Oscillating probe for dual detection of 5'PO4 and 5'OH DNA breaks in tissue sections

Methods Mol Biol. 2006;335:59-69. doi: 10.1385/1-59745-069-3:59.

Abstract

Several types of DNA cuts are used as markers of apoptosis for detection of apoptotic cells in situ. We recently introduced a ligase-based in situ assay that is specific for a single type of DNA damage--a double-strand break of DNase I-type, bearing 5'PO4. Here we describe a vaccinia topoisomerase I-based approach to label another type of DNA damage in situ--a double-strand break of DNase II-type, bearing 5'OH. The assay uses a new type of probe, a molecular oscillator. The probe self-assembles in solution out of a dual-hairpin oligonucleotide and vaccinia topoisomerase I. The enzyme continuously separates and religates two fluorescently labeled hairpins, which can participate in energy transfer. We describe the successful combination of topoisomerase-and ligase-based systems into an in situ assay. The assay uses an oscillating probe for simultaneous detection of two types of DNA cuts in tissue sections.

Publication types

  • Review

MeSH terms

  • DNA Damage*
  • Fluorescent Dyes*
  • In Situ Hybridization / methods*
  • Molecular Probe Techniques*
  • Molecular Probes*
  • Nucleic Acid Hybridization / methods*

Substances

  • Fluorescent Dyes
  • Molecular Probes