R5.4c: Assays For DNA Damage And Repair

Comet Assay

A comet assay is an easily performed test of DNA damage. Single cells are placed on a glass slide, held in suspension by an agarose gel. They are then exposed to radiation (or some other stimulus) before being lysed by an aqueous solution. The DNA is unable to escape the agarose gel, whereas the remainder of the cell is removed by the solution. The DNA occupies this space (the nucleoid).
The slide is then immersed in an electrophoresis solution and has a current applied. Undamaged DNA remains trapped in the nucleoid, whereas damaged DNA is small enough to move through the agarose gel. Once the current has been applied for a specified time, the slide is stained for DNA molecules and visualised under a specialised microscope, often with image analysis software to calculate the presence of DNA damage.
The term comet assay is derived from the appearance of the nucleoid after electrophoresis has taken place. The undamaged DNA remains in the nucleoid in a sphere, the 'head' of the comet. The damaged DNA travels towards the anode, forming the 'tail' of the comet.

Plasmid Based Assay

I can't find any useful information on this type of assay.

Pulsed Field Gel Electrophoresis

Gel Electrophoresis works because fragments of DNA have a negative charge, causing them to migrate towards the anode if a charge is run through the gel containing the DNA molecules. This approach is limited due to poor sensitivity to large (over 50 kbp) fragments of DNA, which tend to move at the same rate through the gel. Pulsed field gel electrophoresis involves three pairs of electrodes, aligned at 0 degrees, 120 degrees, and -120 degrees with respect to the direction of travel. Charge is run through the sample for 10 - 60 seconds between a pair of electrodes, with an equal time spent on each group for a net forward migration. Larger fragments take longer to realign themselves to the changing voltage, and therefore there is increased separation of DNA fragments.

Common uses of PFGE

Apoptosis has a distinct appearance with PGFE, with laddering. Laddering occurs due to the cleavage of DNA into fragments of similar sizes. This is in contrast to necrosis, where DNA fragmentation is variable; this leads to a smear instead of distinct laddering.

Micronucleus Assay

A micronucleus is the aberrant formation of a third, small nucleus during a mitotic division. These micronuclei form when there is a piece of DNA not attached to the mitotic spindle (due to double strand breaks). For this experiment, dividing cells are exposed to a stimuli that causes double strand breaks. Cytokinesis is inhibited by cytochalasin-B. The cells are then stained and examined under a microscope; the number of cells that contain micronuclei are counted. About 1,000 cells need to be counted for an accurate result.

Use of the above methods

DNA damage is assessed by any of the above methods. Cells that are more sensitive to radiation will show increased fragmentation of DNA, detectable with pulsed field gel electrophoresis or the comet assay. The micronucleus assay is capable of detecting DNA breaks by measuring the formation of micronuclei, which occur when DNA fragments are not aligned on the mitotic spindle.
DNA repair can also be assessed using these methods. By allowing cells to survive for some time after radiation exposure, and comparing the fragmentation of DNA with cells immediately sacrificed, the amount of repair that occurs is quantifiable.

External Links

Article by Olive et al which discusses single cell gel electrophoresis (comet assay).

Internal Links