As we know well the genomic DNA is cleaved into nucleosomal sub units by CAD enzyme during Apoptosis ..
If every part of genomic DNA is cleaved to 180bp (Nucleosomal sub unit) ,then how do we get DNA ladder, stacking in DNA ladder assay (GEL PICT )?
To get a clear picture on this, I searched various literature .
Below paragraph from wikipedia helps to agree the formation of stacking in DNA ladder assay.
Where I am in need of clear description for the highlighted statement i.e How 180bp mulitiplies?
What does the multiplies refer to -WE KNOW
But how odes it occur?
Thanking you
Apoptotic DNA fragmentation is a key feature of apoptosis, a type of programmed cell death. Apoptosis is characterized by the activation of endogenous endonucleases with subsequent cleavage of chromatin DNA into internucleosomal fragments of roughly 180 base pairs (bp) and multiples thereof (360, 540 etc.). This effect can be used to detect apoptosis, for example via the DNA laddering assay,
The problem is that electrophoretic migration has a logarithmic relationship with mass. Therefore, the spacing between the DNA fragments upon electrophoresis will not be even, despite the fact that the difference in mass between them is the same.
If for example, a stretch of 900-920 bp genomic DNA sequence was to undergo complete cleavage starting from one end by the enzymes involved in apoptotic cleavage of DNA, it would generate 6 cleavage sites and 5 fragments each of 180 bp. If the enzyme only cleaves 5 times, it will generate 3 fragments of 180 bp and 1 of 360 bp, with 4 cuts it will generate fragments of 540 + 180 +180 etc etc . When resolved on a gel together, the fragments will show 180 bp + 360 bp + 540 bp etc . If apoptosis associated DNA digestion went to total completion you would only see 180 bp products. What we visualise at any one time is the process of on-going cleavage that generates fragments of different sizes of multiples of 180 bp corresponding to partial / incomplete digestion occurring in many different cells. It is not dissimilar to a partial digestion of a plasmid by a restriction enzyme.
The easiest way to visualise it is to draw it out on a piece of paper.
CAD enzyme can cleave the accessible DNA molecule, as DNA is wrapped around nucleosome CAD can cleave at approximately 180 bp. Secondly, we see ladder formation because CAD may not be abundant as histones, so wherever it is possible, depending on CAD expression it cleaves accessible DNA molecule.
Chromosomal DNA is first broken into large fragments (50 - 300 kilobases) before they are cleaved into smaller nucleosomal fragments (approximately 200 base pairs). Extraction of the DNA fragments from apoptosis cells followed by agarose gel electrophoresis results in a distinctive DNA ladder appearance.
By Nature it is must that every apoptotic cell has to undergo cleavage at 180bp.
Bcoz, if it is not cleaved at 180bp .for example if apoptotic tumor cell DNA is cleave around 2kb to 5kb ,then the engulfing phagocyte may take up the DNA that may be tumorogenic ...
That in turn will make phagocyte to become cancerous
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