QUALITATIVE ANALYSIS OF DNA FRAGMENTATION BY AGAROSE GEL ELECTROPHORESIS

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1. Introduction

The present protocol provides a method for DNA separation of fragmented and intact DNA fractions and for their analysis by agarose gel electrophoresis. In apoptotic cells specific DNA cleavage becomes evident in electrophoresis analysis as a typical ladder pattern due to multiple DNA fragments. However, although this protocol is simple and generally able to provide good results, it is only qualitative because of its limitations in DNA recovery and solubilization. In order to obtain a cleaner DNA, other methods for DNA preparation are required (in some cases use of proteinase K for deproteinization is recommended).

Nuclear morphology changes characteristic of apoptosis appear within the cell together with a distinctive biochemical event: the endonuclease-mediated cleavage of nuclear DNA. In fact, formation of DNA fragments of oligonucleosomal size (180-200 bp) is an hallmark of apoptosis in many cell types. 2. Protocol

2.2. Methodology

2. Centrifuge cells at 200xg at 4°C for 10 min.

3. Transfer supernatants carefully in new tubes labeled S (supernatant).

4. Add to the pellet in tubes B 0.5 ml of TTE solution and vortex vigorously. This procedure allows the release of fragmented chromatin from nuclei, after cell lysis (due to the presence of Triton X-100 in the TTE solution) and disruption of the nuclear structure (following Mg++ chelation by EDTA in the TTE solution).

5. To separate fragmented DNA from intact chromatin, centrifuge tubes B at 20,000xg for 10 min at 4°C.

6. Carefully transfer supernatants in new tubes labeled T (top).

7. Add to the small pellet in tubes B 0.5 ml of TTE solution.

8. Add to the 0.5 ml volume present in tubes B, S and T, 0.1 ml of ice-cold 5M NaCl and vortex vigorously. The addition of the salt should be able to remove histons from DNA.

9. Add to each tube 0.7 ml of ice-cold isopropanol and vortex vigorously.

10. Allow precipitation to proceed overnight at -20°C. This step can be shortened by putting samples in a bath of ethanol/dry ice for 1 hr.

11. After precipitation, recover DNA by pelleting for 10 min at 20,000xg at 4°C.

12. Discard supernatants by aspiration or by rapidly inverting tubes and carefully remove any drops or fluid remaining adherent to the wall of the tube with a paper towel corner. This can be a critical step because the pellet could be loosen and transparent, hard to be seen.

13. Rinse the pellets by adding to each tube 0.5-0.7 ml of ice-cold 70% ethanol.

14. Centrifuge tubes at 20,000xg for 10 min at 4°C.

15. Discard supernatants by aspiration or by rapidly inverting tubes. Carefully remove any drops or fluid remaining adherent to the wall of the tube by inverting tubes over an absorbent paper towel for 30 min. Let air dry the tubes in upright position for at least 3 hr before proceeding.

16. Dissolve DNA by adding to each tube 20-50 m l of TE solution and place the tubes at 37°C for 1-3 days. The redissolution of DNA may be a crical step, in fact it depends on the DNA quantity and size present in the samples. Thus, the non-fragmented DNA contained in the B tubes, may need higher volumes of TE and longer incubation times in order to be resuspended.

17. Mix the samples of DNA with loading buffer by adding 10x loading buffer to a final concentration of 1x. The addition of loading buffer to samples allows to load gel wells more easily and to monitor the run of samples.

18. Place samples in a heating block at 65°C for 10 min and immediately load 10-20 m l of them to each well of a standard 1% agarose gel containing ethidium bromide 0.5 mg/ml. Appropriate DNA molecular weight markers should be included. Ethidium bromide is a potential carcinogen: wear gloves and handle with care.

19. Run the electrophoresis in standard TBE buffer after setting the voltage to the desired level. During electrophoresis it is possible to monitor the migration of samples by following the migration of bromophenol blue dye contained in the loading buffer.

20. Stop the electrophoresis when the dye reaches about 3 cm from the end of the gel.

21. To visualize DNA, place the gel on a UV transilluminator and take photos of the gel. Wear eye and skin protection when UV are on.

3. Commentary

Apoptosis is an innate mechanism of eukariotic cell suicide which plays a major role in many physiological and pathological processes. Therefore, the definition of cellular regulatory mechanisms and biochemical processes involved in apoptosis is an important challenge from both theoretical and applied points of view.

During apoptosis a series of reorganisation occur in the cell: chromatin condensation, loss of cell volume and membrane blebbing are some of the most evident morphological changes of apoptotic cells. Although the molecular mechanisms leading to such changes are not completely known, many of them seem to proceed in parallel with biochemical events. This is the case, for example, of chromatin condensation and nuclear envelop breakdown. In fact, in parallel with them occurs DNA fragmentation, a biochemical hallmark of apoptosis in the majority of cells. Responsible for DNA cleavage is believed to be an endogenous Ca++- and Mg++-dependent endonuclease able to break double strand DNA at internucleosomal sites. Therefore, apoptotic DNA cleavage results in characteristic fragments of oligonucleosomal size (180-200 bp). Such phenomenum, described for the first time by Wyllie (1980), can be visualized by an agarose gel electrophoresis analysis. The present protocol provides a method for qualitative determination of DNA fragmentation.

3.1. Background information

3.2. Critical parameters

Moreover, this method is not recommended when different behaviour in DNA fragmentation following apoptotic stimuli is described. In fact, in some cell types where random double-stranded or rare single-stranded DNA fragmentation occur, it cannot be detected by agarose gel electrophoresis assay.

Sometimes, in particular with cells obtained from ex vivo cultures (e.g. thymocytes and lymphocytes), an high background of spontaneous DNA fragmentation could be observed.

