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Primer Extension Reaction at the Gold Electrode Surface
Vidláková, Pavlína ; Havran, Luděk ; Fojta, Miroslav
One option of covalent DNA labeling is incorporation of chemically modified nucleotides using corresponding deoxynucleotide triphosphates into DNA molecules using polymerase-catalyzed primer extension. Primer extension is usually performed in solution and then the product is analyzed, often after a suitable separation. In our study we tested the possibility to prepare oligonucleotides with enzymatically incorporated anthraquinone labeled nucleotides using primer extension on templates immobilize at a gold electrode surface.
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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.
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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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Natural and Synthetic Components of Nucleic Acids as Reactive Sites for DNA Modification by Osmium Tetroxide Complexes
Havran, Luděk ; Špaček, Jan ; Rozkosna, Jana ; Fojta, Miroslav
Natural DNA electroactivity find wide use in electrochemical analysis of its interactions and damage. For some applications is useful applied redox active tag to improve specificity of the analysis. One from utilized labels in this field are complexes of osmium tetroxide with nitrogen ligands (Os, L), which produce with DNA electroactive covalent adducts. Prime reaction site for Os, L is C=C double bond in pyrimidine nucleobases. If is 2,2'-bipyridine used as ligand, reaction is specific for single strand DNA. In this contribution will be presented results of electrochemical analysis of Os, bipy adducts with different ODN containing chemically modified purine bases.
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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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Electrochemical Analysis of Chemically Modified DNA at Gold Electrodes
Vidláková, Pavlína ; Havran, Luděk ; Fojta, Miroslav
DNA labeling is used to increase sensitivity of its electrochemical detection. Covalently bound markers based on a complex of osmium tetroxide with 22-bipyridine(Os,bipy) is one of those frequently applied. Os bipy reacts with pyrimidines (mainly thymines) in single stranded regions of DNA, therefore it represents a useful marker for the analysis of DNA structure or labeling of hybridization probes . Another possibility of DNA labeling is the incorporation of chemically modified dNTPs into DNA molecules with using primer extension. DNA labeling in connection with voltammetry at gold electrodes has application potential in development of sensors for studying of DNA structure and interaction.
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New Ligands for Modification of DNA by Osmium Tetroxide Reagents
Havran, Luděk ; Zenka, Martin ; Fojta, Miroslav
Besides DNA intrinsic electroactivity, modification of DNA by redox active tags find wide use in electrochemical analysis of DNA interactions and damage. Application of these tags can improve specificity of the analysis. One from utilized labels in this field are complexes of osmium tetroxide with nitrogen ligands, which produce with DNA electroactive covalent adducts. These complexes preferentially react with thymine in single strand DNA. Type of used ligand influence electrochemical behavior forming adducts. In this contribution will be presented results which are acquired by testing of set a new ligands based of quinone and substituted 1, 10-phenanthroline.
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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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