Chapter 4 Analysis of DNA Damage and Repair by Comet Fluorescence In Situ Hybridization (Comet-FISH) Michael Glei and Wiebke Schlörmann Abstract A useful tool in the detection of overall and region-specific DNA damage is the Comet-FISH technique. This method combines two well-established methods, the Comet assay (single cell gel electrophoresis), which makes it possible to detect and quantify DNA damage at the single cell level, and FISH (fluorescence in situ hybridization), a technique that allows the specific detection of selected DNA sequences. The influence of specific substances such as water pollutants or food ingredients on individual cells can be measured with the alkaline version of the Comet assay, which involves the embedding of cells in agarose on microscopic slides, lysis of cells, and separation of DNA via electrophoresis. In damaged cells a “comet tail” is formed by fractured DNA migrating from the nucleus (head of the comet) in the electric field. The damaged DNA (DNA strand breaks) correlates with the percentage of DNA in the tail. In combination with the FISH method, DNA damage or repair capacity in single cells can be measured using labelled probes, which hybridize to specific DNA sequences of interest. This protocol exemplarily provides a description of the Comet-FISH technique for the detection of DNA damage using hydrogen peroxide as a genotoxic model substance. Key words Comet-FISH, Comet assay, FISH, DNA damage, Hybridization
1
Introduction
1.1 The Comet-FISH Technique
The Comet assay or single cell gel electrophoresis represents the first of two well-established methods of the Comet-FISH technique. The neutral version of the Comet assay was primarily described by Östling and Johanson [1]. The basic principle of this method is the embedding of single cells in a layer of low-melting agarose which is applied onto microscopic slides. After the cells have been subjected to a lysis step, the damaged DNA is separated from undamaged DNA by electrophoresis. The intact DNA remains within the nucleus representing the head of the comet while damaged DNA migrates through the agarose in the electric field toward the anode forming its tail. The separation of fragmented from non-fragmented DNA and quantification of DNA
Juan C. Stockert et al. (eds.), Functional Analysis of DNA and Chromatin, Methods in Molecular Biology, vol. 1094, DOI 10.1007/978-1-62703-706-8_4, © Springer Science+Business Media New York 2014
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damage by, e.g., genotoxic compounds, can be measured by this relatively simple and fast method. Also the capacity of DNA repair can be assessed in single cells using the Comet assay. It is supposed that the neutral Comet assay mainly detects double-strand breaks whereas the alkaline version, which was described by Singh et al. [2], also detects single-strand breaks and alkali labile sites. But this is discussed controversially [3]. Santos et al. [4] were the first who combined the Comet assay with the fluorescence in situ hybridization (FISH). This combination allows the detection of specifically labelled DNA sequences of interest including whole chromosomes and is a useful tool to detect overall and region-specific DNA damage and repair in individual cells. This modification of the Comet assay inserts a hybridization step with specific fluorescent-labelled probes to special DNA sequences of interest after unwinding and electrophoresis. In this way specific gene sequences or telomeres can be detected. Also site-specific breaks in DNA regions, which are relevant for development of different diseases, can be detected by the combination of both techniques. The detection of DNA sequence modifications as well as the distribution of DNA damage and repair in the complete genome can be detected at the single cell level. In contrast to the Comet assay, which only measures overall DNA damage, Comet-FISH also distinguishes whether the probed sequences are located in the damaged or undamaged part of the comet (tail or head, respectively). Glei et al. [5] provide more information about the CometFISH technique and its modifications for different applications. For more detailed information about evaluation and interpretation of the Comet-FISH results, see also Schaeferhenrich et al. [6], Knöbel et al. [7, 8], Glei et al. [9], Shaposhnikov et al. [10, 11], and Mladinic et al. [12]. 1.2 Application of the Comet-FISH Technique
One of the basic methods in Comet-FISH is the classical alkaline Comet assay, a useful and simple method to detect DNA damage at the single cell level. Potential genotoxic effects of test compounds can be measured. The use of lesion-specific endonucleases makes it also possible to analyze different kinds of DNA damage. Furthermore, the capacity of DNA repair can be studied in single cells. The additional application of fluorescent-labelled probes, which are specific for particular DNA sequences, allows the detection of DNA damage or repair at an even higher level of resolution. By quantification of migrated DNA fragments it offers a unique possibility to study the sensitivity of genes toward genotoxic compounds [13, 14]. Therefore, the combination of the Comet assay with FISH provides further insights in mechanisms of cancer development and also chemoprevention. The question whether the damage and repair are occurring within the vicinity of the probed genes of
Detection of DNA Damage by Comet-FISH
41
