Gel electrophoresis is a fundamental technique in molecular biology used to separate and analyze macromolecules like DNA, RNA, and proteins. The process works by applying an electrical field to move charged molecules through a porous gel matrix. For DNA analysis, this method sorts a mixed population of DNA fragments based primarily on their length and electrical charge. This separation is routinely used in research, forensic science, and clinical diagnostics to determine the size and quantity of specific genetic material.
The Essential Components
The foundation of the setup is the gel matrix, typically made from the polysaccharide agarose. Agarose, derived from seaweed, forms a microscopic, sieve-like network when dissolved in a liquid buffer, heated, and allowed to solidify. The concentration of the agarose directly controls the pore size within this molecular sieve. A higher percentage of agarose creates smaller pores, which is ideal for separating very small DNA fragments with greater resolution.
The gel is submerged in a running buffer, often a formulation like Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE). This buffer conducts the electrical current and maintains a stable pH environment. The buffer’s composition includes components that help stabilize the DNA by chelating metal ions.
The physical apparatus consists of an electrophoresis chamber and a power supply. The chamber holds the submerged gel and the running buffer, with electrodes positioned at opposite ends. The power supply generates the electrical field, creating a negative terminal (cathode) and a positive terminal (anode). This electrical potential drives the movement of the charged DNA molecules through the gel matrix.
Preparing the DNA Sample and Gel
The procedure begins with the preparation of the agarose gel. Agarose powder is mixed with the running buffer and heated until it fully dissolves. This molten solution is poured into a casting tray containing a removable plastic insert called a comb. The comb creates small indentations, or wells, in the gel as it cools and solidifies.
Once the gel has set, the comb is carefully removed, leaving the empty wells that will hold the DNA samples. The purified DNA samples are then mixed with a specialized liquid known as a loading dye. This dye serves a dual function: adding density and providing a visual tracking marker.
The first function is to add density, usually through a high concentration of glycerol or sucrose. This increased density allows the sample to sink neatly into the well at the bottom of the buffer layer rather than floating out. The second function is to provide a visual tracking marker, as the dye contains colored molecules that migrate through the gel alongside the invisible DNA.
The prepared samples are carefully loaded into the wells using a fine-tipped micropipette. One well is reserved for a DNA ladder, a separate sample of DNA fragments of known lengths. With the samples in place, the gel is ready to be connected to the power source.
Running the Separation
With the samples loaded, the gel tray is placed into the electrophoresis chamber, ensuring the wells are positioned near the negative electrode (cathode). The chamber is filled with running buffer to submerge the gel. The power supply is connected, applying the electrical current across the gel from the negative cathode to the positive anode.
The electrical current initiates the movement of the DNA molecules. DNA has a net negative charge due to the phosphate groups in its backbone structure. This negative charge causes the DNA to be repelled by the cathode and attracted to the positive anode, moving the fragments toward the opposite end of the gel.
The separation relies on molecular sieving within the agarose matrix. As the DNA molecules are pulled through the electrical field, they navigate the dense network of agarose fibers. Smaller DNA fragments encounter less resistance, allowing them to travel faster and cover a greater distance. Conversely, larger DNA fragments are slowed down, resulting in a shorter migration distance. The run is monitored using the colored tracking dye and stopped when the fastest dye front reaches the opposite edge of the gel.
Visualization and Interpreting the Results
Once the electrical current is stopped, the DNA fragments are separated by size but remain invisible. To visualize the results, the gel must be stained with a DNA-binding fluorescent dye. Dyes such as ethidium bromide or its safer alternatives, like SYBR Green, are used. These molecules insert themselves, or intercalate, between the bases of the DNA double helix.
After staining, the gel is placed on a UV transilluminator. When exposed to ultraviolet light, the intercalated dye molecules emit visible light. The DNA appears as bright, distinct bands, with each band representing a collection of millions of DNA fragments that are all the same length. The intensity of a band is directly related to the amount of DNA present in that specific group of fragments.
The most important tool for interpretation is the DNA ladder, run in a separate lane alongside the unknown samples. The ladder consists of fragments of known base pair lengths, creating a series of distinct bands that act as a ruler. By comparing the migration distance of an unknown band to the corresponding bands in the ladder, researchers can determine the base pair length of the DNA fragment.