What is the agarose gel?

Introduction

Agarose is a polysaccharide derivative of Agar. While Agar is extracted directly from the cell walls of different species of red algae (Gelidium, Gracilaria...), Agarose is obtained later from the Agar through a complex and costly purification process. Agarose gels are made by heating Agarose in an appropriate buffer. The gel contains microscopic pores that act as a molecular sieve. Specific molecules can also interact with Agarose to varying degrees, affecting their mobility. The higher the concentration of Agarose, the better the resolution. One per cent agarose is commonly used. The agarose gel is kept submerged under a buffer in a horizontal apparatus. For this reason, it is sometimes called submarine electrophoresis, although running agarose gels in a vertical apparatus is possible.

Uses

Agarose is a linear polysaccharide with repeating agarobiose units extracted from red seaweed. Commonly, it is used in agarose gel electrophoresis, but in recent decades, it has drawn the attention of formulation scientists due to its solubility, biocompatible and biodegradable nature, and almost no toxic behaviour. Its gelling capacity at different temperatures makes it more valuable for formulation scientists. In the late 1990s, agarose nanoparticles were prepared to deliver proteins and peptides. Further, Satar et al. have prepared and evaluated the agarose nanoparticles for their antimicrobial effects. Agarose, when compressed with a drug, forms a combination of diffusible and erodible matrix, leading to the sustained release of the drug[1]. An attempt was made to control the release of salbutamol by Agarose in combination with gelatin Saxena et al. However, at the same time, Agarose has limitations when a poorly water-soluble drug is entangled in its matrix. The poor water-soluble drug does not have uniformity due to the high hydrophilic nature of Agarose.

Agarose gel

Nucleic acids are commonly separated by agarose gels, and they are helpful for DNA mapping and apoptosis ladder assays. As DNA and RNA have a constant charge-to-mass ratio (above 400 bp), separation is based on size and shape, not charge. Agarose gels are normally neutral, but alkaline gels are used in certain circumstances, e.g., for the separate nucleic acids as single strands. RNA preparations can be separated using a denaturing agarose gel. Low melting point (LMP) agarose is used for preparative gels. DNA fragments can be cut from the gel, and the LMP agarose can easily be removed without causing strand separation due to its LMP. Very small fragments of DNA (less than 100 bp) are better resolved using the finer mesh of polyacrylamide gels.

Agarose's high gel strength allows handling low percentage gels to separate large DNA fragments. Molecular sieving is determined by the size of pores generated by the bundles of Agarose in the gel matrix. Generally, the higher the concentration of Agarose, the smaller the pore size. Traditional agarose gels are most effective at separating DNA fragments between 100 bp and 25 kb. To separate DNA fragments larger than 25 kb, one will need to use pulse field gel electrophoresis, which involves applying alternating current from two different directions. In this way, sized DNA fragments are separated by the speed at which they reorient themselves to the changes in the current direction[2]. DNA fragments smaller than 100 bp are more effectively separated using polyacrylamide gel electrophoresis. Unlike agarose gels, the polyacrylamide gel matrix is formed through a free radical-driven chemical reaction. These thinner gels are of higher concentration, run vertically, and have better resolution.

References

[1] Pandey, S. et al. “Use of Polymers in Controlled Release of Active Agents.” Basic Fundamentals of Drug Delivery 35 1 (2019): 113-172.

[2] Pei Yun Lee. “Agarose gel electrophoresis for the separation of DNA fragments.” Jove-Journal of Visualized Experiments (2012).