Tail-labelling of DNA probes using modified deoxynucleotide triphosphates and terminal deoxynucleotidyl transferase. Application in electrochemical DNA hybridization and protein-DNA binding assays

Org Biomol Chem. 2011 Mar 7;9(5):1366-71. doi: 10.1039/c0ob00856g. Epub 2011 Jan 4.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Nucleotidylexotransferase / chemistry*
  • DNA Probes / analysis*
  • DNA Probes / chemistry
  • DNA-Binding Proteins / chemistry*
  • Electrochemical Techniques / methods*
  • Molecular Structure
  • Nucleic Acid Hybridization / methods*
  • Purine Nucleotides / chemistry*

Substances

  • DNA Probes
  • DNA-Binding Proteins
  • Purine Nucleotides
  • DNA Nucleotidylexotransferase