In the process of gel electrophoresis, agarose gel is used to trace 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 sorting large molecules by their size and electrical charge. These processes use agarose to separate and analyze proteins and DNA. It is well suited for these applications due to its molecular structure, which allows molecules of different sizes to move at different speeds. The material is obtained from different types of algae and is normally found in laboratories in powder form. To make a suitable medium for a given test, the powder is added to water in the proper concentration, boiled, and then allowed to cool to a gel.
Manufacture
Red algae are a source of agarose gel.
Agarose is extracted in the form of agar from various species of red algae, or seaweeds, found in California and East Asia. Agar, a term derived from the Malay word agar-agar, meaning jelly, is normally obtained from the Gelidium types of seaweed. It is made up of two different substances, known as agarose and agaropectin, and provides support for the cell walls of algae. When removed, the agar can be used as a food thickener, 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 agarose molecules bind strongly to each other while agaropectin freezes weakly. There are several methods to achieve agarose isolation. In one method, carrageenan, another molecule found in red algae, 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.
gel electrophoresis
Agarose gel is most commonly associated with electrophoresis. In this procedure, scientists apply an electric field to a plate of material that contains dissolved fragments of DNA, RNA, or protein. This causes these large molecules to move due to 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 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 the little ones to get through the holes and therefore travel faster. Among larger molecules, shape also plays a role, since more compact ones pass through more easily. The technique is used both to analyze samples and to isolate particular DNA sequences for use in biotechnological applications.
Before electrophoresis, a DNA sample was treated with special enzymes that cut the long chain-like molecules at specific places, forming smaller fragments. The agarose gel is prepared by dissolving the powder in a buffer solution, which resists changes in pH (acidity/alkalinity) that might 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 heated in the microwave and allowed to harden.
DNA samples are placed in small wells in the gel and a direct electrical current is applied through the gel. Molecules of different sizes travel through the gel at different speeds, so after some time they will appear in different positions, with the smaller fragments closer to the positive end. This allows scientists to determine the size of the fragments and to isolate different DNA sequences.
other uses
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 gel beads and a solution containing molecules of different sizes is poured into it. In contrast to electrophoresis, larger molecules move faster down the column to emerge at the bottom, while the progress of smaller ones slows 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. Gel type and concentration can be adjusted to suit the sizes of the molecule to be separated.