interest can be evaluated by this technique [5]. Several probes can be used for Comet-FISH [10], but the size of the probes represents a limitation for the detection of gene-specific effects. The resolution of the Comet assay is limited to approximately 10–800 kB fragments using the standard conditions. Therefore, fragments smaller than 10 kB might get lost [15]. For detection of genespecific loci by FISH probes targeting at least 10 kB or longer are necessary. Probes, which are commercially available, predominantly rank from 30 to 100 kB in size and are assemblies of approximately 300 bases covering unique DNA sequences, spanning the target gene [16]. If the particular gene of interest is located in an undamaged region of DNA, the Comet-FISH method will result in two fluorescence signals in the head of a comet. One spot or several spots in the tail of a comet indicate that a break or breaks has/have occurred in the proximity of the probed gene (see Fig. 1). If certain parts of genes remain in the head, even when there is a DNA break nearby, the presence of a scaffold- or matrix-associated region at or near the gene can be supposed [17]. This was observed, for example, for the dihydrofolate reductase gene (DHFR) from Chinese hamster ovary (CHO) cells upon treatment with H2O2 or a photosensitizer plus light [18]. To meet the specific experimental requirements of DNA embedded in agarose the classical FISH protocol has been adjusted. Kelvey-Martin et al. [14], for example, used thermal codenaturation of the probes and DNA (74 °C, 5 min), whereas Rapp et al. [19] suggested chemical denaturation to separate the target strand DNA. Of the above presented two versions of CometFISH, one is based on the alkaline version, and the other on the neutral version of the Comet assay. The gel matrix, which contains the lysed cells, has to be treated carefully. Therefore, the parameters for denaturation, conditions for washing steps, signal amplification, and microscopic analysis have to be optimized for the Comet-FISH protocol. Hybridization efficiency, which should be always determined [6], can be ameliorated using optimized DNA probes with a higher DNA concentration and a reduced size compared to the probes used for conventional FISH on metaphase chromosomes [19]. To avoid gel matrix damage padlock probes can be used which are very stable and specific. The advantage of such probes is that reactions can be performed at 37 °C [10]. Comet-FISH is a relative simple method to study DNA damage and its repair throughout the genome and in defined chromosomal regions [20] at the level of individual cells, but only a few further developments are described. A new application of Comet-FISH, which increases the output and sample size with a twelve-gel slide format, was presented for example by Shaposhnikov et al. [21]. Hovhannisyan [22] described the combination of FISH, not only
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Fig. 1 Overview of potential fragmentation patterns and resulting signal distributions after Comet-FISH experiments: (a) undamaged DNA; (b) damaged DNA without a break in the vicinity of the specifically labelled DNA sequence; (c, d) damaged DNA with one or two signals located in the tail; (e) damaged DNA with more than two signals resulting from double-strand breaks in the vicinity of the specifically labelled DNA located in the tail
with the Comet assay but also with the micronucleus test. Furthermore, Kwasniewska et al. [23] reported for the first time on the application of FISH to Comet preparations from plants to analyze DNA damage and repair.
Detection of DNA Damage by Comet-FISH
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43
Materials
2.1 Pre-coating of Slides
1. Glass slides (frosted). 2. Glass cover slips. 3. Phosphate-buffered saline (PBS): 8.0 g/L NaCl, 0.2 g/L KCl, 0.2 g/L KH2HPO4, 1.15 g/L Na2HPO4 · 2H2O, pH 7.3. Store at 4 °C. 4. Normal-melting agarose (NMA; 0.5 %): 150 mg NMA in 30 ml PBS (microwave). Store at 4 °C (see Note 1). 5. Water quench. 6. Heating plate. 7. Ice, icebox.
2.2 Treatment of Cells with Test Compounds (e.g., Genotoxic Model Chemicals Like H2O2)
1. Hydrogen peroxide (H2O2) (see Note 2).
2.3 Comet Assay (Detection of DNA Damage)
1. Pre-coated slides from Subheading 2.1.
2. Pipettes and tips. 3. Vortex mixer. 4. Tubes.
2. Low-melting agarose (LMA; 0.7 %): 210 mg LMA in 30 ml of PBS (microwave). Store at 4 °C (see Note 1). 3. Glass cover slips. 4. Lysis stock solution: 146.1 g/L NaCl, 37.2 g/L Na2EDTA, 1.2 g/L Tris-base, 8 g/L NaOH, 10 g/L N-lauroyl sarcosin sodium salt, pH 10. Store at room temperature. 5. 5. Lysis working solution: 1 % Triton-X, 10 % DMSO, 89 % lysis stock solution. Store at 4 °C, prepare freshly. 6. Electrophoresis chamber (e.g., Renner GmbH, Dannstadt, Germany). 7. Electrophoresis stock solutions: NaOH solution, 400 g/L NaOH; Na2EDTA solution, 74.4 g/L Na2EDTA. 8. Electrophoresis working solution: 60 mL NaOH stock solution, 10 mL Na2EDTA stock solution, 1,930 mL distilled water, store at 4 °C, prepare freshly. 9. Neutralization buffer: 4.2 M Tris–HCl, 0.08 M Tris-base, pH 7.2. 10. TE-buffer: 10 mM Tris–HCl, 1 mM EDTA, pH 8. Store at room temperature. 11. Antifade buffer: 2.5 g DABCO, 50 mL TE-buffer, 50 ml glycerol, store at 4 °C, protect from light.
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12. SYBR Green I [1:10.000]: 1 μL SYBR®-Green, 9,999 μL Antifade buffer, store at 4 °C, protect from light. 13. Ethanol (100 %). 2.4 FISH: Preparation of Slides
1. 0.5 M NaOH. 2. PBS. 3. Ethanol (70, 80, 95, 100 %). 4. Hybrisol VI (Oncor, Gaithersburg, UK). 5. Plastic cover slips.
2.5 FISH: Hybridization
1. Digoxigenin-labelled DNA probes (e.g., Oncor, Gaithersburg, UK). 2. Hybrisol VI (Oncor, Gaithersburg, UK). 3. Water quench. 4. Plastic cover slips. 5. Hybridization chamber. 6. Saline sodium citrate (2× SSC): 0.3 M NaCl, 0.03 M sodium citrate, pH 7.2. 7. 1× Phosphate-buffered detergent (PBD) (Oncor, Gaithersburg, UK).
