DNA fragmentation assay

DNA fragmentation detected with agarose gel electrophoresis of DNA extracted from X-ray (6 Gy) irradiated cells. A and B represent with and without glycerol (0.6 M), respectively. C, no irradiation; Lanes 12, 24. 36 and 48, incubation periods (h) after X-ray irradiation. Lane M, øX174 DNA Hae III fragments as size markers.

Ohnishi et al. Molecular Cancer 2002 1:4   doi:10.1186/1476-4598-1-4

Apoptosis DNA Fragmentation is a key feature of programmed cell death and also occurs in certain stages of necrosis. Apoptosis is characterized by the activation of endogenous endonucleases with subsequent cleavage of chromatin DNA into internucleosomal fragments of 180 BP and multiples thereof.

DNA cleavage during apoptosis occurs at sites between nucleosomes, protein-containing structures that occur in chromatin at ~200-BP intervals. This DNA fragmentation is often analyzed using agarose gel electrophoresis to demonstrate a "ladder" pattern at ~200-BP intervals. Necrosis, on the other hand, is characterized by random DNA fragmentation which forms a "smear" on agarose gels.

DNA fragmentation is a secondary consequence, rather than an integral cause, of apoptosis. Endonuclease involved might be similar to DNAse I, a potential indication that the DNA fragmentation might occur after the release of enzymes from cytoplasmic membrane lysis, an event that would potentially occur only after the final lytic event in the apoptotic sequence. More recently, data have shown that specific proteases residing in the cytoplasm mediate the terminal events of apoptosis, including those of nuclear morphology. Even so, the detection of DNA fragmentation and the presence of single strand ends of DNA has continued to be an assay used in many studies to detect apoptotic cells, particularly in intact tissues. This is in spite of the fact that necrosis also produces single-strand DNA ends in cell nuclei. Therefore, the interpretation of these in situ assays of DNA fragmentation [in situ nick-translation (ISNT); terminal transferase (TUNEL) must be carefully assessed together with morphological features of apoptotic cells.

Even though much work has been performed on the analysis of apoptotic events, little information is available to link the timing of morphological features at the cell surface and in the nucleus to the biochemical degradation of DNA in the same cells. Apoptosis can be initiated by a myriad of different mechanisms in different cell types, and the kinetics of these events vary widely, from only a few minutes to several days depending on the cell system.

There are many methods to assess the DNA fragmentation caused by apoptosis and also many commercial kits are available. Some new methods do quantification assays for DNA Fragmentation by flow cytometry and fluorescent assays.





재무 덕분에 정말 많이 배우고 있다.

쪼개진 ladder를 보던 그 순간을 기억하자. ㅎ

Ref.
http://en.wikipedia.org/wiki/Apoptosis_DNA_fragmentation
http://www.molecular-cancer.com/content/1/1/4/figure/F3?highres=y
http://www.enzolifesciences.com/welcome/apoptosis-detection-tools/