Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of the two main components of agar. The proteins may be separated by charge and/or size (isoelectric focusing agarose electrophoresis is essentially size independent), and the DNA and RNA fragments by length. Biomolecules are separated by applying an electric field to move the charged molecules through an agarose matrix, and the biomolecules are separated by size in the agarose gel matrix.
Agarose gel is easy to cast, has relatively fewer charged groups, and is particularly suitable for separating DNA of size range most often encountered in laboratories, which accounts for the popularity of its use. The separated DNA may be viewed with stain, most commonly under UV light, and the DNA fragments can be extracted from the gel with relative ease. Most agarose gels used are between 0.7–2% dissolved in a suitable electrophoresis buffer.
Properties of agarose gel
Agarose gel is a three-dimensional matrix formed of helical agarose molecules in supercoiled bundles that are aggregated into three-dimensional structures with channels and pores through which biomolecules can pass. The 3-D structure is held together with hydrogen bonds and can therefore be disrupted by heating back to a liquid state. The melting temperature is different from the gelling temperature, depending on the sources, agarose gel has a gelling temperature of 35–42 °C and a melting temperature of 85–95 °C. Low-melting and low-gelling agaroses made through chemical modifications are also available.
Agarose gel has large pore size and good gel strength, making it suitable as an anticonvection medium for the electrophoresis of DNA and large protein molecules. The pore size of a 1% gel has been estimated from 100 nm to 200–500 nm, and its gel strength allows gels as dilute as 0.15% to form a slab for gel electrophoresis. Low-concentration gels (0.1–0.2%) however are fragile and therefore hard to handle. Agarose gel has lower resolving power than polyacrylamide gel for DNA but has a greater range of separation, and is therefore used for DNA fragments of usually 50–20,000 bp in size. The limit of resolution for standard agarose gel electrophoresis is around 750 kb, but resolution of over 6 Mb is possible with pulsed field gel electrophoresis (PFGE). It can also be used to separate large proteins, and it is the preferred matrix for the gel electrophoresis of particles with effective radii larger than 5–10 nm. A 0.9% agarose gel has pores large enough for the entry of bacteriophage T4.
The agarose polymer contains charged groups, in particular pyruvate and sulphate. These negatively charged groups create a flow of water in the opposite direction to the movement of DNA in a process called electroendosmosis (EEO), and can therefore retard the movement of DNA and cause blurring of bands. Higher concentration gels would have higher electroendosmotic flow. Low EEO agarose is therefore generally preferred for use in agarose gel electrophoresis of nucleic acids, but high EEO agarose may be used for other purposes. The lower sulphate content of low EEO agarose, particularly low-melting point (LMP) agarose, is also beneficial in cases where the DNA extracted from gel is to be used for further manipulation as the presence of contaminating sulphates may affect some subsequent procedures, such as ligation and PCR. Zero EEO agaroses however are undesirable for some applications as they may be made by adding positively charged groups and such groups can affect subsequent enzyme reactions. Electroendosmosis is a reason agarose is used in preference to agar as the agaropectin component in agar contains a significant amount of negatively charged sulphate and carboxyl groups. The removal of agaropectin in agarose substantially reduces the EEO, as well as reducing the non-specific adsorption of biomolecules to the gel matrix. However, for some applications such as the electrophoresis of serum proteins, a high EEO may be desirable, and agaropectin may be added in the gel used.
Migration of nucleic acids in agarose gel
Factors affecting migration of nucleic acid in gel
A number of factors can affect the migration of nucleic acids: the dimension of the gel pores (gel concentration), size of DNA being electrophoresed, the voltage used, the ionic strength of the buffer, and the concentration of intercalating dye such as ethidium bromide if used during electrophoresis.