2.6 Detection of DigoxigeninLabelled Probes
1. Anti-digoxigenin-AP Germany).
Fab-fragments
(Roche,
Mannheim,
2. HNPP-Fluorescence Detection Kit (Roche, Mannheim, Germany). 3. SYBR®-Green (1 μL/10 mL). 4. Fluorescence microscope. 5. Fluorescence filter (green and red, e.g., ZEISS filter No 09 and 15). 6. Digital CCD camera and imaging software (e.g., MicroMAX, BFI OPTILAS GmbH; Visitron Systems GmbH, Puchheim, Germany). 7. Image analysis system (e.g., Kinetic Imaging, Liverpool, UK or Perceptive Instruments Suffolk, UK).
3
Method
3.1 Pre-coating of Slides
1. Dissolve 0.5 % normal-melting agarose (NMA) at 60 °C (water quench, see Note 1). 2. Drop 50 µL of 0.5 % NMA on one side of each slide and distribute NMA with another slide by dragging it to the opposite side (smear). 3. Dry slides at ~60 °C (heating plate).
Detection of DNA Damage by Comet-FISH
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4. Coat slides with another 200 µL of 0.5 % NMA (see Note 3). 5. Cover slides with cover slips rapidly to allow a homogeneous disposition of NMA. 6. Cool slides for 10 min on an icebox (see Note 4). 7. Store slides at 4 °C (see Note 5). 3.2 Treatment of Cells with Test Compounds (e.g., Genotoxic Model Chemicals Like H2O2)
2. Incubate cell suspension, e.g., HT29 cells (2 × 106 cells/mL) with H2O2 solutions for 5 min at 4 °C.
3.3 Comet Assay (Determination of DNA Damage)
1. Dissolve 0.7 % of low-melting agarose (LMA) at 40 °C (water quench, see Note 1) and resuspend each cell pellet in a volume of 50 μL.
1. Dilute H2O2 in PBS to favored concentrations, vortex.
3. Remove H2O2 solutions by centrifugation at 400 × g for 5 min. 4. Discard the supernatants.
2. Distribute 50 μL of each cell suspension onto pre-coated slides, cover them with cover slips, and allow agarose to solidify for 10 min on an icebox. 3. Remove cover slips and immerse slides in lysis solution for at least 60 min at 4 °C (see Note 6). 4. Place slides into an electrophoresis chamber containing alkaline electrophoresis buffer and incubate them for 20 min. 5. Carry out electrophoresis at 1.25 V/cm and 300 mA for 20 min by adjusting the total volume of electrophoresis buffer. 6. Remove slides from the electrophoresis chamber and wash them three times for 5 min in neutralization buffer. 7. Stain slides with SYBR Green I (in Antifade buffer, 30 μl per slide) and determine DNA damage or continue to step 8. 8. Dehydrate slides in absolute EtOH for at least 3 days. 3.4 FISH: Preparation of slides
1. Rehydrate slides in double-distilled H2O for 10 min. 2. Denature DNA in 0.5 M NaOH for 30 min. 3. Carry out neutralization in PBS for 1 min. 4. Dehydrate slides in an ascending EtOH series: 70, 80, 95 % each for 5 min. 5. Dry slides at room temperature. 6. Drop 30 μL Hybrisol VI onto each slide and spread it with a plastic cover slip.
3.5 FISH: Hybridization
1. Prepare the hybridization mixture containing 10 μL digoxigenin-labelled probe (commercially acquired) and 20 μL Hybrisol VI. 2. Denature hybridization mixture at 37 °C for 5 min (water quench) (see Note 7).
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3. Apply hybridization mixture onto one half of each slide and Hybrisol VI as negative control on the other half and cover it with a plastic cover slip. 4. Incubate slides at 37 °C in hybridization chambers for 24–72 h. 5. Wash slides in 2× SSC at 72 °C for 5 min. 6. Wash slides in 1× PBD for 5 min at room temperature. 3.6 Detection of Digoxigenin-Labelled Probes
1. For detection of digoxigenin-labelled probes use anti-digoxigenin-AP Fab fragments and 2-hydroxy-3-naphthoic acid-20phenylanilide-phosphate (HNPP) fluorescent detection set (Roche, Mannheim, Germany) according to the manufacturer’s instructions. 2. Use SYBR Green I (in Antifade buffer; 30 mL per slide) to counterstain the HNPP-detected probes (see Note 8). 3. Evaluate slides with a fluorescence microscope, equipped with filters to score images stained with SYBR Green I and HNPP or rather HNP/TR (2-hydroxy-3-naphthoic acid-20phenylanilide/Texas Red, see manufacturer’s instructions) (green and red emission, respectively) (see Notes 9–11). 4. For capturing images use a digital CCD camera (e.g., MicroMAX, BFI OPTILAS GmbH, Puchheim, Germany, processed with the Meta View Imaging Software, Visitron Systems GmbH, Puchheim, Germany).
3.7
Evaluation
1. Determine hybridization efficiency [6] (see Note 12). 2. Categorize comets into four degrees of damage ranging from undamaged images (class 1) to severely damaged images (class 4) [24] and/or by determination of the tail intensity with an image analysis system (e.g., Perceptive Instruments Suffolk, UK). 3. For the further Comet-FISH evaluation use only cells with two or more fluorescent spots and record the position of the signals in the comet head or comet tail (see Fig. 1). 4. Record the percentage of damaged cells (belonging to comet classes 2–4), in which it is possible to find a migration of at least one signal into the comet tail as the parameter of migration. 5. Score about 100 cells per slide (see Notes 11 and 12).
4
Notes 1. NMA and LMA are dissolved by heating in a microwave and kept fluid in a water quench. NMA and LMA can be stored for 2–3 weeks at 4 °C.
