QUALITATIVEANALYSISOFDNAFRAGMENTATIONBYAGAROSEGELELECTROPHORESIS2

3. Commentary 
  
 

3.1. Background information

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.

3.2. Critical parameters 
 

• 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.

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. 
 

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

  
Appendix 1 (A1): Stock solutions 
 

Appendix 2 (A2): Reagents 
 

RPMI-1640

• , 500 ml

 

• 42402-016

 


• Gibco BRL

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