Agarose is a natural substance widely used in molecular biology and biochemistry laboratories. It primarily functions to form a porous gel matrix for separating various molecules. Its distinctive attributes make it an indispensable material for scientific research.
Source and Chemical Structure
Agarose is a polysaccharide extracted from the cell walls of certain species of red seaweed, particularly from genera like Gelidium and Gracilaria. It is purified from agar, a mixture of agarose and agaropectin, by removing the latter component. This purification yields a highly refined, linear polymer.
The chemical structure of agarose consists of repeating disaccharide units called agarobiose. Each agarobiose unit is composed of two sugar molecules: D-galactose and 3,6-anhydro-L-galactopyranose. These sugar units are linked by specific glycosidic bonds, forming long, unbranched chains. The linear nature of these chains, along with their minimal inherent charge, is fundamental to agarose’s ability to form a stable gel network.
Physical Properties
Agarose powder dissolves when heated in a liquid, such as water or a buffer solution, forming a clear solution. Upon cooling, this solution transitions into a solid gel, creating a robust, porous matrix.
A notable property of agarose is its thermal hysteresis, where the melting temperature of the gel is higher than its gelling temperature. For instance, an agarose solution might gel upon cooling to approximately 34-38°C, yet the resulting gel may not melt until heated to 85-90°C. This difference allows researchers to handle the gel in its liquid state without premature solidification, while ensuring the gel remains stable and solid at temperatures well above its setting point, which is beneficial for sensitive biological samples.
The concentration of agarose dictates the resulting pore size within the gel matrix. A higher concentration of agarose leads to a denser network of polymer chains, creating smaller pores. Conversely, a lower concentration yields a looser matrix with larger pores, accommodating bigger molecules. This adjustable pore size is a powerful tool, allowing for tailored separation of molecules based on their dimensions. For example, a 0.7% agarose gel might have average pore diameters ranging from 50 to 200 nanometers, suitable for larger molecules, while a 2% gel could have pores as small as 20 nanometers for smaller fragments.
Electroendosmosis (EEO) refers to the movement of liquid through the gel matrix. This occurs due to residual negatively charged groups, such as sulfate and carboxyl groups, that are fixed within the agarose polymer chains. While these fixed negative charges cannot move, their associated positive counter-ions migrate towards the cathode when an electric field is applied, pulling water along. This movement of liquid can impede the migration of negatively charged molecules, causing distortion or blurring of separation results. For most applications, especially those involving nucleic acids like DNA, low-EEO agarose is preferred to minimize this effect, ensuring molecules separate based on their size rather than buffer flow.
Application in Molecular Sieving
The porous gel matrix formed by agarose acts as a molecular sieve, a property used in laboratory techniques like gel electrophoresis. In this procedure, the agarose gel serves as a medium through which charged molecules, such as DNA or RNA, can travel. DNA, being negatively charged due to its phosphate backbone, migrates towards the positive electrode when an electric current is applied across the gel.
The separation mechanism relies on the gel’s network of pores. Smaller molecules can navigate through these pores more easily and travel further through the gel. Larger molecules, however, encounter more resistance from the dense gel matrix, causing them to move more slowly and travel shorter distances. This differential migration rate leads to the separation of molecules by size, allowing scientists to distinguish between different fragments or molecules within a sample.
Scientists control the pore size of the agarose gel by adjusting the agarose concentration during preparation. For instance, a gel with a lower agarose percentage, such as 0.7%, creates larger pores suitable for separating large DNA fragments, potentially several kilobases in length. Conversely, a higher percentage gel, like 2% or 3%, produces smaller pores, ideal for resolving much smaller DNA fragments, often in the range of hundreds of base pairs. This ability to adjust the gel’s sieving properties allows researchers to optimize the separation for a specific range of molecule sizes, making agarose a versatile tool in molecular biology.