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.
- MeSH
- DNA sondy analýza chemie MeSH
- DNA vazebné proteiny chemie MeSH
- DNA-nukleotidylexotransferasa chemie MeSH
- elektrochemické techniky metody MeSH
- hybridizace nukleových kyselin metody MeSH
- molekulární struktura MeSH
- purinové nukleotidy chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
