Form and Function
Agarose is a purified hydrogel isolated from red algae; it is the main component of agar. Structurally, agarose is a linear polymer consisting of alternating beta-D-galactose and 1,4-linked 3,6-anhydro-alpha-L-galactose units.
Agarose can be used for electrophoretic separation in agarose gel electrophoresis or for column-based gel filtration chromatography. Agarose gel electrophoresis is a method used to separate DNA or RNA molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field. Shorter molecules move faster and migrate farther than longer ones.
Figure 1. Structure of agarose polymer.
The most critical characteristics to consider when determining the suitability and performance of an agarose are:
1. Melting/Gelling Temperature:
Agarose exhibits a high hysteresis (difference between melting and gelling temperatures) making it ideal for separations such as electrophoresis and chromatography. The gelling temperature ranges from 32 - 45°C, and the melting temperature range is normally 80 - 95°C depending on the type of Agarose preparation used. Melting and gelling temperatures can be changed by methylation, as well as alkylation and hydroxyalkylation of the polymer chain.
2. Gel Strength:
Gel strength is particularly important when gels must be handled or blotted after electrophoresis. pH and other additives can effect the gel strength of Agarose; in particular, sulfate salts and the sulfate content in the agarose molecules reduce the strength of the gel.
3. Sulphate Content:
Sulfate content may be used as an indicator of purity, since sulfate is the major ionic group present.
4. Electroendosmosis (EEO):
Electroendosmosis (EEO) is one of the most important characteristics to consider when choosing an agarose to meet your electrophoretic needs. EEO is the movement of non-charged molecules through a medium toward the cathode during electrophoresis. If agarose is the medium, ions such as ester sulfate and pyruvate groups impart a net negative charge to the agarose. Though the gel itself cannot move, counter-ions and water associated with the sulfate and pyruvate groups move toward the cathode. As water migrates with the counter-ions, neutral molecules, which normally would not migrate, are pulled along with the water. This movement of molecules towards the cathode is known to slow the separation of DNA, so the lower the EEO, the faster DNA will migrate. In most electrophoretic procedures, EEO is an unwelcome side-effect. Just as the mobility of a charged molecule is a direct function of the voltage gradient, so is EEO. Additionally, lower EEO helps improve the resolution of DNA and RNA as their migration is determined only by their size, not by their charge.
How to measure EEO:
Uncharged molecules are used to measure EEO, materials such as urea, dextran, sucrose, and deoxyribose are typically employed. Blue dextran is frequently used because it can be directly visualized.
EEO is measured by subjecting a mixture of dextran and albumin to electrophoresis, then visualizing them, and measuring their respective distances from the origin. The amount of EEO (-Mr) is calculated by dividing the migration distance of the neutral dextran (OD) by the sum of the migration distances of the dextran and the albumin (OD + OA).
-Mr = OD/(OD + OA)
Type of Agarose -Mr range (Relative Migration Distance)
Low EEO 0.09-0.13
Medium EEO 0.16-0.19
High EEO ~0.20
Agarose Gel Electrophoresis can be used for relative sizing of DNA, RNA and high molecular weight proteins. Agarose gel has the advantages that the gel is easily poured and it does not denature the samples. The samples can also be recovered. The disadvantages are that gels can melt during electrophoresis and different forms of genetic material may run in unpredictable forms.
The most important factor is the length of the DNA molecule, smaller molecules travel farther. But conformation of the DNA molecule is also a factor. To avoid this problem linear molecules are usually separated, usually DNA fragments from a restriction digest, linear DNA PCR products, or RNAs. Increasing the agarose concentration of a gel reduces the migration speed and enables separation of smaller DNA molecules. The higher the voltage, the faster the DNA moves. The voltage is limited by the fact that the gel heats up and ultimately causes it to melt. High voltages 9>5-8V/cm) also decrease the resolution. Different conformations of a DNA plasmid that has not been cut with a restriction enzyme will move with different speeds (slowest to fastest: nicked or open circular, linearized, or supercoiled plasmid).
Agarose gel electrophoresis can be used for the separation of DNA fragments ranging from 50 base pairs to several megabases. The distance between DNA bands of a given length is determined by the percent agarose in the gel. In general, lower concentrations of agarose are better for larger molecules because they result in greater separation between bands that are close in size. The disadvantage of higher concentrations is the long run times. Optionally, high percentage agarose gels can be run with pulsed field electrophoresis (PFE). Most agarose gels are made with between 0.7-2% agarose dissolved in electrophoresis buffer. Up to 3% can be used for separating very tiny fragments. Low percentage gels are very weak and may break when you try to lift them. High percentage gels are often brittle and do not set evenly. 1% gels are common for many applications.
Concentration Molecular Weight
Unknown DNA samples are typically run on the same gel with a "DNA ladder." A ladder is a sample of DNA where the sizes of the bands are known. Direct comparison can be made between the known fragments and the unknown sample to determine the approximate size of the unknown DNA.