Gel electrophoresis

Separating macromolecules with sieves

How electrophoresis of works:

Do you remember our discussion about the phosphate backbone of nucleic acids? The backbone is made up of phosphate ester linkages that are negatively charged. If we place a DNA molecule in an electric field, it will therefore be attracted to the "positive" electrode (the red one), and this is what we call "electrophoresis."

What happens if we partially block the path of migration of the nucleic acids, by placing the DNA in a gel of polymerized agarose or acrylamide? Then the rates of migration are reduced, because the molecules must traverse a complicated obstacle course. The crosslinked matrix of the gel will tend to reduce the rate of migration of molecules with a larger cross-section.

An analogy: for those students who might rather be out fishing.

Imagine ... you are casting a net into the ocean. The boat is going back and forth, back and forth, back and forth, back and forth. Anyway, you've chosen a net with a mesh size that will capture large fish and allow the smaller ones to swim through. What happens to the medium size fish? They can make it through the net, but their rate of progress is slower. Which fish will make it through the net most easily? More fish pictures: National Marine Fisheries Service surf eel spotted eagle ray Obviously, the spotted eagle ray is going to have a bit of trouble! On the other hand, the eel may glide through the net more easily as long as its orientation is favorable. We can imagine therefore, how the size and shape of the fish might affect our catch.

DNA is just like the fish in our example. Larger molecules will be tend to be more delayed in electrophoresis than smaller molecules, because they keep bumping into obstructions. Molecules that are branched (for example, DNA molecules that have a single-stranded "bulge" in the midst of double-stranded DNA) will migrate more slowly than molecules that are perfect homoduplexes.

Molecules that are more compact because of supercoiling will also encounter fewer obstructions, and will migrate more rapidly through a gel matrix.

Progressive digestion of a supercoiled DNA molecule.

When a supercoiled plasmid is digested with a restriction enzyme, the molecule relaxes after the first nick in the phosphodiester backbone. This relaxed circular molecule migrates more slowly that the supercoiled molecules during gel electrophoresis, because it cannot fit through the gel matrix as easily. Once the enzyme has had a bit more time to do its work, linear molecules will be formed (after the phosphodiester backbones of both strands have been severed). The linear molecules migrate more rapidly than nicked circles, but less rapidly than supercoiled circles.

In the schematic diagram above, the DNA molecules migrated from top to bottom in the electric field. In practice, molecules can be visualized on a gel after staining with ethidium bromide or SYBR green I, and viewing the fluorescence pattern under an ultraviolet light.