A modified and automated version of the 'Fluorimetric Detection of Alkaline DNA Unwinding' method to quantify formation and repair of DNA strand breaks.
BACKGROUND
Formation and repair of DNA single-strand breaks are important parameters in the assessment of DNA damage and repair occurring in live cells. The 'Fluorimetric Detection of Alkaline DNA Unwinding (FADU)' method [Birnboim HC, Jevcak JJ. Cancer Res (1981) 41:1889-1892] is a sensitive procedure to quantify DNA strand breaks, yet it is very tedious to perform.


RESULTS
In order (i) to render the FADU assay more convenient and robust, (ii) to increase throughput, and (iii) to reduce the number of cells needed, we have established a modified assay version that is largely automated and is based on the use of a liquid handling device. The assay is operated in a 96-well format, thus greatly increasing throughput. The number of cells required has been reduced to less than 10,000 per data point. The threshold for detection of X-ray-induced DNA strand breaks is 0.13 Gy. The total assay time required for a typical experiment to assess DNA strand break repair is 4-5 hours.


CONCLUSIONS
We have established a robust and convenient method measuring of formation and repair of DNA single-strand breaks in live cells. While the sensitivity of our method is comparable to current assays, throughput is massively increased while operator time is decreased.
Moreno-Villanueva
María
M
Lehrstuhl Molekulare Toxikologie, Fachbereich Biologie, Universität Konstanz, Konstanz, Germany. maria.moreno-villanueva@uni-konstanz.de
Pfeiffer
Ragen
R
Sindlinger
Thilo
T
Leake
Alan
A
Müller
Marcus
M
Kirkwood
Thomas B L
TB
Bürkle
Alexander
A
D004247:DNA / Q000032:analysis* / Q000528:radiation effects; D053904:DNA Breaks, Single-Stranded; D004260:DNA Repair; D005470:Fluorometry / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D010957:Plasmids; D012680:Sensitivity and Specificity
D004247:DNA