Application of FISH for in situ detection and quantification of DNA breakage.
We describe a simple procedure that allows the use of fluorescence in situ hybridization (FISH) for in situ detection of DNA strand breaks in single cells (DBD-FISH: DNA Breakage Detection-FISH). After trapping within an agarose microgel, cells are incubated in an unwinding alkaline solution, deproteinized and dehydrated. Areas of single-stranded DNA are generated by the alkaline solution in proportion to the degree of DNA strand breakage. These then act as targets for FISH of whole genomic or region-specific probes (telomeric, human chromosome 8 painting, human alphoid DXZ1 locus, and human c-erbB-2 cosmid probes). Measurement of the amount and surface of FISH signals provides information on the breakage level in probed areas, permitting the assessment of possible intragenomic differences in sensitivity as well as intercellular heterogeneity in DNA damage induction or repair.
Fernández
J L
JL
Laboratorio de Genética Molecular y Radiobiología, Centro Oncológico de Galicia, La Coruña (Spain). genetica<gcog@jet.es>
Goyanes
V J
VJ
Ramiro-Díaz
J
J
Gosálvez
J
J
D000818:Animals; D016466:CHO Cells; D002467:Cell Nucleus / Q000235:genetics / Q000528:radiation effects; D002898:Chromosomes, Human, Pair 8; D006224:Cricetinae; D004249:DNA Damage; D004252:DNA Mutational Analysis / Q000379:methods; D004307:Dose-Response Relationship, Radiation; D005260:Female; D015894:Genome, Human; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007962:Leukocytes / Q000528:radiation effects