By Aarav Ghate | 1 October 2024
Electrophoresis is a technique used to separate DNA, RNA or protein molecules based on their size, using an electric current. Agarose Gel Electrophoresis uses agarose, a component of agar, as a matrix to filter nucleic acids based on their size. The matrix is made of supercoiled agarose molecules arranged in a three dimensional structure, which has pores and channels for the proteins to pass.
Initially, agarose powder is dissolved in a suitable medium and heated to just below boiling point. The liquid is placed in an appropriate cast and allowed to solidify. The matrix is held together by hydrogen bonds, and liquifies once again if heated.
DNA and RNA carry a negative charge due to the phosphate groups that form their backbone. The samples are placed into wells (holes in the agarose gel) and begin to move towards the positive terminal when a voltage is applied across the gel. Dyes may be placed along with the sample to monitor its progress (called tracking dyes, such as bromophenol blue) and loading buffers may be used to increase the density (so that the sample sinks to the bottom of the well).
Smaller molecules travel faster in the gel because of sieving through the pores in agarose, so after applying voltage for some time, a gradient is seen from the largest molecules to the smallest molecules. Eventually, the molecules separate into groups called bands depending on their size. These DNA in these bands may be separated by cutting, liquefying the agarose gel and then purifying the sample.
Since nucleic acids cannot be viewed with the naked eye, dyes like methylene blue may be added to view the bands. However the most commonly used technique is the incorporation of ethidium bromide in the sample. Ethidium bromide forms a complex with the nucleic acids and fluoresces under UV light. The sample is stained and placed under a UV light with a higher intensity of fluorescence indicating a larger amount of DNA in that band.
Agarose Gel Electrophoresis has many applications ranging from separation of DNA fragments by size, to analyzing the products of PCR.