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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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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Basic Protocol 1: QPCR for Nuclear and Mitochondrial DNA Integrity

  • 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

  • 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

  • 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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Literature Cited

Literature Cited
   Anson, R.M., Mason, P.A., and Bohr, V.A. 2006. Gene‐specific and mitochondrial repair of oxidative DNA damage. Methods Mol. Biol. 314:155‐181.
   Ayala‐Torres, S., Chen, Y., Svoboda, T., Rosenblatt, J., and Van Houten, B. 2000. Analysis of gene‐specific DNA damage and repair using quantitative PCR. In Methods: A Companion to Methods in Enzymology (P.W. Doetsch, ed.) pp. 135‐147. Academic Press, New York.
   Bailey, S.M., Robinson, G., Pinner, A., Chamlee, L., Ulasova, E., Pompilius, M., Page, G.P., Chhieng, D., Jhala, N., Landar, A., Kharbanda, K.K., Ballinger, S., and Darley‐Usmar, V. 2006. S‐adenosylmethionine prevents chronic alcohol‐induced mitochondrial dysfunction in the rat liver. Am. J. Physiol. Gastrointest. Liver Physiol. 291:G857‐G867.
   Ballinger, S.W., Patterson, C., Knight‐Lozano, C.A., Burow, D.L., Conklin, C.A., Hu, Z., Reuf, J., Horaist, C., Lebovitz, R., Hunter, G.C., McIntyre, K., and Runge, M.S. 2002. Mitochondrial integrity and function in atherogenesis. Circulation 106:544‐549.
   Bennetts, L.E. and Aitken, R.J. 2005. A comparative study of oxidative DNA damage in mammalian spermatozoa. Mol. Reprod. Dev. 71:77‐87.
   Bohr, V.A. 2002. Repair of oxidative DNA damage in nuclear and mitochondrial DNA, and some changes with aging in mammalian cells. Free Radic. Biol. Med. 32:804‐812.
   Cakir, Y., Yang, Z., Knight, C.A., Pompilius, M., Westbrook, D., Bailey, S.M., Pinkerton, K.E., and Ballinger, S.W. 2007. Effect of alcohol and tobacco smoke on mtDNA damage and atherogenesis. Free Radic Biol Med. 43:1279‐1288.
   Chandrasekhar, D. and Van Houten, B. 2000. In vivo formation and repair of cyclobutane pyrimidine dimers and 6‐4 photoproducts measured at the gene and nucleotide level in E. coli. Mutat. Res. 450:19‐40.
   Chen, K.H., Srivastava, D.K., Yakes, F.M., Singhal, R.K., Rawson, T.Y., Sobol, R.W., Van Houten, B., and Wilson, S.H. 1998. Up‐regulation of base excision repair correlates with enhanced protection against a DNA damaging agent in mouse cell lines. Nucleic Acids Res. 26:2001‐2007
   Copeland, W.C., Wachsman, J.T., Johnson, F.M., and Penta, J.S. 2002. Mitochondrial DNA alterations in cancer. Cancer Invest. 20:557‐569.
   Cortopassi, G.A. 2002. A neutral theory predicts multigenic aging and increased concentrations of deleterious mutations on the mitochondrial and Y chromosomes. Free Radic. Biol. Med. 33:605‐610.
   De Minicis, S. and Brenner, D.A. 2008. Oxidative stress in alcoholic liver disease: Role of NADPH oxidase complex. J. Gastroenterol. Hepatol. 1:S98‐S103.
   Enns, G.M. 2003. The contribution of mitochondria to common disorders. Mol.Genet. Metab. 80:11‐26.
   Farris, M.W., Chan, C.B., Paiel, M., Van Houten, B., and Orrernius, S. 2005. Role of mitochondria in toxic oxidative stress. Mol. Interv. 5:94‐111.
   Fliss, M.S., Usadel, H., Caballero, O.L., Wu, L., Buta, M.R., Eleff, S.M., Jen, J., and Sidransky, D. 2000. Facile detection of mitochondrial DNA mutations in tumors and bodily fluids. Science 287:2017‐2019.
   Forbes, J.M., Coughlan, M.T., and Cooper, M.E. 2008. Oxidative stress as a major culprit in kidney disease in diabetes. Diabetes 57:1446‐1454.