Detection of DNA Damage by Comet-FISH
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2. H2O2 can be used in a favored genotoxic concentration (usually 75 μM). 3. Pre-coating of the slides is more homogeneous when slides stay on the heating plate at lower temperature (~ 30 °C) while the second agarose coat is applied. 4. A metal box filled with ice is useful to cool down pre-coated slides. Cooling causes a more homogeneous coating of the slides and less background signals. 5. The slides can be stored a maximum of one week at 4 °C in a humidified chamber. 6. The slides can also be lysed overnight at 4 °C. 7. Denaturation procedure depends on DNA probe used, for self made dig-labelled samples denature for 5 min at 72 °C, 4 min on ice, 30 min at 37 °C and mix 5 μl sample with 10 μl Hybrisol VI [7]. 8. If the fluorescence signals are too weak, dilute SYBR Green I 1:1,000 instead of 1:10,000 in Antifade buffer. 9. In the case that the hybridization signals are too weak, reduce SSC concentration to improve the stringency of the washing steps and wash for a longer period of time. 10. Peroxides may induce a fading of SYBR Green. So it could be necessary to use higher concentrations of DABCO Antifade and/or to wash the slides before staining. 11. If there are too much background signals, which disturb evaluation, rehydrate slides for a longer period of time before staining. 12. Improvement of the hybridization efficiency might be achieved by changing the hybridization temperature and duration, as well as by varying the temperature and stringency of the washing steps (15 min in double-distilled H2O). References 1. Östling O, Johanson KJ (1984) Microelectrophoretic study of radiationinduced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 123:291–298 2. Singh NP, McCoy MT, Tice RR et al (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191 3. Collins AR, Oscoz AA, Brunborg G et al (2008) The comet assay: topical issues. Mutagenesis 23:143–151 4. Santos SJ, Singh NP, Natarajan AT (1997) Fluorescence in situ hybridization with comets. Exp Cell Res 232:407–411
5. Glei M, Hovhannisyan G, Pool-Zobel BL (2009) Use of Comet-FISH in the study of DNA damage and repair: review. Mutat Res 681:33–43 6. Schaeferhenrich A, Beyer-Sehlmeyer G, Festag G et al (2003) Human adenoma cells are highly susceptible to the genotoxic action of 4-hydroxy-2-nonenal. Mutat Res 526:19–32 7. Knöbel Y, Glei M, Weise et al (2006) Uranyl nitrilotriacetate, a stabilized salt of uranium, is genotoxic in nontransformed human colon cells and in the human colon adenoma cell line LT97. Toxicol Sci 93:286–297 8. Knöbel Y, Weise A, Glei M et al (2007) Ferric iron is genotoxic in non-transformed and
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9.
10.
11.
12.
13.
14.
15.
Michael Glei and Wiebke Schlörmann preneoplastic human colon cells. Food Chem Toxicol 45:804–811 Glei M, Schaeferhenrich A, Claussen U et al (2007) Comet fluorescence in situ hybridization analysis for oxidative stress-induced DNA damage in colon cancer relevant genes. Toxicol Sci 96:279–284 Shaposhnikov S, Frengen E, Collins AR (2009) Increasing the resolution of the comet assay using fluorescent in situ hybridization: a review. Mutagenesis 24:383–389 Shaposhnikov S, Thomsen PD, Collins AR (2011) Combining fluorescent in situ hybridization with the comet assay for targeted examination of DNA damage and repair. Methods Mol Biol 682:115–132 Mladinic M, Zeljezic D, Shaposhnikov SA et al (2012) The use of FISH-comet to detect c-Myc and TP 53 damage in extended-term lymphocyte cultures treated with terbuthylazine and carbofuran. Toxicol Lett 211:62–69 Bock C, Rapp A, Dittmar H et al (1999) Localisation of specific sequences and DNA single strand breaks in individual UV-A irradiated human lymphocytes by COMET FISH. Prog Biomed Optics 3568:207–217 Kelvey-Martin VJ, Ho ET, McKeown SR et al (1998) Emerging applications of the single cell gel electrophoresis (Comet) assay. I. Management of invasive transitional cell human bladder carcinoma. II Fluorescent in situ hybridization Comets for the identification of damaged and repaired DNA sequences in individual cells. Mutagenesis 13:1–8 Rapp A, Bock C, Dittmar H et al (2000) UV-A breakage sensitivity of human chromosomes as measured by COMET-FISH depends on gene density and not on the chromosome size. J Photochem Photobiol, B 56:109–117
16. Ooi FA (2001) Oncogene amplification detection by fluorescence in situ hybridization (FISH). Acta Histoche Cytochem 34: 391–397 17. Anderson D, Yu TW, Browne MA (1997) The use of the same image analysis system to detect genetic damage in human lymphocytes treated with doxorubicin in the Comet and fluorescence in situ hybridisation (FISH) assays. Mutat Res 390:69–77 18. Horvathova E, Dusinska M, Shaposhnikov S et al (2004) DNA damage and repair measured in different genomic regions using the comet assay with fluorescent in situ hybridization. Mutagenesis 19:269–276 19. Rapp A, Hausmann M, Greulich KO (2005) The comet-FISH technique: a tool for detection of specific DNA damage and repair. Methods Mol Biol 291:107–119 20. Spivak G (2010) The Comet-FISH assay for the analysis of DNA damage and repair. Methods Mol Biol 659:129–145 21. Shaposhnikov S, Azqueta A, Henriksson S et al (2010) Twelve-gel slide format optimised for comet assay and fluorescent in situ hybridisation. Toxicol Lett 195:31–34 22. Hovhannisyan GG (2010) Fluorescence in situ hybridization in combination with the comet assay and micronucleus test in genetic toxicology. Mol Cytogenet 3:17 23. Kwasniewska J, Grabowska M, Kwasniewski M et al (2012) Comet-FISH with rDNA probes for the analysis of mutagen-induced DNA damage in plant cells. Environ Mol Mutagen 53:369–375 24. Wollowski I, Ji ST, Bakalinsky AT et al (1999) Bacteria used for the production of yogurt inactivate carcinogens and prevent DNA damage in the colon of rats. J Nutr 129:77–82
1
Introduction
1.1 The Comet-FISH Technique
The Comet assay or single cell gel electrophoresis represents the first of two well-established methods of the Comet-FISH technique. The neutral version of the Comet assay was primarily described by Östling and Johanson [1]. The basic principle of this method is the embedding of single cells in a layer of low-melting agarose which is applied onto microscopic slides. After the cells have been subjected to a lysis step, the damaged DNA is separated from undamaged DNA by electrophoresis. The intact DNA remains within the nucleus representing the head of the comet while damaged DNA migrates through the agarose in the electric field toward the anode forming its tail. The separation of fragmented from non-fragmented DNA and quantification of DNA
