Tail-labelling of DNA probes using modified deoxynucleotide triphosphates and terminal deoxynucleotidyl transferase. Application in electrochemical DNA hybridization and protein-DNA binding assays
Language English Country England, Great Britain Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
21203615
DOI
10.1039/c0ob00856g
Knihovny.cz E-resources
- MeSH
- DNA Probes analysis chemistry MeSH
- DNA-Binding Proteins chemistry MeSH
- DNA Nucleotidylexotransferase chemistry MeSH
- Electrochemical Techniques methods MeSH
- Nucleic Acid Hybridization methods MeSH
- Molecular Structure MeSH
- Purine Nucleotides chemistry MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- DNA Probes MeSH
- DNA-Binding Proteins MeSH
- DNA Nucleotidylexotransferase MeSH
- Purine Nucleotides MeSH
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.
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