Institute of Biophysics

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2019-07-25
09:13
Use of Monovalent Copper for Sensitive Detection of Methyl Derivatives of Xanthine
Navrátil, R. ; Motlova, D. ; Jelen, F. ; Trnková, L.
This work is concerned with the electroanalysis of xanthine (Xan) and its methylated derivatives (1-, 3-, 7-, and 9-mXan) on a pencil graphite electrode in the presence of copper ions. The main idea of using copper consists in the in situ formation of the complex Cu(I)-mXan on the electrode surface and improvement of oxidative signals of the corresponding mXan during the detection. Linear sweep voltammetry (LSV) in connection with adsorption stripping techniques was used for the determination of mXan. To enhance the oxidation signals the elimination voltammetric procedure (EVP) was applied.

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2019-07-25
09:13
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.

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2019-07-25
09:13
Enzymatic Incorporation of Biotin into DNA for DNA Hybridization Analysis and for Sensitive Detection of PCR-Amplified DNA
Špaček, Jan ; Zenka, Martin ; Haroniková, Lucia ; Havran, Luděk ; Fojta, Miroslav
We present two enzymatical electrochemical assays for DNA analysis. For hybridization analysis we used probes with biotin-14-dC introduced to 3' OH end by terminal transferase. For detection of PCR products we used Deep Vent polymerase to incorporate biotin-14-dCduring PCR. In both cases streptavidin-alkaline phosphatase conjugate was subsequently attached to the incorporated biotins and was used to catalyze dephosphorylation of 1-naphthyl phosphate to 1-naphthol, the electrochemical signal of which was utilized for detection. Compared to the former method, biotin incorporation during PCR offers lower molar detection limits, whereas application of the biotin-tailed probe can provide us with more selective detection.

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2019-07-25
09:13
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.

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2019-07-25
09:13
Electrochemical Detection of p53 Protein Interactions with Plasmid DNAs Modified with Cisplatin Using Immunoprecipitation at Magnetic Microbeads
Pivoňková, Hana ; Tichý, Vlastimil ; Orság, Petr ; Šebest, Peter ; Fojta, Miroslav
Antineoplastic drug [cis-diamminedichloroplatinum(II)] (cisplatin) forms covalent adducts with DNA. Cisplatin-modified DNA can be determined sensitively using square-wave voltammetry at mercury electrodes. Tumor suppressor protein p53 binds to DNA in different modes, including sequence-and structure-specific ones and these interactions are influenced by modification of the DNA with cisplatin. In this contribution we present a simple immunoprecipitation technique with magnetic beads, followed by voltammetric determination of recovered cisplatinated DNA, for the evaluation of p53 protein binding to DNAs containing various target sites differing in their proneness to being internally modified with the platinum complex.

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2019-07-25
09:13

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2019-07-25
09:13
DNA POLYMERASE STOP ASSAY FOR DETECTION OF G-QUADRUPLEXES
Petr, Marek ; Bažantová, Pavla ; Adámik, Matěj ; Kejnovská, Iva ; Dvořáková, Zuzana ; Vorlíčková, Michaela ; Pečinka, Petr ; Brázdová, Marie
In this work we performed DNA polymerase stop assays while using template DNA derived from promoter regions of VEGF and c-Myc proto-oncogenes under variety of experimental conditions. Partial stopping of DNA synthesis along the template strand was observed in the presence of K+ ions due to formation of stable G-quadruplex structures. In contrast, Na+ ions alone were unable to stabilize G-quadruplexes to stop the reaction at their site. These data suggest that K+ and Na+ ions, which are both known to stabilize G-quadruplexes, do this in different manner or extent.

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2019-07-25
09:13
P36 VOLTAMETRIC DETECTION OF DNA DELETION ON PENCIL ELECTRODE
Haroniková, Lucia ; Špaček, Jan ; Fojta, Miroslav
In this work, we present a new qualitative approach of detection of PCR products using electrochemistry on pencil electrodes. PCR products with an incorporated biotin-labeled dNTP (dCTP in this study) are detected via conjugated streptavidinealkaline phospatase. 1-Naphthyl phosphate (1-NP), which is dephosphorylated by alkaline phosphatase to release 1-naphthol, was used as a substrate in electrochemical detection of the PCR product. Voltammetric measurement on disposable pencil electrode is a very cheap and easy to use method. The system was optimized for plasmid DNA PCR product with potential to detect human genome DNA products and possible application in gene deletion monitoring.

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2019-07-25
09:13
ANALYSIS OF SEQUENCE SPECIFIC INTERACTIONS BETWEEN DNA AND P53 FAMILY PROTEINS BY ELISA, SLOT-BLOT AND EMSA
Adámik, Matěj ; Holanová, L. ; Navrátilová, Lucie ; Nygrinova, J. ; Pokorova, J. ; Petr, Marek ; Tichý, Vlastimil ; Brázdová, Marie
DNA-protein interactions of core domains of p53, p63 and p73, members of tumor suppressor p53 family, were investigated by multiple methods with regard to verifying the sequence specificity with which short target oligonucleotides/long DNA fragments can be recognized. The sequence specificity of core domains and wtp53 full protein binding to specific sequence in both types of DNA substrates was confirmed in solution, on surface and in gels by ELISA, slot-blot and EMSA.

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2019-07-25
09:13
TAIL-LABELED OLIGONUCLEOTIDE PROBES FOR A DUAL ELECTROCHEMICAL MAGNETIC IMMUNOPRECIPITATION ASSAY OF DNA-PROTEIN BINDING
Hermanová, Monika ; Špaček, Jan ; Orság, Petr ; Fojta, Miroslav
A novel assay for detection of DNA-protein binding has been developed. Oligonucleotides bearing or lacking specific binding site of the p53 protein were tail-labeled by two different modified deoxynucleotide triphosphates using terminal deoxynucleotidyl transferase. Electrochemical detection enabled to discriminate between sequence-specific and non-specific p53-DNA binding in a competition assay.

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