The race in action
Molecular geneticists are like racing fans. They need to observe the action to gain information about the DNA. The tracking dye provides some information. In addition to helping the geneticist load their DNA sample in the well, the tracking dye will also move in response to electric current and as it move it provides an indicator of how the DNA is moving. In Fig. 16, the tracking dye looks dark purple as it just begins to leave the wells. In Fig. 17, the dye is now seen to consist of a mixture of a faster moving purple dye and a slower moving blue dye. By observing how the tracking dye moves, the geneticist can determine that current is moving through the gel. However, in room light, the DNA is not visible.
Fig. 16. The tracking dye moves along with the DNA sample. (Image by D. Lee)
Fig. 17. The two dyes used to make the tracking dye separate as the dye moves through the gel. (Image by D. Lee)
Because DNA lacks color, another type of dye molecule that binds specifically to DNA is added to the electrophoresis buffer or to the gel. A commonly used dye is ethidium bromide. This dye has a structure that allows it to bind to the DNA helix and stay there. (note: Ethidium bromide’s DNA binding abilities make it a mutagen. Molecular geneticists wear gloves to prevent this dye from binding to the DNA in their own cells.) Ethidium bromide has an orange color in visible light but it’s real power for detection comes in the ultraviolet range of wavelengths. In UV light, the dye fluoresces brightly. Therefore to see the DNA, the gel is placed on a UV light source (Fig. 18).
Fig. 18. The ethidium bromide stained DNA can be observed running through the gel with a UV light source. (Image by D. Lee)
Ethidium bromide will emit a strong fluorescent signal, but detection of DNA depends on one additional factor; copies of the DNA segment. A single molecule of DNA cannot emit a strong enough fluorescent signal to be seen or detected. Instead, the molecular geneticist attempts to have hundreds or thousands of copies of the DNA segments they are trying to detect and load them in the gel well together. If the segments are the same length, they will move at about the same rate through the gel and form a band of DNA. The molecules making up this band collectively bind enough of the ethidium bromide to emit a detectable fluorescent signal. Therefore, when we view an electrophoresis gel that has been run and stained, we can observe bands of DNA. This is called the DNA fragment banding pattern. A band contains many copies of the same length molecule.