PCR-based methods for detecting DNA damage and its repair at the sub-gene and single nucleotide levels in cells |
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Authors: | Keith A Grimaldi Claire J McGurk Peter J McHugh John A Hartley |
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Institution: | (1) CRC Drug-DNA Interactions Research Group, Department of Oncology, Royal Free and University College London Medical School, UCL, 91, Riding House Street, W1W 7BS London |
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Abstract: | Three PCR-based methods are described that allow covalent drug-DNA adducts, and their repair, to be studied at various levels
of resolution from gene regions to the individual nucleotide level in single copy genes. A quantitative PCR (QPCR) method
measures the total damage on both DNA strands in a gene region, usually between 300 and 3000 base pairs in length. Strand-specific
QPCR incorporates adaptations that allow damage to be measured in the same region as QPCR but in a strand-specific manner.
Single-strand ligation PCR allows the detection of adduct formation at the level of single nucleotides, on individual strands,
in a single copy gene in mammalian cells. If antibodies to the DNA adducts of interest are available, these can be used to
capture and isolate adducted DNA for use in single-strand ligation PCR increasing the sensitivity of the assay. |
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Keywords: | DNA damage DNA repair PCR based method drug-DNA adduct single strand ligation PCR quantitative PCR |
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