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 PCR-based assays and are relatively insensitive, relying on decreased PCR amplification arising from damage in template DNA. In this study, a quantitative assay that combines specific immunocapture of damaged DNA by an antiserum specific for thymine dimers (IgG479), with PCR amplification of a 149bp fragment of the human H-ras proto-oncogene was established. 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 UVC-and-UVB-irradiated genomic DNA, as measured by gas chromatography-mass spectrometry and enzyme-linked immunosorbent assay, respectively. This result suggests that global damage assessments do indeed reflect gene-level events. However, preferential repair of UVB-induced T < > T from the human H-ras proto-oncogene than the overall genome of human keratinocyte cells was detected. These findings demonstrate the suitability of this approach to the detection of UVR-induced DNA damage and repair at the level of individual genes.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:409378 |
| Date | January 2004 |
| Creators | Karakoula, Aikaterini |
| Publisher | University of Leicester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/2381/29692 |
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