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The apoptotic DNA fragmentation is being used as a marker of apoptosis and for identification of apoptotic cells either via the DNA laddering assay, [2] the TUNEL assay, [3] [4] or the by detection of cells with fractional DNA content ("sub G 1 cells") on DNA content frequency histograms e.g. as in the Nicoletti assay.
The apoptotic process is accompanied by shrinkage and fragmentation of the cells and nuclei and degradation of the chromosomal DNA into nucleosomal units. DNA fragmentation factor (DFF) is a heterodimeric protein of 40-kD (DFFB) and 45-kD (DFFA) subunits. DFFA is the substrate for caspase-3 and triggers DNA fragmentation during apoptosis.
TUNEL is a method for detecting apoptotic DNA fragmentation, widely used to identify and quantify apoptotic cells, or to detect excessive DNA breakage in individual cells. [3] The assay relies on the use of terminal deoxynucleotidyl transferase (TdT), an enzyme that catalyzes attachment of deoxynucleotides, tagged with a fluorochrome or another ...
The apoptotic process is accompanied by shrinkage and fragmentation of the cells and nuclei and degradation of the chromosomal DNA into nucleosomal units. DNA fragmentation factor (DFF) is a heterodimeric protein of 40-kD (DFFB) and 45-kD subunits. DFFA is the substrate for caspase-3 and triggers DNA fragmentation during apoptosis.
Apoptotic DNA fragmentation is a natural fragmentation that cells perform in apoptosis (programmed cell death). DNA fragmentation is a biochemical hallmark of apoptosis.In dying cells, DNA is cleaved by an endonuclease that fragments the chromatin into nucleosomal units, which are multiples of about 180-bp oligomers and appear as a DNA ladder when run on an agarose gel. [8]
During apoptosis, a cell goes through a series of steps as it eventually breaks down into apoptotic bodies, which undergo phagocytosis.In the context of karyorrhexis, these steps are, in chronological order, pyknosis (the irreversible condensation of chromatin), karyorrhexis (fragmentation of the nucleus and condensed DNA) and karyolysis (dissolution of the chromatin due to endonucleases).
DNA laddering (left) visualised in an agarose gel by ethidium bromide staining. A 1 kb marker (middle) and control DNA (right) are included.. DNA laddering is a feature that can be observed when DNA fragments, resulting from Apoptosis DNA fragmentation are visualized after separation by gel electrophoresis the first described in 1980 by Andrew Wyllie at the University Edinburgh medical school ...
The relative dose administered determines the extent to which DNA fragmentation occurs. In the presence of large BLM concentrations, pseudoapoptosis is observed as rapid DNA fragmentation occurs, resulting in cell death in the absence of typical apoptotic components such as specific endonucleases and proteases. [ 4 ]