Juan C. Stockert et al. (eds.), Functional Analysis of DNA and Chromatin, Methods in Molecular Biology, vol. 1094, DOI 10.1007/978-1-62703-706-8_4, © Springer Science+Business Media New York 2014
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Michael Glei and Wiebke Schlörmann
damage by, e.g., genotoxic compounds, can be measured by this relatively simple and fast method. Also the capacity of DNA repair can be assessed in single cells using the Comet assay. It is supposed that the neutral Comet assay mainly detects double-strand breaks whereas the alkaline version, which was described by Singh et al. [2], also detects single-strand breaks and alkali labile sites. But this is discussed controversially [3]. Santos et al. [4] were the first who combined the Comet assay with the fluorescence in situ hybridization (FISH). This combination allows the detection of specifically labelled DNA sequences of interest including whole chromosomes and is a useful tool to detect overall and region-specific DNA damage and repair in individual cells. This modification of the Comet assay inserts a hybridization step with specific fluorescent-labelled probes to special DNA sequences of interest after unwinding and electrophoresis. In this way specific gene sequences or telomeres can be detected. Also site-specific breaks in DNA regions, which are relevant for development of different diseases, can be detected by the combination of both techniques. The detection of DNA sequence modifications as well as the distribution of DNA damage and repair in the complete genome can be detected at the single cell level. In contrast to the Comet assay, which only measures overall DNA damage, Comet-FISH also distinguishes whether the probed sequences are located in the damaged or undamaged part of the comet (tail or head, respectively). Glei et al. [5] provide more information about the CometFISH technique and its modifications for different applications. For more detailed information about evaluation and interpretation of the Comet-FISH results, see also Schaeferhenrich et al. [6], Knöbel et al. [7, 8], Glei et al. [9], Shaposhnikov et al. [10, 11], and Mladinic et al. [12]. 1.2 Application of the Comet-FISH Technique
One of the basic methods in Comet-FISH is the classical alkaline Comet assay, a useful and simple method to detect DNA damage at the single cell level. Potential genotoxic effects of test compounds can be measured. The use of lesion-specific endonucleases makes it also possible to analyze different kinds of DNA damage. Furthermore, the capacity of DNA repair can be studied in single cells. The additional application of fluorescent-labelled probes, which are specific for particular DNA sequences, allows the detection of DNA damage or repair at an even higher level of resolution. By quantification of migrated DNA fragments it offers a unique possibility to study the sensitivity of genes toward genotoxic compounds [13, 14]. Therefore, the combination of the Comet assay with FISH provides further insights in mechanisms of cancer development and also chemoprevention. The question whether the damage and repair are occurring within the vicinity of the probed genes of
Detection of DNA Damage by Comet-FISH
41
interest can be evaluated by this technique [5]. Several probes can be used for Comet-FISH [10], but the size of the probes represents a limitation for the detection of gene-specific effects. The resolution of the Comet assay is limited to approximately 10–800 kB fragments using the standard conditions. Therefore, fragments smaller than 10 kB might get lost [15]. For detection of genespecific loci by FISH probes targeting at least 10 kB or longer are necessary. Probes, which are commercially available, predominantly rank from 30 to 100 kB in size and are assemblies of approximately 300 bases covering unique DNA sequences, spanning the target gene [16]. If the particular gene of interest is located in an undamaged region of DNA, the Comet-FISH method will result in two fluorescence signals in the head of a comet. One spot or several spots in the tail of a comet indicate that a break or breaks has/have occurred in the proximity of the probed gene (see Fig. 1). If certain parts of genes remain in the head, even when there is a DNA break nearby, the presence of a scaffold- or matrix-associated region at or near the gene can be supposed [17]. This was observed, for example, for the dihydrofolate reductase gene (DHFR) from Chinese hamster ovary (CHO) cells upon treatment with H2O2 or a photosensitizer plus light [18]. To meet the specific experimental requirements of DNA embedded in agarose the classical FISH protocol has been adjusted. Kelvey-Martin et al. [14], for example, used thermal codenaturation of the probes and DNA (74 °C, 5 min), whereas Rapp et al. [19] suggested chemical denaturation to separate the target strand DNA. Of the above presented two versions of CometFISH, one is based on the alkaline version, and the other on the neutral version of the Comet assay. The gel matrix, which contains the lysed cells, has to be treated carefully. Therefore, the parameters for denaturation, conditions for washing steps, signal amplification, and microscopic analysis have to be optimized for the Comet-FISH protocol. Hybridization efficiency, which should be always determined [6], can be ameliorated using optimized DNA probes with a higher DNA concentration and a reduced size compared to the probes used for conventional FISH on metaphase chromosomes [19]. To avoid gel matrix damage padlock probes can be used which are very stable and specific. The advantage of such probes is that reactions can be performed at 37 °C [10]. Comet-FISH is a relative simple method to study DNA damage and its repair throughout the genome and in defined chromosomal regions [20] at the level of individual cells, but only a few further developments are described. A new application of Comet-FISH, which increases the output and sample size with a twelve-gel slide format, was presented for example by Shaposhnikov et al. [21]. Hovhannisyan [22] described the combination of FISH, not only
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Fig. 1 Overview of potential fragmentation patterns and resulting signal distributions after Comet-FISH experiments: (a) undamaged DNA; (b) damaged DNA without a break in the vicinity of the specifically labelled DNA sequence; (c, d) damaged DNA with one or two signals located in the tail; (e) damaged DNA with more than two signals resulting from double-strand breaks in the vicinity of the specifically labelled DNA located in the tail
with the Comet assay but also with the micronucleus test. Furthermore, Kwasniewska et al. [23] reported for the first time on the application of FISH to Comet preparations from plants to analyze DNA damage and repair.
