Tail labelled oligonucleotide probes for the detection of DNA-protein interactions

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)

Electrochemical Detection of p53 Protein Interactions with Plasmid DNAs Modified with Cisplatin Using Immunoprecipitation at Magnetic Microbeads

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

Marketing Analysis of the Company STAMAT Plzeň Ltd.

Předložená diplomová práce je zaměřena na marketingovou analýzu firmy STAMAT Plzeň spol. s r.o zpracovávanou z důvodu dlouhodobé absence jejího marketingového řízení. V práci jsou nejdříve uvedeny teoretické základy pro formulování marketingové strategie následované základními informacemi o společnosti. Cílem práce je na základě vybraných analýz podniku vybrat optimální marketingovou strategii. Toho je dosaženo vypracováním finanční analýzy, analýzy mikroprostředí, makroprostředí a hodnocením internetového konkurenčního prostředí a marketingového mixu. Výsledky veškerého zkoumání jsou použity pro výběr vhodné strategie pomocí metod vycházejících ze SWOT analýzy. V závěru práce jsou navržena vhodná opatření vedoucí k dosažení této strategie.Katedra financí a účetnictvíObhájenoThe submitted diploma thesis focuses on the marketing anylysis of the company STAMAT Plzeň ltd. The reason of working up this thesis is long-lasting absence of marketing management. Initially, theoretical basis for forming the marketing strategy is given, followed by basic informations concerning the company. The aim of the work is to choose the optimal marketing strategy on the grounds of selected analyses, by working of financial analysis, macroanalysis, analysis of micro-environment, by evaluating of internet competitive environment and marketing mix. The outcomes of all research are used for the choice of suitable strategy by the methods flowing from SWOT analysis. In the end of the work, there were given several recommendations leading to achieve the strategy

Differential Salt-Induced Dissociation of the p53 Protein Complexes with Circular and Linear Plasmid DNA Substrates Suggest Involvement of a Sliding Mechanism

A study of the effects of salt conditions on the association and dissociation of wild type p53 with different ~3 kbp long plasmid DNA substrates (supercoiled, relaxed circular and linear, containing or lacking a specific p53 binding site, p53CON) using immunoprecipitation at magnetic beads is presented. Salt concentrations above 200 mM strongly affected association of the p53 protein to any plasmid DNA substrate. Strikingly different behavior was observed when dissociation of pre-formed p53-DNA complexes in increased salt concentrations was studied. While contribution from the p53CON to the stability of the p53-DNA complexes was detected between 100 and 170 mM KCl, p53 complexes with circular DNAs (but not linear) exhibited considerable resistance towards salt treatment for KCl concentrations as high as 2 M provided that the p53 basic C-terminal DNA binding site (CTDBS) was available for DNA binding. On the contrary, when the CTDBS was blocked by antibody used for immunoprecipitation, all p53-DNA complexes were completely dissociated from the p53 protein in KCl concentrations ≥200 mM under the same conditions. These observations suggest: (a) different ways for association and dissociation of the p53-DNA complexes in the presence of the CTDBS; and (b) a critical role for a sliding mechanism, mediated by the C-terminal domain, in the dissociation process

Mercury and mercury electrodes in the electrochemistry of biopolymers: Are they really inevitable in the decade of non-mercury sensors?

In this article, electrochemical properties of nucleic acids and proteins at mercury electrodes are briefly reviewed. We focus on structure sensitive DNA and protein sensing and/or techniques based on the utilization of catalytic hydrogen evolution i.e., analyses that are inherently connected with the mercury electrodes. Advantages of these approaches are briefly summarized and discussed towards answering the title question regarding necessity of mercury electrodes in biopolymer electroanalysis

Genosensing on a 3D-printed nanocarbon electrode

In this paper we present the characterization of 3D-printed nanocarbon electrodes (3DnCes) and their application in electrochemical enzyme-linked detection of DNA hybridization. The approach takes advantage of a facile procedure based on adsorption of target DNA on the electrode surface followed by hybridization with a biotinylated probe and binding of streptavidin–alkaline phosphatase conjugate. The alkaline phosphatase converts 1-naphthyl phosphate in the background electrolyte into electrochemically oxidizable 1-naphthol, which is subsequently detected using linear sweep voltammetry. The preparation, characterization, and analytical performance of the 3DnCes are reported. The results show the applicability of such 3DnCes in detection of target DNA hybridization specifically with the complementary biotinylated probe, and indicate the potential of 3D printed electrodes for use in various bioanalytical approaches.Web of Science151art. no. 10750

Enzyme-linked electrochemical detection of DNA fragments amplified by PCR in the presence of a biotinylated deoxynucleoside triphosphate using disposable pencil graphite electrodes

WOS: 000353219700015In this report, we present a simple electrochemical detection protocol for the detection of specific PCR-amplified DNA fragments, based on incorporation of biotin tags into DNA amplicons during PCR run in the presence of a biotinylated nucleoside triphosphate. For detection, an enzyme-linked electrochemical system involving streptavidin-alkaline phosphatase conjugate attached to the biotinylated DNA, adsorbed at the surface of a disposable pencil graphite electrode, is used. The enzyme converts an inactive indicator, 1-naphthyl phosphate, into electrochemically oxidizable indicator 1-naphthol that is subsequently detected. Excellent selectivity of this fast, facile, and inexpensive analysis not requiring any sophisticated electrode modification and its applicability for off-line monitoring of DNA amplification is demonstrated. Applications of the technique include detection of the presence of specific nucleotide sequences in biological samples, such as sequences related to pathogenic microorganism or transgenes. [GRAPHICS] .Czech Science FoundationGrant Agency of the Czech Republic [P206/11/1638, P206/11/P739]; ASCRCzech Academy of Sciences [RVO 68081707]; Turkish Scientific and Technological Research CouncilTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [111T050]; ASCR (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [111T050]This work was supported by the Czech Science Foundation (Grant P206/11/1638 to M. F. and P206/11/P739 to P. H.) and by the ASCR (RVO 68081707). A. E and M. F acknowledges to the grant of international joint project through between Turkish Scientific and Technological Research Council and the ASCR (TUBITAK Project No. 111T050)