Analysis of Oxidative Damage by Gene‐Specific Quantitative PCR

Olga A. Kovalenko1, Janine H. Santos1

1 University of Medicine and Dentistry of New Jersey, Newark, New Jersey
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 19.1
DOI:  10.1002/0471142905.hg1901s62
Online Posting Date:  July, 2009
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Abstract

This unit describes the gene‐specific quantitative PCR‐based (QPCR) assay, which is used to measure DNA integrity of both nuclear and mitochondrial genomes based on amplification of long DNA targets. QPCR can be used to quantify the formation of DNA damage and the kinetics of DNA repair by following restoration of amplification of the target DNA over time after removal of the damaging agent. A detailed protocol to set up QPCR in any laboratory, highlighting critical parameters for successful establishment of the assay and interpretation of the results, is provided here. Advantages (e.g., the use of nanogram amounts of DNA) and limitations (e.g., the inability to define the specific type of lesion present on the DNA) of using QPCR to assay DNA damage in human cells are also described. Curr. Protoc. Hum. Genet. 62:19.1.1‐19.1.13. © 2009 by John Wiley & Sons, Inc.

Keywords: mitochondrial and nuclear DNA integrity; oxidative stress; reactive oxygen species; DNA damage and repair; QPCR

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: QPCR for Nuclear and Mitochondrial DNA Integrity
  • Basic Protocol 2: Estimation of DNA Damage
  • Support Protocol 1: Sample Preparation and DNA Quantification
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: QPCR for Nuclear and Mitochondrial DNA Integrity

  Materials
  • Sample genomic DNA (see protocol 3)
  • GeneAmp XL PCR kit (Applied Biosystems cat. no. N808‐0188), including:
    • rTth DNA polymerase XL (2 U/µl)
    • 3.3× XL PCR buffer
  • 1 mg/ml bovine serum albumin (BSA, Roche cat. no. 10711454001)
  • dNTPs: 10 mM of total solution (2.5 mM of each nucleotide, GE cat. no. 27‐2035‐01) diluted in water, in 100‐µl aliquots, and frozen at −80°C
  • Primers (see Table 19.1.1 and ): dilute in water for a working concentration of 10 µM
  • Magnesium (Mg2+)
  • Dedicated PCR hood for setting up reactions, e.g., Purifier PCR Enclosure (Labconco cat. no. LC‐LO‐3970302) or a tissue culture hood
  • 200‐µl PCR tubes
  • Thermal cyclers (e.g., Biometra)

Basic Protocol 2: Estimation of DNA Damage

  Materials
  • 20× TE buffer: 200 mM Tris⋅Cl, 20 mM EDTA, pH 7.5
  • PCR product samples and blank (see protocol 1)
  • 5 µl/ml Quant‐iT PicoGreen dsDNA reagent (Molecular Probes, Invitrogen cat. no. P7581) in 1× TE buffer
  • 96‐well plates
  • Aluminum foil
  • Plate reader (e.g., Victor3 Multilabel reader, PerkinElmer) with excitation wavelength at 485 nm and emission wavelength at 535 nm
  • Data collection software, e.g., Excel

Support Protocol 1: Sample Preparation and DNA Quantification

  Materials
  • Cell culture or tissue
  • Genomic DNA buffer set (Qiagen cat. no. 19060)
  • 20× TE buffer: 200 mM Tris⋅Cl, 20 mM EDTA, pH 7.5
  • Lambda (λ)/HindIII DNA (GIBCO cat. no. 15612‐013) diluted in 1× TE buffer to make the following 20, 10, 5, 2.5, and 1.25 ng/µl DNA standards
  • 5 µl/ml Quant‐iT PicoGreen dsDNA reagent (Molecular Probes, Invitrogen cat. no. P7581) in 1× TE buffer
  • Genomic‐tips (Qiagen cat. no. 10223)
  • 50°C water bath
  • 96‐well plates
  • Aluminum foil
  • Plate reader (e.g., Victor3 Multilabel reader, PerkinElmer) with excitation wavelength at 485 nm and emission wavelength at 535 nm
  • Data collection software, e.g., Excel
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Figures

Videos

Literature Cited

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