   Grishko, V.I., Rachek, L.I., Spitz, D.R., Wilson, G.L., and LeDoux, S.P. 2005. Contribution of mitochondrial DNA repair to cell resistance from oxidative stress. J Biol. Chem. 280:8901‐8905.
   Jennerwein, M.M. and Eastman, A. 1991. A polymerase chain reaction‐based method to detect cisplatin adducts in specific genes. Nucleic Acids Res. 19:6209‐6214.
   Jin, G.F., Hurst, J.S., and Godley, B.F. 2001. Rod outer segments mediate mitochondrial DNA damage and apoptosis in human retinal pigment epithelium. Curr. Eye Res. 23:11‐19.
   Kalinowski, D., Illenye, S., and Van Houten, B. 1992. Analysis of DNA damage and repair in murine leukemia L1210 cells using a quantitative polymerase chain reaction assay. Nucleic Acids Res. 20:3485‐3494.
   Karthikeyan, G., Santos, J.H., Graziewicz, M.A., Copeland, W.C., Isaya, G., Van Houten, B., and Resnick, M.A. 2003. Reduction in frataxin causes progressive accumulation of mitochondrial damage. Hum. Mol. Genet. 24:3331‐3342.
   Khurana, R.N., Parikh, J.G., Saraswathy, S., Wu, G.S., and Rao, N.A. 2008. Mitochondrial oxidative DNA damage in experimental autoimmune uveitis. Invest. Ophthalmol. Vis. Sci. 49:3299‐3304.
   Maynard, S., Schurman, S.H., Harboe, C., de Souza‐Pinto, N.C., and Bohr, V.A. 2009. Base excision repair of oxidative DNA damage and association with cancer and aging. Carcinogenesis 30:2‐10.
   Mandavilli, B.S., Ali, S.F., and Van Houten, B. 2000. DNA damage in brain mitochondria caused by aging and MPTP treatment. Brain Res. 885:45‐52.
   Moon, S.K., Thompson, L.J., Madamanchi, N., Ballinger, S.W., Papaconstantinou, J., Horaist, C., Runge, M.S., and Patterson, C. 2001. Aging, oxidative stress, and proliferative capacity in cultured mouse aortic smooth muscle cells. Am. J. Physiol. Heart Circ. Physiol. 280:2779‐2788.
   Ng, F., Berk, M., Dean, O., and Bush, A.I. 2008. Oxidative stress in psychiatric disorders: Evidence base and therapeutic implications. Int. J. Neuropsychopharmacol. 6:851‐876.
   O'Brien, T., Xu, J., and Patierno, S.R. 2001. Effects of glutathione on chromium‐induced crosslinking and DNA polymerase arrest. Mol. Cell Biochem. 222:173‐182.
   Papanikolaou, G. and Pantopoulos, K. 2005. Iron metabolism and toxicity. Toxicol. Appl. Pharmacol. 202:199‐211.
   Pessayre, D. 2007. Role of mitochondria in non‐alcoholic fatty liver disease. J. Gastroenterol. Hepatol. 1:S20‐S27.
   Polyak, K., Li, Y., Zhu, H., Lengauer, C., Wilson, J.K., Markowitz, S.D., Trush, M.A., Kinzler, K.W., and Vogelstein, B. 1998. Somatic mutations of the mitochondrial genome in human colorectal tumours. Nat. Genet. 20:291‐293.
   Ponti, M., Forrow, S.M., Souhami, R.L., D'Incalci, M., and Hartley, J.A. 1991. Measurement of the sequence specificity of covalent DNA modification by antineoplastic agents using Taq DNA polymerase. Nucleic Acids Res. 19:2929‐2933.
   Quan, T. and States, J.C. 1996. Preferential DNA damage in the p53 gene by benzo[a]pyrene metabolites in cytochrome P4501A1‐expressing xeroderma pigmentosum group A cells. Mol. Carcinog. 16:32‐43.
   Rezin, G.T., Amboni, G., Zugno, A.I., Quevedo, J., and Streck, E.L. 2008. Mitochondrial dysfunction and psychiatric disorders. Neurochem. Res. Epub ahead of print. DOI 10.1007/s11064‐008‐9865‐9868.
   Richter, C., Park, J.W., and Ames, B.N. 1988. Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proc. Natl. Acad. Sci. U.S.A. 85:6465‐6467.
   Richter, C. 1995. Oxidative damage to mitochondrial DNA and its relationship to ageing. Int. J. Biochem. Cell Biol. 27:647‐653.
