Hana ; Vosáhlová, J. ; Schwarzová-Pecková, K.
Catalysis of hydrogen evolution (CHE) at mercury in the presence of proteins was discoverd shortly after the introduction of polarography. In contrast, unmodified nucleic acids have not been reported to produce distinct signals due to the CHE to date. Chemically modified nucleic acids bearing certain extrinsic groups produce analytically useful signals due to hydrogen evloution catalyzed by the respective modifications. These species include (a) transition metal complexes, and (b) non-metal catalytically active organic moieties. In addition, the CHE has been reported to be invoved in guanine reduction process at the mercury-based electrodes.
2D Condensation of 5-Fluorocytosine in Different Electrolytes
Fojt, Lukáš ; Fojta, Miroslav
Two-dimensional (2D) condensation and adsorption of 5-fluorocytosine on hanging mercury drop electrode in differnt electrolytes was investigated. We have used different sodium halide salts as basic electrolytes. The influence of electrolytes composition was displayed using differential capacitance measurements. Effects of ions present in used electolytes solution was remarkable. We observed huge change in 2D condensed layers in dependence on ions type.
A Preliminary Study of Modification of 7-Deazaadenine with a Complex of Osmium Tetroxide with 2,2 '-Bipyridine
Vítová, Lada ; Havran, Luděk ; Fojta, Miroslav ; Šedo, O. ; Zdráhal, Z. ; Vespalec, Radim
Reactivity of a purine nucleic base analogue 7-deazaadenine (A*) in short oligodeoxynucleotide (ODN) towards an osmium tetroxide complex with 2,2'-bipyridine (Os, bipy) was investigated by means of electrochemistry, capillary electrophoresis and MALDI-TOF mass spectrometry. Electrochemical measurement, electrophoretic analyses and mass spectrometric analyses of reaction mixture proved that ODN with an A* residue yields an osmium adduct with properties similar to those exhibited by the well-known adduct of thymine.
Detection of Single Nucleotide Polymorphisms Using Selective Incorporation of Biotin in DNA Strand and Subsequent Enzymatic Detection at Pencil Electrode
Plucnara, Medard ; Ecsin, E. ; Erdem, A. ; Fojta, Miroslav
Enzyme-linked electrochemical assay using DNA labeling by biotin followed by binding of enzyme-streptavidin conjugates has been found to be a potent tool for DNA diagnostic. This approach brings a special advantage of signal amplification due to the fact, that only very small number of enzymatic labels can produce a number of molecules of an electrochemically detectable product from an inactive substrate to obtain sufficiently strong signal. A new way of using of this technique combined with selective primer extension reaction designed for the detection of single nucleotide polymorphism is presented here. The assay was combined with measurements at a pencil graphite electrode, which is a very practical tool for potential clinical applications due to its cheapness and disposability.
Study on the Electrophoretic Behaviour of Short Oligodeoxyribonucleotides in Fused Silica Capillaries. A Preliminary Communication
Vítová, Lada ; Fojta, Miroslav ; Vespalec, Radim
Effects of background electrolyte pH, composition and concentration on the electrophoretic transport of short oligodeoxyribonucleotides (ODNs) in fused silica capillaries have been investigated in the presented study. Mobilities and separation efficiencies of ODNs peaks varied with all three background electrolyte characteristics. Just changes in the background electrolyte characteristics and acid-base properties of ODNs are not sufficient for a systematic explanation of the observed variations. Despite the indicated complexity of ODNs electrophoretic behaviour, we show that very high separation efficiencies of ODNs peaks can be achieved in background electrolytes of physiological pH.
Possibility of Using Purine Oxidation Signals for DNA Sequence Analysis
Špaček, Jan ; Cahová, Kateřina ; Havran, Luděk ; Fojta, Miroslav
Signals provided by electrochemical oxidation of single stranding DNA on the surface of pyrolytic graphite electrode, which correspond to the oxidation of adenine and guanine (A and G) are known for over forty years. The mechanism of oxidation of free A and G as well as nucleoside mono-, and triphosphates of these bases is know but the mechanism of oxidation of purines in the DNA still remains unexplained. Ratiometry of A and G signals could be a new tool for DNA sequence analysis, if signals of A and G oxidation obtained from single-stranded DNA adsorbed at the electrode surface are proportional to the number of oxidized As and Gs in studied samples. Our preliminary results suggest that there is a correlation, yet we encountered problems, which yet has to be addressed and explained before this method could be applied for DNA analysis.
