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Tail labelled oligonucleotide probes for the detection of DNA-protein interactions
Pivoňková, Hana ; Němcová, Kateřina ; Horáková Brázdilová, Petra ; Havran, Luděk ; Macíčková-Cahová, Hana ; Hocek, Michal ; Fojta, Miroslav
DNA-protein interactions can be monitored via different ways. We introduce novel, fast and simple approaches in DNA-protein interaction detection based on electrochemical measurements of DNA alone (structure-sensitive DNA sensing), or DNA modified with osmium tetroxide bearing nitrogenous ligands, or measurements of redox-active moieties enzymatically attached to the end of a DNA substrate thus forming a labeled tail (by terminal transferase).
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Redox labelling of nucleic acids for analyzing nucleotide sequences and monitoring DNA-protein interactions
Fojta, Miroslav ; Havran, Luděk ; Horáková Brázdilová, Petra ; Pivoňková, Hana ; Kostečka, Pavel ; Macíčková-Cahová, Hana ; Raindlová, Veronika ; Vrábel, Milan ; Hocek, Michal
Nucleobase labelling of DNA for electrochemical sensing was attained through chemical modification of thymine bases with osmium tetroxide in the presence of nitrogenous ligands, or via enzymatic incorporation of nucleotide conjugates with redox-active moieties using labelled deoxynucleoside triphosphates. DNA hybridization, primer extension and PCR techniques were used for sequence-specific DNA assays. Tail-labelled DNA substrates were applied to monitor DNA binding by tumour suppressor p53 protein.
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The Use of Electrochemical Stripping Methods for Lead and Cadmium Speciation in Soil Solutions
Šestáková, Ivana ; Jaklová Dytrtová, J. ; Jakl, M. ; Navrátil, Tomáš ; Fojta, Miroslav
Soil solution is an aqueous phase of soil which is affected by plant root systems. The soil solution contains nutrients, organic and inorganic soil substances and also exudates – substances released by plant roots (e.g. low molecular weight organic acids). For the bioaccumulation (transport from soil into plants) the free metal ions and labile complexes contents (usually in trace amounts) in soil solution are important. That is why the electrochemical methods using working electrode preconcentration step, i.e. differential pulse anodic stripping voltammetry, anodic stripping square-wave voltammetry and stripping chronopotentiometry with constant current, are applicable. Mentioned methods combined with the hanging mercury drop electrode were tested for the cadmium and lead speciation possibilities in model and also real soil solutions.
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The use of prototype of multichannel device for electrochemical detection of DNA hybridization
Brázdilová, P. ; Navrátil, Tomáš ; Josypčuk, Bohdan ; Fojta, Miroslav
The article presents newly constructed 8-channel device, which enables sequential analysis realized on up to 8 different or equal sensors. The device is computer controlled. The screen-printed carbon electrodes (SPCE) were connected to the device. DNA hybridization was detected. DNA present in the sample spontaneously adsorbed on the surface of SPCE and free surface was subsequently blocked using beef serum albumin. The adsorbed DNA was then hybridized with the probe marked with biotin and streptavidin conjugated with enzyme alkaline phosphatase (SALP). This enzyme catalyzes the transformation of electrochemically inactive 1-naphtylphosphateone 1-naphtol, which anodic signal was detected DC voltammetry (DCV).
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