Assays for DNA Damage

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

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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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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Materials

Basic Protocol 1: Detection of Single‐stranded DNA Breaks Using the Single‐cell Microgel Electrophoresis Assay (Comet Assay) Under Alkaline Conditions

Materials

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

Materials

Cells of interest
[Methyl ³ H]thymidine (20 Ci/mmol) or [2‐¹⁴ C]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 KH₂ PO₄
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

Materials

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 A₃₃₈ = 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 ³² P, purification of DNA probes, and autoradiography ( appendix 3A )

Alternate Protocol 5: Detection of Double‐strand DNA Breakage

Materials

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 CaCl₂
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 [³ H]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% CO₂ 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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Figures

Figure 3.5.1 Untreated lymphocytes processed as controls in the comet assay. From McKelvey‐Martin et al. (); reprinted with permission from Elsevier Science Publishers.

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Figure 3.5.2 Human lymphocyte following treatment with 172.5 µM H₂ O₂ for 1 hr at 4°C and processed in the comet assay. From McKelvey‐Martin et al. (); reprinted with permission from Elsevier Science Publishers.

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Figure 3.5.3 Protein‐dependent (A ) and protein‐independent (B ) DNA cross‐linking in L 1210 cells by bis (2‐chloroethyl) methyl amine (HN₂ ). Cells, prelabeled with [¹⁴ C]thymidine (20 hr) were treated with 0.1 µM HN₂ (0.5 hr). Tests were conducted with and without proteinase K and with and without 300 red of X ray. Symbols: open circles, no X ray or proteinase K; open triangle, X ray, no proteinase K; filled circle, no X ray with proteinase K; filled triangle, X ray with proteinase K. From Ewig and Kohn ; reprinted with permission from American Association for Cancer Research (AACR).

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Figure 3.5.4 DNA‐protein cross‐links (mean ± std. dev.) induced by cis‐platinum (cis‐Pt; 16 hr) in EBV‐transformed human Burkitt's lymphoma cells. From Costa et al. (). Reprinted with permission from Elsevier Science Publishers.

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Figure 3.5.5 Unscheduled DNA synthesis (UDS) in human amnion (AV₃ ) cells following culture in arginine‐deficient medium, treatment with 5 mM hydroxyurea (1 hr), irradiation with 254 nm ultraviolet light and exposure to [³ H]TdR (5 µCi/ml medium; 2 or 4 hr). (A ) Control (unirradiated) cells pulsed for 4 hr. (B ) Cells irradiated with 15 ergs/mm² and pulsed for 2 hr. From Trasko and Yager ; reprinted with permission from Academic Press.

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Videos

Literature Cited

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Assays for DNA Damage

Abstract

Table of Contents

Materials

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

Figures

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Literature Cited