15.3 : Eletroforese em Gel de Agarose de DNA

Agarose gel electrophoresis is a technique commonly used to separate DNA molecules by their size.

Agarose — a polysaccharide derived from red algae —  is added to a buffer such as Tris-acetate EDTA (TAE) or Tris-borate EDTA (TBE) and dissolved by heating.

Commonly, 1 gram of agarose is dissolved in 100 milliliters of buffer to form a 1% agarose solution, though different amounts of agarose can be added depending on the application.

To stain the gel, a fluorescent dye, such as ethidium bromide, is often added to the agarose solution. The dye intercalates between the DNA bases and fluoresces under UV light.

The solution can then be poured into horizontal molds. A comb is inserted into the gel cast to create wells into which DNA samples can be loaded.

The agarose solution is left undisturbed to solidify into a gel.

The gel consists of hydrogen-bonded agarose molecules forming a porous matrix through which the DNA molecules move.

DNA samples are mixed with a loading buffer and loaded on the gel. The loading buffer contains glycerol, which increases the density of the DNA samples and helps them settle at the bottom of the wells.

The buffer also contains dyes, such as xylene cyanol and bromophenol blue, which help monitor the progress of the migrating bands of DNA.

When an electric potential is applied along the length of the gel, the negatively charged DNA migrates towards the positive electrode, traveling through the layers of agarose pores.

Larger DNA molecules move more slowly through the pores than smaller molecules, allowing the molecules to be separated according to their size.

The size of the pores depends on the concentration of agarose in the solution. The higher the agarose concentration, the smaller the pore size, which helps separate shorter DNA fragments.

Under UV light, the stained DNA molecules appear as fluorescent bands. The position of the bands can be compared with DNA fragments of known sizes, called a DNA ladder.

The bands of a ladder serve as a reference to determine the size of the DNA molecules of interest.