In the process of gel electrophoresis, agarose gel is used to track DNA and RNA molecules.
Agarose gel is a substance used in biochemistry and biotechnology for gel electrophoresis and size exclusion chromatography, which are methods of classifying large molecules by their size and electrical charge. These processes use agarose to separate and analyze proteins and DNA. It is very suitable for these applications because of its molecular structure, which allows molecules of different sizes to move at different speeds. The material is obtained from different types of seaweed and is normally found in laboratories in the form of powder. To make a suitable medium for a given test, the powder is added to water at the appropriate concentration, boiled and then allowed to cool in a gel.
Red seaweed is a source of agarose gel.
Agarose is extracted in the form of agar from several species of red seaweed, or seaweed, found in California and East Asia. Agar, a term derived from the Malay word agar-agar, meaning jelly, is normally obtained from the types of Gelidium seaweed. It is made up of two different substances, known as agarose and agaropectin, and it provides support for the cell walls of seaweed. When removed, agar can be used as a food thickener, much like gelatin, or as a laxative. If purified, it can be used as a medium for the cultivation of bacteria, fungi or other microorganisms.
Agarose gel is used to help separate and analyze proteins and DNA.
It is quite easy to separate agarose from agaropectin in agar because the agarose molecules bind tightly to each other while agaropectin freezes weakly. There are several methods to achieve isolation of agarose. In one method, carrageenan, another molecule found in red seaweed, and a salt are added to the agar. This causes the agaropectin to precipitate or form a solid that can be removed from the agar solution. Another method adds the enzyme pectinase, a chemical that breaks down agaropectin, allowing it to dissolve in water.
Agarose gel is most commonly associated with electrophoresis. In this procedure, scientists apply an electric field to a plate of material containing dissolved DNA, RNA or protein fragments. This causes these large molecules to move because of their electrical charges: the positively charged types will move to the negative side and vice versa. DNA and RNA fragments have a negative charge and will therefore move towards the positive end, while protein fragments can be either negative or positive.
The speed at which molecules move depends on their size and the amount of charge they carry. The agarose gel is structured to form a kind of mesh, with holes through which other molecules can pass. It is easier for smaller ones to get through the holes and therefore travel faster. Among larger molecules, shape also plays a role, as the more compact ones pass through more easily. The technique is used both to analyze samples and to isolate particular DNA sequences for use in biotechnology applications.
Before electrophoresis, a DNA sample would be treated with special enzymes that cut the long, strand-like molecules at specific locations, forming smaller fragments. Agarose gel is prepared by dissolving the powder in a buffer solution, which resists changes in pH – acidity/alkalinity – that could result from electrochemical effects. Different amounts of powder are used for different ranges of molecules, but generally the concentration is between 0.7 and 1.2%. A fluorescent dye called ethidium bromide is usually added at this point, as it stains the DNA and makes it easily visible under ultraviolet light. This mixture is then microwaved and allowed to harden.
DNA samples are placed in small wells in the gel and a direct electrical current is applied through the gel. Different sized molecules travel through the gel at different speeds, so after a certain time they will appear in different positions, with the smaller fragments closer to the positive end. This allows scientists to determine the sizes of the fragments and isolate different DNA sequences.
Agarose gel is sometimes used in a related technique that does not involve electricity, known as size exclusion chromatography. In this method, a glass column is filled with beads made of gel and a solution containing molecules of different sizes is poured. In contrast to electrophoresis, larger molecules move faster down the column to emerge at the bottom, while the progress of smaller ones is slowed down in the beads. This is because small molecules tend to be absorbed into the pores of the gel, while larger ones are too large to enter these pores and tend to flow between the granules instead. The gel type and concentration can be adjusted to suit the sizes of the molecule to be separated.