Detection of DNA Damage by Comet-FISH
2
43
Materials
2.1 Pre-coating of Slides
1. Glass slides (frosted). 2. Glass cover slips. 3. Phosphate-buffered saline (PBS): 8.0 g/L NaCl, 0.2 g/L KCl, 0.2 g/L KH2HPO4, 1.15 g/L Na2HPO4 · 2H2O, pH 7.3. Store at 4 °C. 4. Normal-melting agarose (NMA; 0.5 %): 150 mg NMA in 30 ml PBS (microwave). Store at 4 °C (see Note 1). 5. Water quench. 6. Heating plate. 7. Ice, icebox.
2.2 Treatment of Cells with Test Compounds (e.g., Genotoxic Model Chemicals Like H2O2)
1. Hydrogen peroxide (H2O2) (see Note 2).
2.3 Comet Assay (Detection of DNA Damage)
1. Pre-coated slides from Subheading 2.1.
2. Pipettes and tips. 3. Vortex mixer. 4. Tubes.
2. Low-melting agarose (LMA; 0.7 %): 210 mg LMA in 30 ml of PBS (microwave). Store at 4 °C (see Note 1). 3. Glass cover slips. 4. Lysis stock solution: 146.1 g/L NaCl, 37.2 g/L Na2EDTA, 1.2 g/L Tris-base, 8 g/L NaOH, 10 g/L N-lauroyl sarcosin sodium salt, pH 10. Store at room temperature. 5. 5. Lysis working solution: 1 % Triton-X, 10 % DMSO, 89 % lysis stock solution. Store at 4 °C, prepare freshly. 6. Electrophoresis chamber (e.g., Renner GmbH, Dannstadt, Germany). 7. Electrophoresis stock solutions: NaOH solution, 400 g/L NaOH; Na2EDTA solution, 74.4 g/L Na2EDTA. 8. Electrophoresis working solution: 60 mL NaOH stock solution, 10 mL Na2EDTA stock solution, 1,930 mL distilled water, store at 4 °C, prepare freshly. 9. Neutralization buffer: 4.2 M Tris–HCl, 0.08 M Tris-base, pH 7.2. 10. TE-buffer: 10 mM Tris–HCl, 1 mM EDTA, pH 8. Store at room temperature. 11. Antifade buffer: 2.5 g DABCO, 50 mL TE-buffer, 50 ml glycerol, store at 4 °C, protect from light.
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Michael Glei and Wiebke Schlörmann
12. SYBR Green I [1:10.000]: 1 μL SYBR®-Green, 9,999 μL Antifade buffer, store at 4 °C, protect from light. 13. Ethanol (100 %). 2.4 FISH: Preparation of Slides
1. 0.5 M NaOH. 2. PBS. 3. Ethanol (70, 80, 95, 100 %). 4. Hybrisol VI (Oncor, Gaithersburg, UK). 5. Plastic cover slips.
2.5 FISH: Hybridization
1. Digoxigenin-labelled DNA probes (e.g., Oncor, Gaithersburg, UK). 2. Hybrisol VI (Oncor, Gaithersburg, UK). 3. Water quench. 4. Plastic cover slips. 5. Hybridization chamber. 6. Saline sodium citrate (2× SSC): 0.3 M NaCl, 0.03 M sodium citrate, pH 7.2. 7. 1× Phosphate-buffered detergent (PBD) (Oncor, Gaithersburg, UK).
2.6 Detection of DigoxigeninLabelled Probes
1. Anti-digoxigenin-AP Germany).
Fab-fragments
(Roche,
Mannheim,
2. HNPP-Fluorescence Detection Kit (Roche, Mannheim, Germany). 3. SYBR®-Green (1 μL/10 mL). 4. Fluorescence microscope. 5. Fluorescence filter (green and red, e.g., ZEISS filter No 09 and 15). 6. Digital CCD camera and imaging software (e.g., MicroMAX, BFI OPTILAS GmbH; Visitron Systems GmbH, Puchheim, Germany). 7. Image analysis system (e.g., Kinetic Imaging, Liverpool, UK or Perceptive Instruments Suffolk, UK).
