Seed Dispersal: Definition, Types, Importance, Adaptations & Examples

Separation of molecules based on their charges and size using agarose gel as a supporting medium, is called agarose gel electrophoresis.

Principle

If a direct current is passed through an agarose gel block containing charged molecules, the positively charged cations (+) will migrate towards the cathode (-) and the negatively charged anions (-) will move towards the anode (+). Proteins and nucleic acids are negatively charged molecules, with the result that they tend to move towards the anode. Mobility of the molecules depends on the net charge, size and shape of the molecule, pH and composition of the suspending medium and electric current. Agarose is a linear polymer of D-galactose and L-galactose. When it is mixed with water, it undergoes cross-linking between the agarose units and forms a porous matrix called gel. If a solution is dispersed within the gel, the pores have sieving capacity. As a result, smaller molecules move towards the anode faster than the larger ones. The pore size is determined by the concentration of agarose. DNA and mRNA are negatively charged molecules. So, the electric field helps them to move towards the anodic end.

Procedure:

1. Agarose (0.8g/100ml of 1× TAE buffer) is dissolved and heated by keeping it in a microwave oven. It is then transferred into the trough of electrophoresis apparatus upto the height of 75mm thickness. Prior to solidification, a comb is fixed at one end to prepare loading wells. The comb is removed to have loading wells in the gel.
2. Buffer reservoirs are filled with 1×TAE buffer.
3. The DNA sample is digested with a restriction enzyme. The DNA sample is heated to 65°C for 2 minutes to remove aggregates into individual fragments of DNA. To observe the progress of electrophoresis, a dye bromophenol blue of known migration rate, is mixed with the DNA sample.
4. The sample is filled in the wells and suitable molecular markers are loaded in one well. The electrophoresis unit is connected with a power supply and is switched on to start the movement of DNA fragments from the cathodic end to anodic end.
5. After about 1 hour, the current is switched off and the gel is transferred to a container containing ethidium bromide. The stained gel is washed with distilled water to remove unbound ethidium bromide from the gel.
6. This gel is exposed to UV light by keeping it under an UV transilluminator to observe light bands that indicate DNA bands. The size of the DNA fragments can be estimated by comparing their locations in relation to the positions of the molecular markers.

Applications 

1. It is used for the separation of DNA fragments in a sample.
2. It is used to detect the purity of DNA fragments. 
3. It is used to determine the size of DNA fragments.
4. It is also used in the separation of proteins in a solution. 
5. Estimation of the size of DNA molecules.
6. Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting.
7. Separation of restricted genomic DNA prior to Southern analysis, or of RNA prior to Northern analysis.
8. Agarose gels allow purification of DNA fragments.