DNA laddering

DNA laddering is a feature that can observed when DNA fragments, resulting from apoptotic DNA fragmentation, are visualised after separation by gel electrophoresis as first described in 1980 by Andrew Wyllie at the University Edinburgh medical school. DNA fragments can also be detected in cells that have undergone necrosis. When these DNA fragments, after separation, are subjected to gel electrophoresis - this results in a characteristic ladder pattern.

DNA degradation

DNA laddering is a distinctive feature of DNA degraded by caspase-activated DNase (CAD), which is a key event during apoptosis. CAD cleaves genomic DNA at internucleosomal linker regions, resulting in DNA fragments that are multiples of 180–185 base-pairs in length. Separation of the fragments by agarose gel electrophoresis and subsequent visualization, for example by ethidium bromide staining, results in a characteristic "ladder" pattern. A simple method of selective extraction of fragmented DNA from apoptotic cells without the presence of high molecular weight DNA sections, generating the laddering pattern, utilizes pretreatment of cells in ethanol.

Apoptosis and necrosis

While most of the morphological features of apoptotic cells are short-lived, DNA laddering can be used as final state read-out method and has therefore become a reliable method to distinguish apoptosis from necrosis.
DNA laddering can also be used to see if cells underwent apoptosis in the presence of a virus. This is useful because it can help determine the effects a virus has on a cell.

DNA laddering can only be used to detect apoptosis during the later stages of apoptosis. This is due to DNA fragmentation taking place in a later stage of the apoptosis process. DNA laddering is used to test for apoptosis of many cells, and is not accurate at testing for only a few cells that committed apoptosis. To enhance the accuracy in testing for apoptosis, other assays are used along with DNA laddering such as TEM and TUNEL. With recent improvements to DNA laddering, DNA laddering has become a more reliable, and reasonable technique to use when detecting apoptosis. It is also important to note that DNA laddering occurs differently depending on the type of cell, so there may be slight changes in the process of DNA laddering depending on the cell that is being investigated.

Apoptosis DNA fragmentation analysis protocol

find out the producedure for apoptosis is the fragmentation in useful step by steps ,

individual Biochemical features of the apoptosis is the fragmentation of DNA by specific nuclese called Caspase-activated, DNase CAD ,the activation of CAD by the Caspase leads to specific cleavage of the DNA at the internucleosomal linker sites the fragmentation of -200 base pair know as DNA ladders

the method to detect DNA ladders to examine fragmented genomic DNA on the agarose gel this semi quantitative method is simple Technique

harvest cells

* pellet cells

* lyse cells in 0.5 ml detergent buffer:10mm tris PH 7.4MM EDTA 0.2 Triton
* vortex
* incubate on ice for 30 min
* centrifuge at 27,000 for 30 min
* divide supernatant into two 250ul aliquot

* add 50ul ice-cold 5m Nacl to each aliquot and vortex

precipitate DNA

* Add 600ul ethanol and 150 ul 3m sodium acetate pH 5.2 and mix by pipetting up and down
* incubate tubes at 80°c one hours
* centrifuge 20,000 for 20 min and discard
* pool DNA extract together by re-dissolving the pellets in a total of 400ul extraction buffer 10mM Triton 5mM EDTA.
* Add 2ul of 10mg/ml DNase-free RNase and incubate for 5hrs at 37°C
* Add 25ul proteinase at 20mg/ml and 40 ul of buffer 100mM Tris pH 8,0 100mM EDTA 250mM Nacl
* incubate overnight at 65°C
* Extract DNA with phenol chloroform alcohol and precipitate with ethanol,

* carefully discard supernatant trying not to disturb the pellet as it is quite loose

load DNA in agarose gel

* Air dry pellet and rsusupend in 20ul tris-acetate EDTA buffer supplemented with 2ul sample buffer 0.25% bromophenol blue 30% glycerol
* separate DNA electrophoretically on a 2% agarose gel Containing 1ug/ml Ethidium bromide and visualize by ultraviolet transillumination

protocol tips

* the DNA will make the sample very viscous and sticky, use the DNA sample loading buffer at a higher Concentration than you normally would to ensure
* prepare an agarose gel with 1.8-2% agarose content, the high agarose Concentration provided the necessary resolution to see the steps in the ladder
* run the gel at a low voltage for a longer time than you normally would to avoid overnight the subsequent deformation of the DNA bands,

See also

*Caspase-activated DNase
* Nicoletti assay
*Apoptotic DNA fragmentation
*TUNEL assay

References

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Apoptosis
Biological techniques and tools
Cell biology
Electrophoresis
Laboratory techniques
Programmed cell death