3
Method
3.1 Pre-coating of Slides
1. Dissolve 0.5 % normal-melting agarose (NMA) at 60 °C (water quench, see Note 1). 2. Drop 50 µL of 0.5 % NMA on one side of each slide and distribute NMA with another slide by dragging it to the opposite side (smear). 3. Dry slides at ~60 °C (heating plate).
Detection of DNA Damage by Comet-FISH
45
4. Coat slides with another 200 µL of 0.5 % NMA (see Note 3). 5. Cover slides with cover slips rapidly to allow a homogeneous disposition of NMA. 6. Cool slides for 10 min on an icebox (see Note 4). 7. Store slides at 4 °C (see Note 5). 3.2 Treatment of Cells with Test Compounds (e.g., Genotoxic Model Chemicals Like H2O2)
2. Incubate cell suspension, e.g., HT29 cells (2 × 106 cells/mL) with H2O2 solutions for 5 min at 4 °C.
3.3 Comet Assay (Determination of DNA Damage)
1. Dissolve 0.7 % of low-melting agarose (LMA) at 40 °C (water quench, see Note 1) and resuspend each cell pellet in a volume of 50 μL.
1. Dilute H2O2 in PBS to favored concentrations, vortex.
3. Remove H2O2 solutions by centrifugation at 400 × g for 5 min. 4. Discard the supernatants.
2. Distribute 50 μL of each cell suspension onto pre-coated slides, cover them with cover slips, and allow agarose to solidify for 10 min on an icebox. 3. Remove cover slips and immerse slides in lysis solution for at least 60 min at 4 °C (see Note 6). 4. Place slides into an electrophoresis chamber containing alkaline electrophoresis buffer and incubate them for 20 min. 5. Carry out electrophoresis at 1.25 V/cm and 300 mA for 20 min by adjusting the total volume of electrophoresis buffer. 6. Remove slides from the electrophoresis chamber and wash them three times for 5 min in neutralization buffer. 7. Stain slides with SYBR Green I (in Antifade buffer, 30 μl per slide) and determine DNA damage or continue to step 8. 8. Dehydrate slides in absolute EtOH for at least 3 days. 3.4 FISH: Preparation of slides
1. Rehydrate slides in double-distilled H2O for 10 min. 2. Denature DNA in 0.5 M NaOH for 30 min. 3. Carry out neutralization in PBS for 1 min. 4. Dehydrate slides in an ascending EtOH series: 70, 80, 95 % each for 5 min. 5. Dry slides at room temperature. 6. Drop 30 μL Hybrisol VI onto each slide and spread it with a plastic cover slip.
3.5 FISH: Hybridization
1. Prepare the hybridization mixture containing 10 μL digoxigenin-labelled probe (commercially acquired) and 20 μL Hybrisol VI. 2. Denature hybridization mixture at 37 °C for 5 min (water quench) (see Note 7).
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Michael Glei and Wiebke Schlörmann
3. Apply hybridization mixture onto one half of each slide and Hybrisol VI as negative control on the other half and cover it with a plastic cover slip. 4. Incubate slides at 37 °C in hybridization chambers for 24–72 h. 5. Wash slides in 2× SSC at 72 °C for 5 min. 6. Wash slides in 1× PBD for 5 min at room temperature. 3.6 Detection of Digoxigenin-Labelled Probes
1. For detection of digoxigenin-labelled probes use anti-digoxigenin-AP Fab fragments and 2-hydroxy-3-naphthoic acid-20phenylanilide-phosphate (HNPP) fluorescent detection set (Roche, Mannheim, Germany) according to the manufacturer’s instructions. 2. Use SYBR Green I (in Antifade buffer; 30 mL per slide) to counterstain the HNPP-detected probes (see Note 8). 3. Evaluate slides with a fluorescence microscope, equipped with filters to score images stained with SYBR Green I and HNPP or rather HNP/TR (2-hydroxy-3-naphthoic acid-20phenylanilide/Texas Red, see manufacturer’s instructions) (green and red emission, respectively) (see Notes 9–11). 4. For capturing images use a digital CCD camera (e.g., MicroMAX, BFI OPTILAS GmbH, Puchheim, Germany, processed with the Meta View Imaging Software, Visitron Systems GmbH, Puchheim, Germany).
3.7
Evaluation
1. Determine hybridization efficiency [6] (see Note 12). 2. Categorize comets into four degrees of damage ranging from undamaged images (class 1) to severely damaged images (class 4) [24] and/or by determination of the tail intensity with an image analysis system (e.g., Perceptive Instruments Suffolk, UK). 3. For the further Comet-FISH evaluation use only cells with two or more fluorescent spots and record the position of the signals in the comet head or comet tail (see Fig. 1). 4. Record the percentage of damaged cells (belonging to comet classes 2–4), in which it is possible to find a migration of at least one signal into the comet tail as the parameter of migration. 5. Score about 100 cells per slide (see Notes 11 and 12).
4
Notes 1. NMA and LMA are dissolved by heating in a microwave and kept fluid in a water quench. NMA and LMA can be stored for 2–3 weeks at 4 °C.
