Quantification of UVR-induced DNA damage: global- versus gene-specific levels of thymine dimers

Karakoula, A, Evans, MD, Podmore, I ORCID: https://orcid.org/0000-0002-6417-0151, Hutchinson, PE, Lunec, J and Cooke, MS 2003, 'Quantification of UVR-induced DNA damage: global- versus gene-specific levels of thymine dimers' , Journal of Immunological Methods, 277 (1-2) , pp. 27-37.

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Abstract

The induction and repair of DNA damage has been shown to occur heterogeneously throughout the mammalian genome. As a consequence, analysis of these parameters at a global genome level may not reflect important gene-level events. Few techniques have been established to explore quantitatively gene-specific DNA damage and repair. Most of these are polymerase chain reaction (PCR)-based assays and are relatively insensitive, relying on decreased PCR amplification arising from damage in template DNA. We have developed a quantitative assay that combines specific immunocapture of damaged DNA by an antiserum specific for thymine dimers (IgG479), with PCR amplification of a 149 bp fragment of the human H-ras proto-oncogene. Quantification of DNA damage was based upon proportionality between the amount of the PCR product and the initial amount of damage. Detection of thymine dimers was possible with nanogram amounts of genomic DNA and increased in a linear, dose-responsive manner. Using this assay, gene-level induction of thymine dimers was shown to be directly proportional to levels induced in the global genome of ultraviolet radiation (UVR)-exposed, extracted DNA as measured by gas chromatography-mass spectrometry (GC-MS). This result suggests that global damage assessments do indeed reflect gene-level events although we predict that this relationship may not be maintained when applied to a cellular system. These findings demonstrate the suitability of this approach to the detection of UVR-induced DNA damage at the level of individual genes.

Item Type:	Article
Themes:	Subjects / Themes > Q Science > Q Science (General) Subjects outside of the University Themes
Schools:	Schools > School of Environment and Life Sciences Schools > School of Environment and Life Sciences > Biomedical Research Centre
Journal or Publication Title:	Journal of Immunological Methods
Publisher:	Elsevier
Refereed:	Yes
ISSN:	00221759
Depositing User:	H Kenna
Date Deposited:	08 Aug 2007 10:48
Last Modified:	27 Aug 2021 21:59
URI:	https://usir.salford.ac.uk/id/eprint/170

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