Gel electrophoresis is a fundamental laboratory technique used to separate charged biological molecules, such as proteins and nucleic acids, based on their size and electrical charge using an electric field. Samples are driven through a porous medium, or matrix, which provides the physical resistance necessary for separation. For the routine separation of large molecules like deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in molecular biology, agarose is the most widely adopted material for creating this separation matrix.
The Agarose Matrix: Creating a Molecular Sieve
Agarose is a natural polysaccharide purified from agar, which is extracted from certain species of red algae or seaweed. To form the matrix, agarose powder is dissolved in a buffer solution and heated until it melts into a clear liquid. As this solution cools, the linear agarose polymer chains associate non-covalently, forming a complex, three-dimensional mesh structure.
This process of heating and cooling transforms the solution into a semi-solid gel, much like gelatin, with a network of interconnected channels and pores. The resulting gel acts as a molecular sieve, which is the physical basis for separating molecules by size during electrophoresis. When an electric current is applied, charged molecules are pulled through these pores.
Smaller molecules can navigate the mesh relatively unimpeded, moving quickly toward the electrode of opposite charge. Conversely, larger molecules encounter more resistance from the fibrous network, causing them to migrate more slowly. This difference in migration speed allows a mixed population of molecules to be effectively separated based on their size.
Researchers can precisely control the average pore size of the gel by adjusting the initial concentration of agarose powder used to prepare the solution. A higher agarose concentration creates a denser mesh with smaller pores, ideal for resolving very small molecules. Conversely, a lower concentration yields larger pores, allowing for the effective separation of much larger molecules.
Ideal Properties for Nucleic Acid Separation
The physical characteristics of the agarose matrix are well-suited for the separation of nucleic acid fragments, the primary targets of this technique. Agarose is the preferred choice for separating DNA and RNA fragments because it effectively separates molecules ranging from approximately 100 base pairs (bp) up to 25 kilobases (kb).
This range of separation is significantly greater than that offered by other common gel matrices, which are often limited to much smaller molecules. The chemical composition of agarose further contributes to its suitability for biomolecules because it is largely inert and non-reactive. Agarose contains very few charged chemical groups, which prevents it from binding to or chemically altering the nucleic acid samples during the separation process.
The stability of the agarose gel is another factor for reliable separation, as the process generates heat from the electric current. Agarose remains stable under these mild thermal conditions, ensuring the matrix structure does not degrade or melt during the electrophoresis run. This inert nature and thermal stability maintain the integrity of the nucleic acids, ensuring that the separation is based purely on size and charge, leading to accurate analytical results.
Practical Benefits for Laboratory Use
Beyond its optimal sieving mechanics, agarose is favored in the laboratory due to its straightforward preparation. Casting a gel involves melting the powder in a buffer and pouring the liquid into a mold, where it sets quickly as it cools to room temperature. This physical gelling process, which relies on temperature change rather than a chemical reaction, makes preparation fast and reproducible.
The resulting gel is robust, allowing for easy manipulation and transfer without tearing or breaking. This structure simplifies loading samples and moving the gel from the casting tray to the electrophoresis chamber. Furthermore, agarose is considered non-toxic, offering a safety advantage over alternative matrices that may require handling neurotoxic chemicals during preparation.
Once the separation is complete, the transparent nature of the agarose gel makes the visualization of the separated molecules highly effective. The gel is easily stained with fluorescent dyes that bind to the DNA or RNA. When illuminated with ultraviolet (UV) light, these dyes make the separated fragments visible as distinct bands, allowing researchers to document and analyze the results clearly.