Two essential modifications strongly improve the performance of the Fast Micromethod to identify DNA single- and double-strand breaks.
To identify DNA single- or double-strand breaks, various techniques have been described. One of them, the Fast Micromethod, is an easy-to-perform 96-well microplate assay. Cells treated with chemicals are loaded with the fluorescent dye PicoGreen, which binds to double-stranded DNA with a high specificity. Following DNA denaturation in an alkaline buffer, DNA unwinding occurs and PicoGreen is released. The amount of PicoGreen released over a certain period of time reflects the extent of DNA damage. To maximize the throughput of the procedure and to minimize DNA damage due to the analytical procedures used, the Fast Micromethod was improved in two essential points. First, the very time-consuming cell-counting was substituted by a simple protein measurement. Second, the cell lysis step was omitted. By introducing the two above mentioned modifications, a high number of samples can now be analyzed within a much shorter period of time.
Ullmann
Kristina
K
Institute of Nutritional Science, University of Potsdam, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany.
Müller
Carsten
C
Steinberg
Pablo
P
D000818:Animals; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis* / Q000187:drug effects; D004249:DNA Damage / Q000187:drug effects*; D004277:DNA, Single-Stranded / Q000032:analysis* / Q000187:drug effects; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D051381:Rats; D013997:Time Factors
D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C099476:PicoGreen; D004247:DNA