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For the detection of DNA fragmentation – a hallmark of late apoptotic cells
Apoptosis, one of the main forms of programmed cell death, is undertaken in very distinct stages characterized by specific morphologies. The process is initiated by the activation of the caspase cascade through either the intrinsic or extrinsic apoptosis pathway.
One of the later stages of apoptosis is DNA fragmentation (karyorrhexis), which starts upon completion of the nuclear condensation (pyknosis) stage. DNA fragmentation is mediated by caspase activated endonucleases such as CAD (caspase activated DNAse), which cleave or break the genomic DNA into oligonucleosome fragments or multiples of approximately 200 base pairs. When the genomic DNA from such late apoptotic cells is extracted and run on agarose gels, a “DNA ladder” pattern can be observed (Compton 1992). An alternative method to observing DNA fragmentation on agarose gels are TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) assays.
DNA breaks result in the display of a hydroxyl group at the 3′ end of the DNA fragment. In TUNEL assays, this characteristic is used to label the fragments with Br-dUTP (bromolated deoxyuridine triphosphate nucleotides). The incorporation or labeling is performed by the enzyme TdT (Terminal deoxynucleotidyl Transferase), which adds the Br-dUPT to the 3′ end. The incorporated Br-dUTP can then be detected with the help of anti-BrdU antibodies. In most cases the anti-BrdU antibodies are directly labeled with biotin or a fluorophore.
We offer two TUNEL assay kits:
Compton MM (1992). A biochemical hallmark of apoptosis: internucleosomal degradation of the genome. Cancer Metastasis Rev 11:105-119.
Apo-BrdU™ is a trademark of Phoenix Flow Systems, USA.
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