Apoptosis DNA fragmentation analysis protocol

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.