Protocol

Analysis of DNA Fragmentation Using Agarose Gel Electrophoresis

This protocol was adapted from “Apoptosis Assays,” Chapter 15, in Cells (eds. Spector et al.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1998. This three-volume set is now out of print; however, some of the microscopy methods were republished in Basic Methods in Microscopy, by David L. Spector and Robert D. Goldman.

INTRODUCTION

This protocol provides a qualitative method for assessing cell death by detecting DNA fragments using agarose gel electrophoresis. One of the classic features of apoptosis is the cleavage of the genomic DNA into oligonucleosomal fragments represented by multiples of 180-200 bp. Visualizing these fragments can aid in characterizing an apoptotic event. This method should always be combined with more quantitative methods in order to compare the degree of apoptosis among the experimental samples.

MATERIALS

Reagents

Agarose

6X gel-loading buffer

DNA size markers

Ethidium bromide

Proteinase K (20 mg/ml; Ambion)

RNase Cocktail (RNase A and T1 at 500 units/ml and 20,000 units/ml, respectively; Ambion)

TAE

TES lysis buffer

Equipment

Standard gel electrophoresis equipment

UV transilluminator

Wide-bore pipette tips

Wide-bore pipette tips are recommended to avoid shearing of the DNA. They can be obtained commercially or can be easily made by cutting off the end of standard 200-μl tips. Tips should be autoclaved to avoid DNase contamination.

METHOD

  • 1. Transfer 5 × 105 cells to 1.5-ml sterile microcentrifuge tubes. Centrifuge at 2000 rpm in an Eppendorf table top centrifuge for 5 minutes at 4°C and remove supernatant.

    Be careful not to use much higher numbers of cells because subsequent enzymatic digestion might not be complete, resulting in very viscous DNA solutions.

  • 2. Add 20 μl of TES lysis buffer. Mix cell pellet with TES lysis buffer by stirring with a wide-bore pipette tip.

    Do not apply vigorous vortexing or extensive pipetting because high-molecular-weight DNA might be sheared.

  • 3. Add 10 μl of RNase Cocktail and mix well by flipping the tip of the tube. Do not vortex. Incubate for 30-120 minutes at 37°C.

  • 4. Add 10 μl of proteinase K, mix by flipping the tip of the tube, and incubate at 50°C for at least 90 minutes or, if preferred, overnight.

  • 5. Add 5 μl of 6X DNA loading buffer and load DNA samples into dry wells of a 1-1.5% agarose gel in TAE containing 0.5 μg/ml ethidium bromide.

    To avoid loss of DNA samples due to the viscosity of some of the preparations, it is recommended that the loading should be done into dry wells (before running buffer is added to the reservoirs). For size reference, also load a 100-bp size ladder.

    Instead of adding ethidium bromide directly into the agarose gel, DNA can also be visualized by staining the gel after electrophoresis in 1 μg/ml ethidium bromide-containing TAE buffer for 1 hour and destaining in H2O.

  • 6. Run the gel at a low voltage, which improves resolution of DNA fragments (i.e., 35 V for ~4 hours or until loading dye has run two-thirds of the way down the gel).

  • 7. DNA ladders are finally visualized by a UV light source and documented by photography. Apoptotic cells will form a distinct DNA ladder, whereas necrotic samples may generate a smear pattern (or no DNA fragmentation). DNA from viable cells will stay on the top of the gel as a high-molecular-weight band.

    Apoptosis does not always result in DNA ladders; this depends on the cell type under study. In addition, necrosis can, in some cases, produce ladders.

    During apoptosis, DNA fragmentation (if it occurs) usually precedes loss of membrane integrity. If ladder formation is significantly delayed with respect to loss of viability, it is unlikely to represent an apoptotic event.

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