title: Tail-labelling of DNA probes using modified deoxynucleotide triphosphates and terminal deoxynucleotidyl transferase. Application in electrochemical DNA hybridization and protein-DNA binding assays.
abstract: A simple approach to DNA tail-labelling using terminal deoxynucleotidyl transferase and modified deoxynucleoside triphosphates is presented. Amino- and nitrophenyl-modified dNTPs were found to be good substrates for this enzyme giving 3'-end stretches of different lengths depending on the nucleotide and concentration. 3-Nitrophenyl-7-deazaG was selected as the most useful label because its dNTP was efficiently incorporated by the transferase to form long tail-labels at any oligonucleotide. Accumulation of many nitrophenyl tags per oligonucleotide resulted in a considerable enhancement of voltammetric signals due to the nitro group reduction, thus improving the sensitivity of electrochemical detection of the tail-labelled probes. We demonstrate a perfect discrimination between complementary and non-complementary target DNAs sequences by tail-labelled hybridization probes as well as the ability of tumour suppressor p53 protein to recognize a specific binding site within tail-labelled DNA substrates, making the methodology useful in electrochemical DNA hybridization and DNA-protein interaction assays.
authors.affiliation: Institute of Biophysics, v.v.i., Academy of Sciences of the Czech Republic, Kralovopolska 135, CZ-612 65, Brno, Czech Republic.
authors.forename: Petra
authors.initials: P
authors.lastname: Horáková
authors.forename: Hana
authors.initials: H
authors.lastname: Macíčková-Cahová
authors.forename: Hana
authors.initials: H
authors.lastname: Pivoňková
authors.forename: Jan
authors.initials: J
authors.lastname: Spaček
authors.forename: Luděk
authors.initials: L
authors.lastname: Havran
authors.forename: Michal
authors.initials: M
authors.lastname: Hocek
authors.forename: Miroslav
authors.initials: M
authors.lastname: Fojta
mesh_terms: D004253:DNA Nucleotidylexotransferase / Q000737:chemistry*; D015342:DNA Probes / Q000032:analysis* / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D055664:Electrochemical Techniques / Q000379:methods*; D015394:Molecular Structure; D009693:Nucleic Acid Hybridization / Q000379:methods*; D011685:Purine Nucleotides / Q000737:chemistry*
chemical_list: D015342:DNA Probes; D004268:DNA-Binding Proteins; D011685:Purine Nucleotides; D004253:DNA Nucleotidylexotransferase