Agarose gel is a translucent, jelly-like substance widely used in biological laboratories. Originating from certain red algae, this versatile material forms a stable, porous matrix. Its unique properties enable the separation and analysis of large biomolecules across many scientific disciplines.
What Agarose Is and How it Forms a Gel
Agarose is a polysaccharide, a complex carbohydrate extracted primarily from red algae like Gelidium and Gracilaria. It is a linear polymer composed of repeating disaccharide units, forming helical fibers. Agarose is purified from agar by removing agaropectin, its other component.
When powdered agarose dissolves in hot water (typically above 90°C), its long molecular chains unwind into a viscous solution. As it cools (usually below 45°C), hydrogen bonds form between chains, causing them to aggregate into a three-dimensional network. This process solidifies the liquid into a gel with a mesh-like structure of interconnected channels and pores.
The pore size of this matrix is inversely related to the agarose concentration; higher concentrations result in smaller pores. For example, a 1% agarose gel can have an average pore size of about 150 nanometers. Notably, the gelation temperature is different from its melting temperature.
The Science of Molecular Separation
Agarose gel functions as a molecular sieve, separating biomolecules by size and charge through gel electrophoresis. An electric field is applied across the gel, which is immersed in a buffer solution. This buffer conducts electricity and maintains a stable pH, allowing molecules to retain their charge.
Molecules like DNA and RNA have a net negative charge due to their phosphate backbones, causing them to migrate towards the positively charged electrode. The gel’s porous structure impedes molecular movement, acting like a filter; this is known as the sieving effect. Smaller molecules navigate these pores more easily, traveling faster and further. Larger molecules encounter more resistance, slowing their migration. This difference in speed separates molecules by size, with smaller fragments moving closer to the positive electrode.
The agarose concentration can be adjusted to optimize separation for specific molecule sizes. For instance, a 0.7% gel is suitable for large fragments up to 12 kilobases, while a 2% gel is better for fragments between 50 and 2,000 base pairs.
Key Uses in Biology
Agarose gel electrophoresis is a fundamental tool in molecular biology. One primary application is sizing DNA and RNA fragments, determining their length in base pairs. Researchers use a “DNA ladder” (known fragment sizes) to estimate the size of unknown samples, verifying experimental results like PCR amplification or assessing DNA quality.
Another use is purifying specific DNA or RNA fragments. After separation, desired bands are excised, and nucleic acids extracted for downstream applications such as cloning, DNA sequencing, or restriction enzyme analysis.
Agarose gels also aid diagnostics and research by identifying genes, analyzing mutations, studying gene expression, and in forensic DNA identification. Since DNA and RNA are not visible, fluorescent dyes like ethidium bromide or SYBR Green are incorporated or used to stain the gel. These dyes bind to nucleic acids and fluoresce under ultraviolet (UV) light, making separated molecules visible as distinct bands.
Advantages and Practical Considerations
Agarose gel offers several advantages. It is simple and quick to prepare, dissolving in hot water and cooling without polymerization catalysts. Agarose is non-toxic, making it safe to handle.
Adjusting the agarose concentration allows precise control over the gel’s pore size, tailoring it to separate different size ranges of molecules effectively. This provides a wide range of separation for nucleic acids, typically from approximately 50 base pairs up to 25 kilobases. Once set, the gel is robust and stable, facilitating easy handling and manipulation.