Assays for DNA Damage

Jessica E. Sutherland1, Max Costa1

1 New York University School of Medicine, New York, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 3.5
DOI:  10.1002/0471140856.tx0305s02
Online Posting Date:  May, 2001
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This unit describes several assays for detecting several kinds of DNA damage (strand breaks, internal crosslinking, DNA/protein crosslinks) and repair activity following exposure to genotoxic agents. The methods include single‐cell electrophoresis (comet assay), filter eluting, K‐SDS precipitation, and measurement of unscheduled DNA synthesis.

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Table of Contents

  • Basic Protocol 1: Detection of Single‐stranded DNA Breaks Using the Single‐cell Microgel Electrophoresis Assay (Comet Assay) Under Alkaline Conditions
  • Alternate Protocol 1: Detection of Double‐stranded DNA Breaks Using the Single‐cell Microgel Electrophoresis Assay Under Neutral Conditions
  • Basic Protocol 2: Detection of DNA Damage with Filter Elution
  • Alternate Protocol 2: Detection of Interstrand Cross‐links
  • Alternate Protocol 3: Detection of DNA‐protein Cross‐links
  • Alternate Protocol 4: Detection of Oxidative DNA Base Modifications
  • Alternate Protocol 5: Detection of Double‐strand DNA Breakage
  • Alternate Protocol 6: Simultaneous Detection of DNA Double‐ and Single‐strand Breaks
  • Alternate Protocol 7: Alkaline Elution Using 96‐well Plates
  • Basic Protocol 3: Detection of DNA‐protein Cross‐links by K‐SDS Precipitation
  • Alternate Protocol 8: Using a 96‐well Plate to Determine DNA Concentrations for K‐sds Precipitations
  • Alternate Protocol 9: Using Slot Blotting to Determine DNA Amounts
  • Basic Protocol 4: Detection of DNA Repair Using the Unscheduled DNA Synthesis (UDS) Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Detection of Single‐stranded DNA Breaks Using the Single‐cell Microgel Electrophoresis Assay (Comet Assay) Under Alkaline Conditions

  • Cells of interest
  • Phosphate‐buffered saline (PBS; see recipe)
  • Test chemical
  • Normal‐melting‐point (NMP) agarose
  • Low‐melting‐point (LMP) agarose
  • Alkaline lysis solution, pH 10 (see recipe), 4°C
  • Alkaline electrophoresis buffer (see recipe)
  • 400 mM Tris⋅Cl, pH 7.4 ( appendix 2A)
  • 20 µg/ml ethidium bromide (or other fluorescent DNA stain; e.g., propidium iodide, ethidium bromide, YOYO‐1, TOTO‐1)
  • Metal baking tray
  • Microscope slides, fully frosted
  • 24 × 50–mm no. 1 coverslips
  • 45°C water bath
  • 200‐µl pipet tips with 5 mm cut off the small end
  • Coplin jars or slide staining apparatus
  • Horizontal electrophoresis unit
  • Power supply capable of delivering 300 mA of current at 25 V
  • Fluorescence microscope with excitation and barrier filters appropriate for the selected DNA stain, and camera
  • Eyepiece micrometer
  • Digital calipers
  • Transparencies with mm2 grids
  • Image analysis software (optional)

Alternate Protocol 1: Detection of Double‐stranded DNA Breaks Using the Single‐cell Microgel Electrophoresis Assay Under Neutral Conditions

  • Neutral lysis solution, pH 8.3 (see recipe), or alkaline lysis solution, pH 10 (see recipe)
  • 10 µg/ml RNase A in alkaline lysis solution (see recipe for the alkaline lysis solution; omit Triton X‐100 and adjust pH to 7)
  • 1 mg/ml proteinase K in alkaline lysis solution (see recipe for the alkaline lysis solution; omit Triton X‐100 and adjust pH to 7)
  • Neutral electrophoresis buffer: 300 mM sodium acetate/100 mM Tris⋅Cl, pH 8.5 (see appendix 2A for Tris⋅Cl)
  • 300 mM NaOH

Basic Protocol 2: Detection of DNA Damage with Filter Elution

  • Cells of interest
  • [Methyl 3H]thymidine (20 Ci/mmol) or [2‐14C]thymidine (50 mCi/mmol) for radioactive determination or Hoechst 33258 (bis‐benzamide; for fluorometric determination)
  • Unlabeled thymidine
  • Appropriate growth medium for cells
  • Test compound
  • Phosphate‐buffered saline (PBS; see recipe), ice‐cold
  • Cell lysis solution, pH 9.7 (see recipe)
  • Cell lysis solution (see recipe) containing 0.5 mg/ml proteinase K
  • Alkaline elution solution, pH 12.3 (see recipe)
  • 1 N HCl
  • 0.4 N NaOH
  • Scintillation fluid (e.g., Ecolume, ICN Biomedicals) containing 0.7% (v/v) acetic acid
  • 0.2 M tetrasodium EDTA, pH 10 (pH adjusted with 1 N NaOH)
  • 1 M potassium dihydrogen phosphate
  • 17 mM KH 2PO 4
  • 150 µM Hoechst 33258 stock solution
  • Tabletop centrifuge
  • 2.0‐µm polycarbonate filter (25‐mm diameter; Nucleopore, Whatman)
  • 1‐liter vacuum flask
  • Alkaline elution funnel apparatus (e.g., Millipore)
  • Aluminum foil or black paper cylinder
  • Multichannel peristaltic pump and suitable Tygon tubing
  • Fraction collector
  • Glass scintillation vials
  • 60°C oven or water bath
  • Liquid scintillation counter or fluorometer
  • Additional reagents and equipment for assessing cell viability by trypan blue exclusion ( appendix 3B)

