Abstract
This chapter describes the use of real-time qPCR to quantify damages in genomic DNA. The method is based on the ability of a lesion in one strand to inhibit restriction enzyme digestion of double-stranded DNA. Subsequent amplification of the complementary strand after restriction cleavage gives a quantitative measure of the damage content in that site (Real-time qPCR Analysis of Damage Frequency; RADF). We compare the RADF assay with the commonly used technique to assess damages by their ability to inhibit amplification of a large PCR fragment relative to a short PCR fragment. The RADF method described here is quick, accurate and allows the detection of nuclear and mitochondrial DNA damage in detailed regions.
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Acknowledgements
The work was supported by the Norwegian Research Council.
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Wang, W., Scheffler, K., Esbensen, Y., Eide, L. (2016). Quantification of DNA Damage by Real-Time qPCR. In: McKenzie, M. (eds) Mitochondrial DNA. Methods in Molecular Biology, vol 1351. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3040-1_3
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DOI: https://doi.org/10.1007/978-1-4939-3040-1_3
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3039-5
Online ISBN: 978-1-4939-3040-1
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