Gel electrophoresis is a foundational technique in molecular biology used to separate charged biological molecules, such as DNA and RNA, based on size and electrical charge. Agarose, a purified component of agar, is the most frequently used medium for this process. Agarose allows researchers to analyze DNA and RNA fragments across a broad range of sizes quickly and reliably.
The Physical Structure of Agarose Gel
Agarose is a linear polysaccharide derived from certain species of red seaweed. To create the separation medium, the dry agarose powder is mixed with a buffer solution and heated until dissolved. As the solution cools below 45 degrees Celsius, the agarose chains associate through hydrogen bonding. This self-assembly creates a robust, three-dimensional mesh or lattice structure that traps the liquid buffer inside.
The resulting gel matrix is highly porous, resembling a molecular sponge with interconnected channels. The size of these pores is directly controlled by the concentration of the agarose powder used. For example, a low concentration (0.7%) produces large pores, while a high concentration (2%) yields smaller pores. This ability to adjust pore size by changing the concentration allows researchers to optimize separation for different molecular sizes.
Molecular Sieving and Separation Mechanism
The separation of nucleic acids relies on the principle of molecular sieving combined with an electric field. DNA and RNA molecules possess a uniform negative electrical charge due to the phosphate groups present in their backbones. When the gel is placed in an electrophoresis chamber and an electrical current is applied, these negatively charged molecules are pulled through the gel matrix toward the positive electrode, known as the anode.
The gel itself functions as a sieve, physically impeding the movement of the migrating molecules. Smaller DNA fragments are able to navigate the pores and channels of the mesh more easily and quickly. Conversely, larger fragments encounter more friction and resistance, causing them to migrate at a slower rate. This difference in migration speed separates the mixed population of nucleic acids by size, with the smallest fragments traveling the farthest distance in a given time.
To determine the size of the separated fragments, a DNA ladder or marker is loaded into one of the gel’s lanes. This marker is a mixture of DNA fragments of known lengths, measured in base pairs or kilobase pairs. By comparing the distance traveled by a sample fragment to the corresponding bands in the ladder, researchers estimate the size of the unknown molecules.
Suitability for Analyzing Large Nucleic Acid Fragments
Agarose is the preferred matrix for nucleic acid separation because its pore size range is well-suited for analyzing large DNA and RNA molecules. Standard agarose gels can effectively resolve DNA fragments ranging from approximately 100 base pairs up to 25 kilobases. This size range covers the products of many common molecular biology procedures, such as restriction enzyme digests and PCR amplification.
Other common matrices, such as polyacrylamide gels, have finer and more uniform pore structures. Polyacrylamide is reserved for applications requiring high resolution of very small molecules, such as proteins or short DNA fragments under 1,000 base pairs. For the large fragments typical of routine DNA work, the tight mesh of polyacrylamide would impede migration, making separation impractical.
The practicality of agarose contributes to its widespread use. Agarose powder is non-toxic, and the gel is prepared simply by dissolving the powder and cooling the solution in a mold. In contrast, preparing polyacrylamide gels involves handling unpolymerized acrylamide, which is a neurotoxin, and the polymerization process is more complex.
Practical Applications and Visualization
Once the electrical current is stopped, the nucleic acid fragments remain within the clear gel matrix. Because DNA and RNA are colorless, they must be stained. The most common method involves adding a fluorescent dye, such as ethidium bromide or a safer commercial alternative like SYBR Green, either to the gel solution before casting or after the run.
These dyes bind directly to the nucleic acid molecules. The gel is then placed on an apparatus that exposes it to ultraviolet (UV) light. When illuminated by UV light, the DNA-dye complexes fluoresce brightly, appearing as distinct bands against a dark background. The pattern of these bands provides the data for molecular analysis.
Visualization allows researchers to confirm the success of experiments, such as verifying gene amplification by PCR. Furthermore, separated DNA fragments can be physically cut out of the gel using a scalpel. This process, called gel extraction, is used to purify a specific fragment of interest for use in downstream molecular techniques, such as gene cloning or sequencing.