DNA Damage Detection Technique Applying Time-Resolved Fluorescence Measurements

Abstract

A novel DNA damage detection technique based on the characteristic fluorescence lifetimes exhibited by PicoGreen−single-stranded DNA and −double-stranded DNA complexes is employed to establish the damage produced on DNA isolated from sheep white blood cells following γ radiation. This technique, which incorporates key concepts such as alkaline unwinding buffers and higher unwinding rates of damaged DNA compared to undamaged DNA, allows for the differentiation of DNA damage resulting from doses of γ radiation in the 0−100-Gy range, with the potential of analyzing samples consisting of as little as 10⁴ cells. Experiments were carried out using commercial DNA sources as well as DNA isolated from sheep white blood cells, suggesting its potential for use with isolated DNA from virtually any eukaryotic cell.