Apoptosis DNA fragmentation analysis protocol
Find out the procedure for apoptosis DNA fragmentation in our useful step-by-step guide.
Last edited Tue 14 Feb 2023
A distinctive biochemical feature of apoptosis is the fragmentation of DNA by a specific nuclease called caspase-activated DNase (CAD). Activation of CAD by the caspase cascade leads to specific cleavage of DNA at internucleosomal linker sites, generating fragments of ~200 base pairs known as DNA ladders.
The classical method to detect DNA ladders is to examine fragmented genomic DNA on an agarose gel. This semi-quantitative method is a simple technique that provides a robust answer.
As an alternative, to gel based analysis, consider using a TUNEL assay kit for the ability to analyze DNA fragmentation by flow cytometry or microscopy.
Stage 1 - Harvest cells
Steps
Pellet cells.
Lyse cells in 0.5 mL detergent buffer: 10 mM Tris (pH 7.4), 5 mM EDTA, 0.2% Triton (0.2% NP-40 can be used instead of Triton X-100).
Vortex.
Incubate on ice for 30 min.
Centrifuge
- 27,000 x g for 30 min
Divide supernatants into two 250 µL aliquots.
Add 50 µL ice-cold 5 M NaCl to each aliquot and vortex.
Stage 2 - Precipitate DNA
Steps
Add ethanol and sodium-acetate and mix by pipetting up and down.
- 600 µL ethanol and 150 µL 3 M sodium-acetate, pH 5.2.
Incubate tubes at -80°C for 1 h.
Centrifuge at 20,000 x g for 20 min then discard supernatants carefully.
Pool DNA extracts together by re-dissolving the pellets in a total of 400 µL extraction buffer (10 mM Tris and 5 mM EDTA).
Add DNase-free RNase.
- Add 2 µL of 10 mg/mL DNase-free RNase.
- Incubate for 5 h at 37°C.
Add 25 µL proteinase K at 20 mg/mL and 40 µL of buffer (100 mM Tris pH 8.0, 100 mM EDTA, 250 mM NaCl. Incubate overnight at 65°C.
Extract DNA with phenol/chloroform/isoamyl alcohol (25:24:1) and precipitate with ethanol.
Carefully discard supernatant.
Stage 3 - Load DNA in agarose gel
Steps
Air-dry pellet.
Once dry, resuspend in 20 µL Tris-acetate EDTA buffer supplemented with 2 µL of sample buffer (0.25% bromophenol blue, 30% glycerol).
Separate DNA electrophoretically on a 2% agarose gel containing 1 µg/mL ethidium bromide.
Visualize by ultraviolet transillumination.