Quantification of DNA Damage by Real-Time qPCR

Methods Mol Biol. 2016:1351:27-32. doi: 10.1007/978-1-4939-3040-1_3.

Abstract

This chapter describes the use of real-time qPCR to quantify damages in genomic DNA. The method is based on the ability of a lesion in one strand to inhibit restriction enzyme digestion of double-stranded DNA. Subsequent amplification of the complementary strand after restriction cleavage gives a quantitative measure of the damage content in that site (Real-time qPCR Analysis of Damage Frequency; RADF). We compare the RADF assay with the commonly used technique to assess damages by their ability to inhibit amplification of a large PCR fragment relative to a short PCR fragment. The RADF method described here is quick, accurate and allows the detection of nuclear and mitochondrial DNA damage in detailed regions.

Keywords: DNA damage; DNA repair; mtDNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cells, Cultured
  • DNA Damage / genetics*
  • DNA Primers / genetics
  • DNA, Mitochondrial / analysis*
  • DNA, Mitochondrial / genetics
  • Humans
  • Mitochondria / genetics
  • Nucleic Acid Amplification Techniques / methods*
  • Oxidation-Reduction
  • Oxidative Stress / genetics
  • Reactive Oxygen Species / metabolism
  • Real-Time Polymerase Chain Reaction / methods*

Substances

  • DNA Primers
  • DNA, Mitochondrial
  • Reactive Oxygen Species