Detection of DNA Damage by Comet-FISH
47
2. H2O2 can be used in a favored genotoxic concentration (usually 75 μM). 3. Pre-coating of the slides is more homogeneous when slides stay on the heating plate at lower temperature (~ 30 °C) while the second agarose coat is applied. 4. A metal box filled with ice is useful to cool down pre-coated slides. Cooling causes a more homogeneous coating of the slides and less background signals. 5. The slides can be stored a maximum of one week at 4 °C in a humidified chamber. 6. The slides can also be lysed overnight at 4 °C. 7. Denaturation procedure depends on DNA probe used, for self made dig-labelled samples denature for 5 min at 72 °C, 4 min on ice, 30 min at 37 °C and mix 5 μl sample with 10 μl Hybrisol VI [7]. 8. If the fluorescence signals are too weak, dilute SYBR Green I 1:1,000 instead of 1:10,000 in Antifade buffer. 9. In the case that the hybridization signals are too weak, reduce SSC concentration to improve the stringency of the washing steps and wash for a longer period of time. 10. Peroxides may induce a fading of SYBR Green. So it could be necessary to use higher concentrations of DABCO Antifade and/or to wash the slides before staining. 11. If there are too much background signals, which disturb evaluation, rehydrate slides for a longer period of time before staining. 12. Improvement of the hybridization efficiency might be achieved by changing the hybridization temperature and duration, as well as by varying the temperature and stringency of the washing steps (15 min in double-distilled H2O). References 1. Östling O, Johanson KJ (1984) Microelectrophoretic study of radiationinduced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 123:291–298 2. Singh NP, McCoy MT, Tice RR et al (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191 3. Collins AR, Oscoz AA, Brunborg G et al (2008) The comet assay: topical issues. Mutagenesis 23:143–151 4. Santos SJ, Singh NP, Natarajan AT (1997) Fluorescence in situ hybridization with comets. Exp Cell Res 232:407–411
5. Glei M, Hovhannisyan G, Pool-Zobel BL (2009) Use of Comet-FISH in the study of DNA damage and repair: review. Mutat Res 681:33–43 6. Schaeferhenrich A, Beyer-Sehlmeyer G, Festag G et al (2003) Human adenoma cells are highly susceptible to the genotoxic action of 4-hydroxy-2-nonenal. Mutat Res 526:19–32 7. Knöbel Y, Glei M, Weise et al (2006) Uranyl nitrilotriacetate, a stabilized salt of uranium, is genotoxic in nontransformed human colon cells and in the human colon adenoma cell line LT97. Toxicol Sci 93:286–297 8. Knöbel Y, Weise A, Glei M et al (2007) Ferric iron is genotoxic in non-transformed and
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9.
10.
11.
12.
13.
14.
15.
Michael Glei and Wiebke Schlörmann preneoplastic human colon cells. Food Chem Toxicol 45:804–811 Glei M, Schaeferhenrich A, Claussen U et al (2007) Comet fluorescence in situ hybridization analysis for oxidative stress-induced DNA damage in colon cancer relevant genes. Toxicol Sci 96:279–284 Shaposhnikov S, Frengen E, Collins AR (2009) Increasing the resolution of the comet assay using fluorescent in situ hybridization: a review. Mutagenesis 24:383–389 Shaposhnikov S, Thomsen PD, Collins AR (2011) Combining fluorescent in situ hybridization with the comet assay for targeted examination of DNA damage and repair. Methods Mol Biol 682:115–132 Mladinic M, Zeljezic D, Shaposhnikov SA et al (2012) The use of FISH-comet to detect c-Myc and TP 53 damage in extended-term lymphocyte cultures treated with terbuthylazine and carbofuran. Toxicol Lett 211:62–69 Bock C, Rapp A, Dittmar H et al (1999) Localisation of specific sequences and DNA single strand breaks in individual UV-A irradiated human lymphocytes by COMET FISH. Prog Biomed Optics 3568:207–217 Kelvey-Martin VJ, Ho ET, McKeown SR et al (1998) Emerging applications of the single cell gel electrophoresis (Comet) assay. I. Management of invasive transitional cell human bladder carcinoma. II Fluorescent in situ hybridization Comets for the identification of damaged and repaired DNA sequences in individual cells. Mutagenesis 13:1–8 Rapp A, Bock C, Dittmar H et al (2000) UV-A breakage sensitivity of human chromosomes as measured by COMET-FISH depends on gene density and not on the chromosome size. J Photochem Photobiol, B 56:109–117
16. Ooi FA (2001) Oncogene amplification detection by fluorescence in situ hybridization (FISH). Acta Histoche Cytochem 34: 391–397 17. Anderson D, Yu TW, Browne MA (1997) The use of the same image analysis system to detect genetic damage in human lymphocytes treated with doxorubicin in the Comet and fluorescence in situ hybridisation (FISH) assays. Mutat Res 390:69–77 18. Horvathova E, Dusinska M, Shaposhnikov S et al (2004) DNA damage and repair measured in different genomic regions using the comet assay with fluorescent in situ hybridization. Mutagenesis 19:269–276 19. Rapp A, Hausmann M, Greulich KO (2005) The comet-FISH technique: a tool for detection of specific DNA damage and repair. Methods Mol Biol 291:107–119 20. Spivak G (2010) The Comet-FISH assay for the analysis of DNA damage and repair. Methods Mol Biol 659:129–145 21. Shaposhnikov S, Azqueta A, Henriksson S et al (2010) Twelve-gel slide format optimised for comet assay and fluorescent in situ hybridisation. Toxicol Lett 195:31–34 22. Hovhannisyan GG (2010) Fluorescence in situ hybridization in combination with the comet assay and micronucleus test in genetic toxicology. Mol Cytogenet 3:17 23. Kwasniewska J, Grabowska M, Kwasniewski M et al (2012) Comet-FISH with rDNA probes for the analysis of mutagen-induced DNA damage in plant cells. Environ Mol Mutagen 53:369–375 24. Wollowski I, Ji ST, Bakalinsky AT et al (1999) Bacteria used for the production of yogurt inactivate carcinogens and prevent DNA damage in the colon of rats. J Nutr 129:77–82
