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

Mol Biotechnol. 2002 Feb;20(2):181-96. doi: 10.1385/MB:20:2:181.

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 3,000 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.

Publication types

  • Comparative Study

MeSH terms

  • Base Sequence
  • DNA Damage / physiology*
  • DNA Repair / physiology*
  • DNA, Single-Stranded / isolation & purification*
  • DNA, Single-Stranded / physiology
  • Models, Chemical
  • Molecular Sequence Data
  • Nucleotides / isolation & purification*
  • Nucleotides / physiology
  • Polymerase Chain Reaction / instrumentation
  • Polymerase Chain Reaction / methods*
  • Taq Polymerase / metabolism
  • Time Factors

Substances

  • DNA, Single-Stranded
  • Nucleotides
  • Taq Polymerase