   Santos, J.H., Mandavilli, B.S., and Van Houten, B. 2002. Measuring oxidative mtDNA damage and repair using quantitative PCR. Methods Mol. Biol. 197:159‐176.
   Santos, J.H., Hunakova, L., Chen, Y., Bortner, C., and Van Houten, B. 2003. Cell sorting experiments link persistent mitochondrial DNA damage with loss of mitochondrial membrane potential and apoptotic cell death. J. Biol. Chem. 278:1728‐1731.
   Santos, J.H., Meyer, J.N., Skorvaga, M., Annab, L.A., and Van Houten, B. 2004. Mitochondrial hTERT exacerbates free radical‐mediated mtDNA damage. Aging Cell 6:399‐411.
   Santos, J.H., Meyer, J.N., and Van Houten, B. 2006a. Mitochondrial localization of hTERT as a determinant for hydrogen peroxide‐induced mtDNA damage and apoptosis. Hum. Mol. Genet. 15:1757‐1768.
   Santos, J.H., Meyer, J.N., Mandavilli, B.S., and Van Houten, B. 2006b. Quantitative PCR‐based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells. Methods Mol. Biol. 314:183‐199.
   Sawyer, D.E., Mercer, B.G., Wiklendt, A.M., and Aitken, R.J. 2003. Quantitative analysis of gene‐specific DNA damage in human spermatozoa. Mutat. Res. 529:21‐34.
   Shukla, A., Jung, M., Stern, M., Fukagawa, N.K., Taatjes, D.J., Sawyer, D., Van Houten, B., and Mossman, B.T. 2003. Asbestos induces mitochondrial DNA damage and dysfunction linked to the development of apoptosis. Am. J. Physiol. Lung Cell Mol. Physiol. 285:L1018‐L1025.
   Sobol, R.W., Watson, D.E., Nakamura, J., Yakes, F.M., Hou, E., Horton, J.K., Van Houten, B., Swenberg, J.A., Tindall, K.R., Gold, B., Samson, L.D., and Wilson, S.H. 2002. Mutator phenotype associated with a gene‐environment interaction: Effect of base excision repair deficiency and methylation‐induced genotoxic stress. Proc. Natl. Acad. Sci. U.S.A. 99:6860‐6865.
   Sorolla, M.A., Reverter‐Branchat, G., Tamarit, J., Ferrer, I., Ros, J., and Cabiscol, E. 2008. Proteomic and oxidative stress analysis in human brain samples of Huntington disease. Free Radic. Biol. Med. 45:667‐678.
   Termini, J. 2000. Hydroperoxide‐induced DNA damage and mutations. Mutat. Res. 450:107‐124.
   Thomas, D.C., Morton, A.G., Bohr, V.A., and Sancar, A. 1988. General method for quantifying base adducts in specific mammalian genes. Proc. Natl. Acad. Sci. U.S.A. 85:3723‐3727.
   Van Houten, B., Chen, Y., Nicklas, J.A., Rainville, I.R., and O'Neill, J.P. 1998. Development of long PCR techniques to analyze deletion mutations of the human hprt gene. Mutat. Res. 403:171‐175.
   Van Houten, B. and Friedberg, E.C. 1999. Mitochondrial DNA damage and repair. Mutat. Res. 434:133‐254.
   Van Houten, B., Woshner, V., and Santos, J.H. 2006. Role of mitochondrial DNA in toxic responses to oxidative stress. DNA Repair 5:145‐152.
   Wallace, D.C. 2001. Mitochondrial defects in neurodegenerative disease. Ment. Retard. Dev. Disabil. Res. Rev. 7:158‐166.
   Wang, A.L., Lukas, T.J., Yuan, M., and Neufeld, A.H. 2008. Increased mitochondrial DNA damage and down‐regulation of DNA repair enzymes in aged rodent retinal pigment epithelium and choroid. Mol. Vis. 14:644‐651.
   Yanez, J.A., Teng, X.W., Roupe, K.A., Fariss, M.W., and Davies, N.M. 2003. Chemotherapy induced gastrointestinal toxicity in rats: Involvement of mitochondrial DNA, gastrointestinal permeability and cyclooxygenase‐2. J. Pharm. Pharm. Sci. 6:308‐314.
   Yang, J.L., Weissman, L., Bohr, V.A., and Mattson, M.P. 2008. Mitochondrial DNA damage and repair in neurodegenerative disorders. DNA Repair 7:1110‐1120.
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