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Molecular Biotechnology

, Volume 20, Issue 2, pp 181–196 | Cite as

PCR-based methods for detecting DNA damage and its repair at the sub-gene and single nucleotide levels in cells

  • Keith A. Grimaldi
  • Claire J. McGurk
  • Peter J. McHugh
  • John A. Hartley
Protocol

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.

Index Entries

DNA damage DNA repair PCR based method drug-DNA adduct single strand ligation PCR quantitative PCR 

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Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Keith A. Grimaldi
  • Claire J. McGurk
  • Peter J. McHugh
  • John A. Hartley
    • 1
  1. 1.CRC Drug-DNA Interactions Research Group, Department of OncologyRoyal Free and University College London Medical School, UCLLondon

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