Help! There is a comet in my computer! 3
1 Fluorescence microscopy
We are running a comet experiment. We prepared the slides and ran electrophoresis. Now
we have to measure the amount of DNA that migrated out of nuclei. The most important
thing to remember is this: Measurement is based on a known relationship (e.g. linear,
logarithmic) between the amount of DNA in the sample and the parameter used for
detection of DNA. In all steps of the measurement procedure, we have to take care not to
compromise this relationship.
The first thing we need to do is stain DNA to make it detectable. Staining has to be
quantitative: the amount of stain that binds to DNA must be stoichiometrically proportional
to the amount of DNA present in the sample. In other words, the relationship between the
amount of DNA and the amount of the staining substance bound to DNA must be
linear.
The quantitative DNA dye usually used in the comet assay is ethidium bromide. During
incubation in the staining solution, ethidium bromide binds to DNA on the comet slide.
Ethidium bromide is an intercalator with little or no sequence preference and binds to DNA
at stoichiometry of one molecule per 4-5 base pairs of DNA. Hence, the relationship
between the amount of DNA and the amount of ethidium bromide bound to DNA is
linear.
Ethidium bromide is a molecule with special properties - a fluorophore. Fluorophores are
molecules that absorb the energy of light (photons) of specific wavelengths. This process is
called excitation. Fluorophores then release the absorbed energy in the form of light
energy (photons). This process is called emission. Because some energy is “lost” during
the excitation-emission cycle, the energy of emitted photons is lower than the energy of
absorbed photons. In other words, the wavelength of emitted light is longer than the
wavelength of absorbed light. Fluorescence is thus emission of light or other
electromagnetic radiation at longer wavelengths by matter as a result of absorption of
shorter wavelengths. Emission lasts only as long as the stimulating irradiation is present.
Each fluorophore absorbs and emits light of specific wavelengths. For example, DNA-
bound ethidium bromide absorbs green light and emits orange light (Figure 1A). Once
ethidium bromide is bound to DNA, its fluorescence increases about 10-fold (compared to
free molecules).
To be able to see the comets under a microscope, we need to illuminate the ethidium
bromide-stained slide with intense green light. The source of high intensity light is usually
a mercury lamp. Because a mercury lamp emits light of different wavelengths (including
UV), we need to place an excitation filter between the lamp and the slide. This filter only
passes green light and blocks all other wavelengths (Figures 1, 2A).
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