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A modified and automated version of the 'Fluorimetric Detection of Alkaline DNA Unwinding' method to quantify formation and repair of DNA strand breaks

Lookup NU author(s): Ragen Pfeiffer, Alan Leake, Dr Michelle Muller, Emeritus Professor Thomas Kirkwood, Professor Alexander Burkle

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Abstract

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. Conclusion: 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.


Publication metadata

Author(s): Moreno-Villanueva M, Pfeiffer R, Sindlinger T, Leake A, Muller M, Kirkwood TBL, Burkle A

Publication type: Article

Publication status: Published

Journal: BMC Biotechnology

Year: 2009

Volume: 9

Issue: 1

Pages: 39

Date deposited: 08/02/2010

ISSN (print): 1472-6750

ISSN (electronic):

Publisher: BioMed Central Ltd.

URL: http://dx.doi.org/10.1186/1472-6750-9-39

DOI: 10.1186/1472-6750-9-39


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