The most critical point of DNA electrophoretical analysis is its inability of quantitative measurement of apoptosis. In fact, due to problems linked to the insolubility of large DNA and thus to its final agarose gel analysis, the method is strictly qualitative.

3.3. Troubleshooting

Solubilization of chromosome-length DNA collected in tubes B is generally difficult. Increase of TE volumes and extension of incubation time may be needed for the redissolution of DNA following precipitation. The use of limited number of cells (less than 5x106) will be helpful to limit this problem. However, since the method is exclusively qualitative, the analysis of fragmented DNA present in tubes T is the main interest of the assay.

3.4. Anticipated results

The assay of DNA agarose gel electrophoresis provides good results for the definition of cell apoptosis. A typical ladder pattern of DNA fragmentation should be observed in most apoptotic cells. 3.5. Time considerations

Preparation of DNA for agarose gel electrophoresis analysis depends on the DNA size of samples: generally from 2 to 7 days are required. Additional 4-8 hr are needed for performing electrophoresis. 3.6. Key references 1. Wyllie, A.H. 1980. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activity. Nature 284: 555.

2. Duke, R.C., and Cohen, J.J. 1986. Endogenous endonuclease-induced DNA fragmentation: an early event in cell-mediated cytolisis. Proc. Natl. Acad. Sci. U.S.A. 80: 6361.

3. Arends, M.J., Morris, R.J., and Wyillie, A.H. 1990. Apoptosis. The role of the endonuclease. Am. J. Pathol. 136: 593.

4. Bortner, C.D., Oldenburg, N.B.E., and Cidlowski, J.A. 1995. The role of DNA fragmentation in apoptosis. Trends Cell Biol. 5: 21.

5. Sellins, K.S., and Cohen, J.J. 1991. Cytotoxic T lymphocytes induce different types of DNA damage in target cells of different origin. J. Immunol. 147: 795.

Appendix 1 (A1): Stock solutions
<center> <table> <tbody> <tr> <td> <center> </center> <center> Solution </center> <center> </center> </td> <td> <center> </center> <center> Preparation </center> </td> <td> <center> </center> <center> Storage </center> </td> </tr> <tr> <td> Complete RPMI medium </td> <td> RPMI-1640 medium supplemented with 5% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 25 mM HEPES buffer, 50 µg/ml gentamicin sulfate. L-glutamine is labile, thus it does not last at 4°C for more than one day. </td> <td> <center> 4°C</center> </td> </tr> <tr> <td> TE buffer</td> <td> 10 mM Tris.Cl pH 7.4 (prepare by diluting stock solution), 1 mM EDTA.</td> <td> <center> RT</center> </td> </tr> <tr> <td> Tris.Cl stock solution (1 M)</td> <td> Dissolve 121 g Tris base in 800 ml H2O, adjust to desired pH with concentrated HCl, mix and add H2O to 1 liter. CAUTION: Adjust pH of the Tris buffer at the same temperature at which it will be used, as the pH varies with temperature (about 0.028 pH units per 1°C). </td> <td> <center> RT</center> </td> </tr> <tr> <td> Loading buffer 10x </td> <td> Prepare concentrated stock solution of loading buffer by adding the following reagents at the indicated final concentrations: 20% Ficoll 400, 0.1 M EDTA (pH 8.0), 1% SDS, 0.25% bromophenol blue, 0.25% xylene cyanol (optional).</td> <td> <center> RT</center> </td> </tr> <tr> <td> TBE buffer stock solution </td> <td> Dissolve in 800 ml of H2O 108 g Tris base (89 mM), 55 g boric acid (89 mM), 40 ml 0.5M EDTA, pH 8.0 (2mM); bring to 1 liter with H2O. Use diluted 1:10. </td> <td> <center> RT</center> </td> </tr> <tr> <td> Ethidium bromide stock solution</td> <td> Dissolve 50 mg of ethidium bromide in 100 ml of H2O. Use diluted 1:1000.</td> <td> <center> 4°C</center> <center> Protect from light.</center> </td> </tr> <tr> <td> Agarose gel</td> <td> Dissolve 1% agarose in 1x TBE buffer (in the presence of 0.5 m g/ml ethidium bromide) by heating until melted. </td> <td> <center> Prepare just before use.</center> </td> </tr> </tbody> </table> </center> <center> </center>

Appendix 2 (A2): Reagents

Gentamicin sulfate, solution G-1522
Sigma

L-glutamin 20 mM , 200 ml 25030-024
Gibco BRL

FCS A-1111-L
Hyclone

EDTA disodium salt, dihydrate E-5134
Sigma

TRIZMA base (Tris) T-1503
Sigma

Triton X-100 115291A
BioRad

Sodium Chloride S-9888
Sigma

Isopropyl alcohol 412421
Carlo Erba

Ethanol 1170
Riedel-deHaen

Lauryl sulphate, sodium salt (SDS) L-4390
Sigma

Ficoll 400 F-4375
Sigma

Bromophenol blue B-5525
Sigma

Xylene Cyanol X-4126
Sigma

Boric acid B-0394
Sigma

Ethidium bromide E-7637
Sigma

DNA molecular weight markers IX 1449460
Boehringer Mannheim

Agarose standard 18054
Eurobio

Polaroid film type 667 F-4638
Sigma

Appendix 3 (A3): Equipment

Multi-block Heater Model 2094
Lab-line Instruments, Inc.

Refrigerated cell centrifuge Model GS-56R
Beckman

Refrigerated microcentrifuge Model 5417R
Eppendorf

Vortex Model MT 135
Carlo Erba

Water bath Model 1002
GFL

Gel electrophoresis apparatus Model Horizon 58
Gibco BRL

Power supply Model 1000/500
BioRad

UV Transilluminator Model T2202
Sigma

Direct screen instant camera Model DS34
Polaroid