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.  

• 2.1. Materials Cell suspension at 1-5x106 cells/ml in complete RPMI medium (A1) TTE solution: TE buffer pH 7.4 (A1) with 0.2% Triton X-100 (store at 4°C) NaCl 5M, ice cold Isopropanol, ice cold Ethanol at 70%, ice cold TE buffer pH 7.4 (A1) Loading buffer 10x (A1) TBE buffer for electrophoresis (A1) Ethidium bromide solution (A1) Electrophoresis-grade agarose DNA molecular weight markers Refrigerated cell centrifuge (A3) Microfuge (A3) Heating block (A3) Gel electrophoresis apparatus (A3) DC power supply (A3) UV transilluminator (A3) Polaroid Camera + films (A3)  
• • 1. Dispense 0.5 ml of cell suspension (no less than 5x105, otherwise DNA will not be detectable by photography of ethidium bromide stained gel, and no more than 5x106, to avoid difficult handling of too high amounts of insoluble DNA) in tubes labeled B (bottom).
   

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  

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  

• RPMI-1640 , 500 ml 42402-016 Gibco BRL