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Apoptotic cells exhibit several biochemical modifications such as protein cleavage, protein cross-linking, DNA breakdown, and phagocytic recognition that together result in the distinctive
structural pathology described previously Hengartner 2000. Caspases are widely expressed in an inactive proenzyme form in most cells and once activated can often activate other procaspases,
allowing initiation of a protease cascade. Some procaspases can also aggregate and autoactivate. This proteolytic cascade, in which one caspase can activate other caspases, amplifies the apoptotic
signaling pathway and thus leads to rapid cell death Elmore 2007. Although DNA fragmentation into oligonucleosomal ladders is characteristic of apoptosis,
recent evidence indicates that not all cells undergo such extensive DNA fragmentation Cohen, et. al1. 1992 in Chen and Ioannou 1996. In fact, fragmentation of DNA into kilobase-size fragments
appears to be an early event in apoptosis, preceding the complete digestion of DNA into multiples of nucleosomal size fragments Cohen and Sun 1994 in Chen and Ioannou 1996.
D. DNA LADDER ASSAY
One of the most easily measured features of apoptotic cells is the break-up of the genomic DNA by cellular nucleases. These DNA fragments can be extracted from apoptotic cells and result
in the appearance of “DNA laddering” when the DNA is analyzed by agarose gel electrophoresis Arends, Morris, and Wyllie 1990. Principle of this assay is apoptotic DNA binds quickly to glass
fiber fleece in the presence of a chaotropic salt, guanidine hydrochloride guanidine HCl. After cellular impurities are washed off the fleece, the DNA is released from the fleece with a low salt
buffer Wyllie, et. al 1998. DNA ladder is asolution of DNA molecules of different lengths used in agarose gel
electrophoresis. it is applied to an agarose gel as a reference to estimate the size of unknown DNA molecules. In addition it can be used to approximate the mass off a band by comparison to a
special mass ladder. Different DNA ladders are commercially available depending on expected DNA length. The 1 kb ladder with fragment ranging from about 0.5 kbp to 10 or 12 kbp and the
100 bp ladder with fragment ranging from 100 bp to just above 1000 bp are the most frequent. DNA ladders are often produced by a suitable restriction digest of plasmid.
DNA electrophoresis is an analytical technique used to separate DNA fragments by size. An electric field forces the fragments to migrate through a gel. DNA molecules normally migrate from
negative to positive potential due to the net negative potential due to the net negative charge of the phosphate backbone of the DNA chain. At the scale of the length of DNA molecules, the gel looks
much like a random, intricate network. Longer molecules migrate more slowly because they are more easily „trapped‟ in the network. After the separation is completed, the fractions of DNA
fragments of different length are often visualized using a fluorescent dye spesific for DNA, such as ethidium bromide. The gel shows bands corresponding to different DNA molecules populations
with different molecular weight. Fragment size i s usually reported in “nucleotides”, “base pairs” or
“kb” for 1000‟a of base pairs depending upon wheter single- or double-stranded DNA has been
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separated. Fragment size determination is typically done by comparison to commercially available DNA ladders containing linear DNA fragments of known length.
The types of gel most commonly used for DNA electrophoresis are agarose for relatively long DNA molecules and polyacrylamide for high resolution of short DNA molecules, for
example in DNA sequencing. Electrophoresis techniques used in the assessment of DNA damage include alkaline gel electrophoresis and pulsed field gel elctrophoresis. The measurement and
analysis are mostly done with a specialized gel analysis software. DNA molecules must be separated from other cellular material before they can be examined.
Cellular proteins that package and protect DNA in the environment of the cell can inhibit the ability to analyze the DNA. Therefore, DNA extraction methods have been developed to separate
proteins and other cellular materials from the DNA molecules. The quantity and quality of DNA often need to be measured prior to proceeding further with analytical procedures to ensure optimal
results. Organic extraction was for many years the most widely used methods for DNA extraction. Organic extraction involves the serial addition of several chemicals. First sodium dodecylsulfate
SDS and proteinase K are added to break open the cell walls and to break down the proteins that protect the DNA molecules while they are in chromosomes Butler 2010. NA sample purity and
molecular weight must be controlled by agarose gel electrophoresis with 0.4-0.8 ethidium bromide stain. The DNA can be suspended in TE buffer Tris-HCL and EDTA pH 8.0 or double-
distilled water Walker and Rapley 2008.
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III. MATERIALS AND METHODS
A. MATERIALS AND EQUIPMENTS