Examples of Using of Electrochemical Detection at Pencil Graphite Electrode with Enzymatic Labeling for Analysis of Nucleotide Sequence
Plucnara, Medard ; Haroniková, Lucia ; Špaček, Jan ; Havran, Luděk ; Horáková, Petra ; Pivoňková, Hana ; Ecsin, E. ; Erdem, A. ; Fojta, Miroslav
Many examples of utilization of enzymatic labeling for DNA sequence analysis has been described in literature so far. Some of them involve hybridization with complementary biotinylated probe, while others use incorporation of biotinylated nucleotides into DNA strand by DNA polymerases. Common approach is then binding of streptavidine-enzyme conjugates to biotin tags and incubation with substrate, which is converted to detectable product. Here, two recent applications using this principle are described for the detection of PCR amplicons and for SNP typing. Both techniques are combined with detection at pencil graphite electrodes.
Studies of Protein-DNA Interactions using Immunoprecipitation with DNA Probes Labelled with Electroactive Groups
Hermanová, Monika ; Špaček, Jan ; Pivoňková, Hana ; Fojta, Miroslav
Different electrochemically active species were tested for labelling of DNA probes and detection of DNA-protein binding. As the DNA probes, oligonucleotides bearing or lacking specific binding site of the p53 protein were chosen. They were tail-labelled either via chemical modification of single-stranded (ss) oligo(dT) DNA tails with an oxoosmium complex, or via enzymatic synthesis of the ss tail by terminal deoxynucleotidyl transferase and deoxynucleoside triphosphates modified with electrochemically reducible moieties. Electrochemical detection enabled to discriminate between sequence-specific and non-specific p53-DNA binding.
Electrochemical Analysis of Sybr Green I and its Interaction with DNA
Dudová, Zdenka ; Pivoňková, Hana ; Havran, Luděk ; Fojta, Miroslav
There are used many methods such real-time PCR and electrophoresis in molecular biology and biochemistry which are based on utilization of a fluorescent dye Sybr Green I (SG) for selective detection of double stranded (ds) DNA and its quantification. SG is a planar molecule (see Fig. 1) that intercalates into the DNA double helix and simultaneously can bind into minor groove of dsDNA. In our work we focused on electrochemical behavior of SG on a pyrolytic graphite electrode (PGE) using square-wave voltammetry (SWV). The voltammetric method was used in measurements of SG interactions with DNA at the PGE.
Voltammetric determination of 1-hydroxy-N-(4-nitrophenyl)naphthalene-2-carboxamide by voltammetry at a glassy carbon electrode in microvolumes of dimethyl sulfoxide
Gajdar, J. ; Goněc, T. ; Jampílek, J. ; Brázdová, Marie ; Bábková, Zuzana ; Fojta, Miroslav ; Barek, J. ; Fischer, J.
Voltammetric reduction and oxidation of 1-hydroxy-N-(4-nitrophenyl) naphthalene-2-carboxamide was investigated at glassy carbon electrode in dimethyl sulfoxide. Cyclic voltammetry was used to investigate the mechanism of reduction of nitro group and oxidation of hydroxyl group. The analyte was successfully determined in dimethyl sulfoxide by differential pulse voltammetry and the whole voltammetric procedure was miniaturised. Square wave voltammetry was employed to reduce the interference from dissolved oxygen. Determination in one drop (20 mu L) of 0.1 mol L-1 tetrabutyl-ammonium tetrafluoroborate in dimethyl sulfoxide provided very similar results compared to determination in the bulk solution. Limits of quantification were 5.0 mu mol L-1 for cathodic voltammetry and 5.3 mu mol L-1 for anodic voltammetry.

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