Agarose gel is a foundational laboratory tool used in molecular biology to analyze large biological molecules. This technique separates complex mixtures into individual components. The gel serves as the medium, providing a framework that sorts molecules based on their physical characteristics. It is an indispensable method for assessing sample contents before moving on to more complex experiments.
Composition and Structure
Agarose is a natural, linear polysaccharide, purified from certain types of red seaweed. To create the gel, the purified agarose powder is dissolved in a buffered solution and heated until it becomes a clear liquid. As the solution cools, the agarose chains associate through hydrogen bonds, forming a three-dimensional mesh structure.
This network creates a porous matrix that functions like a molecular sieve. The size of the pores is determined by the initial concentration of the agarose powder used. A lower concentration yields larger pores for separating very large molecules, while a higher concentration creates a denser mesh suitable for resolving smaller molecules.
The Mechanism of Separation
The process that drives the separation of molecules through this gel matrix is known as gel electrophoresis. Once the gel has solidified, samples are loaded into wells cast at one end. The gel is then submerged in a conductive buffer solution, and an electrical current is applied across the system. The electrical field causes charged molecules to migrate toward the oppositely charged end of the gel. Nucleic acids (DNA and RNA) have a negative charge, causing them to move toward the positive electrode.
This movement is governed by the sieving effect of the agarose structure. As the charged molecules travel, they must navigate the dense network of pores, which impedes their progress. Smaller nucleic acid fragments encounter less resistance and move swiftly through the mesh, traveling farther down the gel. Larger fragments are forced to reorient and twist through the tiny openings, significantly slowing their migration rate. Consequently, molecules are separated by size, with the smallest fragments positioned farthest from the wells.
Primary Uses in Biology
The successful separation of molecules by size allows scientists to perform a variety of crucial analyses in the laboratory. One primary use is determining the size of DNA or RNA fragments by comparing their travel distance to a molecular ladder of known lengths. This comparison is routine after procedures like using restriction enzymes or amplifying specific sections through polymerase chain reaction (PCR).
Agarose gel electrophoresis is also used for several other applications:
- Checking the purity and integrity of a sample.
- Serving as a foundational part of DNA fingerprinting in forensic science.
- Identifying specific genetic markers or verifying the presence of an infectious agent’s DNA for diagnostic purposes.
- Isolating and purifying a specific piece of genetic material for subsequent procedures like gene cloning.
Visualizing the Results
The nucleic acid fragments are invisible to the naked eye despite the precise separation. To make the separated bands apparent, scientists use specialized fluorescent stains or dyes. These dyes bind specifically to the DNA or RNA molecules, either by being mixed into the gel or applied afterward.
Once the dye is bound, the gel is exposed to ultraviolet (UV) light. The UV light excites the dye molecules, causing them to fluoresce and reveal the location of the separated fragments as bright, distinct bands. The intensity of a band correlates with the amount of nucleic acid present. Because UV light and some chemical dyes can be hazardous, researchers must wear protective equipment, including gloves and UV-blocking face shields, when examining the results.