Journal
ORGANIC & BIOMOLECULAR CHEMISTRY
Volume 9, Issue 5, Pages 1366-1371Publisher
ROYAL SOC CHEMISTRY
DOI: 10.1039/c0ob00856g
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Funding
- Academy of Sciences of the Czech Republic [Z4 055 0506, Z5 004 0507, Z5 004 0702]
- Ministry of Education [LC06035, LC512]
- Grant Agency of the Academy of Sciences of the Czech Republic [IAA400040901]
- Gilead Sciences, Inc. (Foster City, CA, U. S. A.)
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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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