Molecular Techniques - Isolation of Genetic Material and Gel Electrophoresis

Gel electrophoresis is the standard way to separate, visualise and size DNA (or other charged biomolecules) after any of the cutting, cloning or PCR steps described earlier in the chapter. The underlying principle is simple: apply a direct-current electric field across a gel, and charged molecules migrate through it toward the oppositely charged electrode - positively charged cations move toward the cathode, negatively charged anions move toward the anode. Because DNA's phosphate backbone carries a strong negative charge, DNA molecules placed in wells at one end of an agarose gel and exposed to an electric field will migrate steadily toward the positive electrode, with smaller fragments moving faster through the gel's porous matrix than larger ones - so after enough time, fragments of different sizes end up spread out and sorted by size along the gel. Agarose gel electrophoresis specifically handles DNA fragments from a few hundred base pairs up to roughly 20,000 base pairs; for separating smaller fragments than that, polyacrylamide gel is used instead, since it has a finer pore structure. To actually determine an unknown fragment's size, a lane of the same gel is loaded with a 'marker' - DNA fragments of already-known sizes - and the unknown fragment's position is compared against it by interpolation. Finally, DNA itself is invi …