Alternate Protocol 2: Detection of Interstrand Cross‐links

  • Culture of monolayer cells
  • Phosphate‐buffered saline (PBS; see recipe), ice‐cold
  • K‐SDS cell lysis solution (see recipe)
  • 4 mg/ml bovine serum albumin (BSA; molecular biology grade)
  • Precipitation/washing solution (see recipe)
  • Proteinase K solution (see recipe)
  • 1 mg/ml Hoechst 33258 dye (freshly prepared 1:20 dilution gives A 338 = 1.12)
  • 20 mM Tris⋅Cl, pH 7.5 ( appendix 2A)
  • λ DNA of known concentration
  • 3‐ml syringe with 21‐G needle
  • 50° and 65°C water baths
  • Centrifuge with swinging‐bucket rotor capable of 3300 × g with adapters for 1.5‐ml microcentrifuge tubes (optional)
  • 15‐ml polystyrene centrifuge tubes with caps, sterile
  • 12 × 75–mm borosilicate glass culture tubes of size appropriate for fluorometer
  • Centrifuge with swinging‐bucket rotor capable of 1500 × g with adapters for 15‐ml tubes
  • Fluorometer

Alternate Protocol 3: Detection of DNA‐protein Cross‐links

  • Pico green (Molecular Probes)
  • TE buffer, pH 7.5: 10 mM Tris⋅Cl ( appendix 2A) containing 1 mM EDTA
  • 1 mg/ml DNA stock solution
  • Polystyrene 96‐well plates (Corning Costar no. 3603)
  • Phosphorimager or microtiter plate reader

Alternate Protocol 4: Detection of Oxidative DNA Base Modifications

  • 5 M NaCl
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (prepared with buffered phenol; unit 3.5)
  • 24:1 chloroform/isoamyl alcohol
  • DNA from test species
  • TE buffer, pH 8: 10 mM Tris⋅Cl, pH 8 ( appendix 2A) containing 1 mM EDTA
  • 3 M NaOH
  • 2 M ammonium acetate, pH 7
  • 6× SSPE (see recipe for 10×)
  • Human Alu DNA or purified DNA from other species for use as a probe
  • Random priming kit (e.g., Prime‐A‐Gene Labeling System; Promega)
  • Prehybridization solution (see recipe)
  • 2× and 0.1× SSC (see recipe for 20×)
  • 2× SSC (see recipe for 20×) containing 1% (w/v) SDS
  • 45°, 65°, and 70°C water bath
  • Slot blot apparatus
  • UV transilluminator
  • Sephadex G‐50 spin column
  • X‐ray film
  • Additional reagents and equipment for DNA labeling with 32P, purification of DNA probes, and autoradiography ( appendix 3A)

Alternate Protocol 5: Detection of Double‐strand DNA Breakage

  • Male 200 to 250 g Fisher 344 rats (Charles River Laboratories, Harlan Sprague‐Dawley)
  • General anesthetic (e.g., sodium pentobarbital)
  • 70% and 90% (v/v) and absolute ethanol
  • Liver perfusion buffer 1 (see recipe)
  • Liver perfusion buffer 2 (see recipe)
  • Collagenase A
  • 769 mM CaCl 2
  • Williams medium E‐complete medium (WE‐C medium; see recipe)
  • Williams medium E‐incomplete medium (WE‐I medium; see recipe)
  • 0.4% (w/v) trypan blue stain
  • 1 mCi/ml [3H]thymidine
  • Phosphate‐buffered saline (PBS; see recipe)
  • 1:3 (v/v) glacial acetic acid/absolute ethanol, freshly prepared
  • Permount mounting medium (Fisher)
  • Glycerol
  • Kodak NTB‐2 autoradiography emulsion
  • Desiccant (e.g., silica gel or Drierite)
  • Kodak D19 developer
  • Kodak Fixer
  • Mayers hemalum stain (see recipe)
  • 1% (w/v) aqueous eosin Y
  • Xylene
  • Sterile cotton surgical gauze
  • Dissection scissors
  • Suture thread
  • 18‐G and 14‐G catheters
  • Surgical tubing ∼3 mm o.d. (suitable for a peristaltic pump, e.g., high‐pressure tubing with male and female Luer‐Lok connectors; Harvard Apparatus)
  • Variable‐flow peristaltic pump
  • 6‐well multi‐well plates or 30‐mm Petri dishes, sterile
  • Cheesecloth or gauze, sterile
  • 50‐ml plastic centrifuge tubes, sterile
  • Sterile glass beaker
  • Hemacytometer
  • Microscope with 100× objective interfaced to a colony counter
  • 25‐mm‐diameter round plastic coverslips, sterile (e.g., Thermanox; Fisher)
  • Microscope slides
  • 50‐ml plastic graduated cylinders cut to 30‐ml mark
  • 43°C water bath
  • Plastic teaspoons
  • Light‐proof slide boxes
  • Staining troughs
  • 22 × 50–mm glass coverslips
  • Additional reagents and equipment for counting cells and assessing viability with trypan blue ( appendix 3B)
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All solutions and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.
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Literature Cited

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