title: Oscillating probe for dual detection of 5'PO4 and 5'OH DNA breaks in tissue sections.
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.
authors.affiliation: Molecular and Cell Biology Laboratory, Department of Neurosurgery, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Didenko
mesh_terms: D004249:DNA Damage; D005456:Fluorescent Dyes; D017403:In Situ Hybridization / Q000379:methods*; D015336:Molecular Probe Techniques; D015335:Molecular Probes; D009693:Nucleic Acid Hybridization / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D015335:Molecular Probes