A microplate assay for DNA damage determination (Fast micromethod) in cellsuspensions and solid tissues

A rapid and convenient procedure for DNA damage determination in cell suspe nsions and solid tissues on single microplates was developed. The procedure is based on the ability of commercially available fluorochromes to interac t preferentially with dsDNA in the presence of ssDNA, RNA, and proteins at high pH (>12.0), thus allowing direct measurements of DNA denaturation with out sample handling or stepwise DNA separations. The method includes a simp le and rapid 40-min sample lysis in the presence of EDTA, SDS, and high ure a concentration at pH 10, followed by time-dependent DNA denaturation at pH 12.4 after NaOH addition. The time course and the extent of DNA denaturati on is followed in a microplate fluorescence reader at room temperature for less than 1 h. The method requires only 30 ng DNA per single well and could conveniently be used whenever fast analysis of DNA integrity in small samp les has to be done, e.g., in patients' lymphocytes after irradiation or che motherapy (about 3000 cells per sample), in solid tissues or biopsies after homogenization (about 25 mu g tissue per well), or in environmental sample s for genotoxicity assessment. (C) 1999 Academic Press.