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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.
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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.
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Electrochemical Reduction and Oxidation of Nucleic Acids Bases and their Analogues: a Brief Overview
Fojta, Miroslav ; Špaček, Jan ; Dudová, Zdenka ; Pivoňková, Hana ; Daňhel, Aleš ; Fojt, Lukáš ; Havran, Luděk
Nucleic acids are known as electroactive biomacromolecules containing electrochemically reducible or oxidizable constituents. Nucleobases cytosine, 5-methylcytosine, adenine and guanine can be reduced in aqueous media on mercury or silver amalgam electrodes. Oxidation of all natural nucleic acids bases (in addition to the above mentioned ones, also uracil and thymine) was demonstrated using various types of carbon electrodes. Some of synthetic nucleobases or nucleotide analogues (e g., 7-deazapurines, cytidine analogues used as epigenetic modulators, etc.) exhibit specific electrochemical properties that differ from those of the parent bases and can be utilized to determine the given substance in the presence of natural nucleic acids or their components.
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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.
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Analysis of Denatured PCR Products Modified with 7-deazapurine Bases at Hanging Mercury Drop Electrode
Dudová, Zdenka ; Špaček, Jan ; Havran, Luděk ; Pivoňková, Hana ; Fojta, Miroslav
7-deazapunines are synthetic analogues of natural purine nucleobases which can pair with pyrimidines, retaining pairing complementarity of their parent purines. There is replaced N7 atom by CH group in 7-deazapurines so that DNA modified with them don't participate in Hoogsteen basepairing and don't form alternative structures. Here is a study of 7-deazapurines incorpotated into DNA measured at HMDE by CV and ACV. While 7-deazaadenine was reduced at the HMDE, giving rise to a similar irreversible cathodic peak as the natural adenine, 7-deazaguanine didn't yield any peak analogous to the peak G due to Guanine, in Agreement with a loss of corresponding redox in 7-deazaguanine.
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Magnetic Beads-Based Electrochemical Techniques for DNA-Protein Interaction Monitoring
Fojta, Miroslav ; Pivoňková, Hana ; Němcová, Kateřina ; Horáková Brázdilová, Petra ; Havran, Luděk ; Orság, Petr ; Vidláková, Pavlína ; Macíčková-Cahová, Hana ; Balintová, Jana ; Hocek, Michal
Electrochemical techniques, in connection with separation of nucleoprotein complexes at magnetic beads, are suitable for the monitoring of DNA-protein interactions. For the detection of complexes captured at the beads it is possible to utilize intrinsic electrochemical activity of the protein, intrinsic structure-selective signals of the DNA, or indicator DNA substrates tail-labeled with electroactive moieties.
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Utilisation of enzymatic labelling with 4-aminophtalimide and 4-hydroxybenzylideneimidazolinone fluorescent derivates for monitoring of DNA-protein interaction
Orság, Petr ; Pivoňková, Hana ; Riedl, Jan ; Hocek, Michal ; Fojta, Miroslav
The 5’-substituted deoxycytosine triphosphates with conjugated solvatochromic derivates of 4-aminophtalimide (API) and derivates of the green fluorescent protein, 4-hydroxybenzylideneimidazolinone (HBI) were synthetized and successfully tested for enzymatic incorporation using primer extension assay. Site specifically labelled oligonucleotide probes were prepared and tested for interaction with p53 and SSB proteins, displaying distinct DNA-binding properties. The incorporation of multiple fluorescent labels did not interfere with natural protein binding and protein interaction leaded in both cases the to the gradual ratiometric increase of the fluorescence intensity moreover accompanied with the changes of the fluorescence emission spectra profile. Neither effect was observed after incubation with BSA, non-DNA binding protein, confirming the specificity of the interaction. Modified nucleoside triphosphates with conjugated fluorescence labels derivates of API and HBI can be used as substrates for preparation of the specific oligonucleotide labelled probes and provide the novel tool for studying and monitoring the DNA-protein interaction.
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