A Fluorometric Assay for Quantitating DNA Strand Breaks during Apoptosis

Abstract

Few techniques exist for quantitating DNA fragmentation during apoptosis. Our aim was to develop a quantitative assay for DNA fragmentation in apoptosis by enzymatically labeling DNA with a fluorescent dideoxynucleotide. Terminal deoxynucleotidyl transferase was used to enzymatically label 3′-OH DNA ends with fluorescein-12-dideoxyuridine triphosphate in an assay referred to as fluorophore end-labeling. Because only one labeled dideoxynucleotide can be added per 3′-OH end of DNA, the fluorescence intensity is directly proportional to the number of DNA strand breaks. The sensitivity and validation of this approach were first established in isolated calf thymus DNA treated with the endonuclease, DNase I; an excellent correlation was observed between fluorophore end-labeling and an isotopic approach to quantitate 3′-OH ends of DNA. Quantitation of DNA strand breaks was then obtained in nuclei isolated from hepatocytes undergoing apoptosis using fluorescent digitized microscopy, flow cytometry, and fluorometry. In addition to its quantitative aspects, fluorophore end-labeling proved to be quite sensitive as it detected DNA strand breaks prior to the morphologic changes of apoptosis or the development of the hypodiploid state as assessed by fluorescence microscopy and flow cytometry, respectively. This assay should prove useful for studying the molecular mechanisms leading to DNA cleavage during apoptosis.