Gel electrophoresis is a technique used to separate macromolecules such as DNA, RNA, and proteins. The size of DNA fragments is used to separate them. Proteins can be sorted based on their size and charge (different proteins have different charges).
Also Read: Electrophoresis and It’s Types
Gel electrophoresis can be used for a range of purposes, for example:
- To get a DNA fingerprint for forensic purposes
- To get a DNA fingerprint for paternity testing
- To get a DNA fingerprint so that you can look for evolutionary relationships among organisms
- To check a PCR reaction.
- To test for genes associated with a particular disease.
Also Read: What is Electrophoresis Test?
Gels can be prepared from either agarose or a polyacrylamide combination. A vertical apparatus is used to run polyacrylamide gels. These are the gels used in manual DNA sequencing. They can handle vast quantities of DNA and are quite fast. They can distinguish DNA fragments that differ in size by only one base pair.
Agarose gels are used for a number of purposes, including determining the yield of an experiment aimed to digest, extract, isolate, or duplicate DNA, sorting by size fragments of DNA being evaluated using DNA fingerprinting, and isolating and purifying a specific sequence of DNA. They may be used to separate DNA fragments ranging in length from 200bp to 10,000,000bp (10,000kb). To separate big fragments, employ a gel with a larger concentration of agarose than would be optimal for separating smaller fragments. To separate the biggest pieces, pulsed-field gel electrophoresis would be used, which requires a different sort of apparatus than the one we’ll be using today.
Polyacrylamide gels, on the other hand, have a considerably narrower range of efficacy than agarose gels; they operate best with DNA fragments ranging from five to 500 base pairs (bp), which is fairly short. Because they are so thin and delicate, these gels are challenging to manufacture and handle. They also include acrylamide, a strong, cumulative neurotoxic that is absorbed via the skin.
Also Read: Immunoelectrophoresis
At room temperature, purified agarose is insoluble in water (or buffer). However, it dissolves in hot water. When it begins to cool, it goes through a process called as polymerization. Rather of being dissolved in water or evaporating, the sugar polymers crosslink with one another, causing the solution to “gel” into a semi-solid matrix similar to “Jello” but more rigid. The more agarose that is dissolved in hot water, the harder the gel. While the solution is still hot, pour it into a mould known as a “casting tray” so that it may polymerize into the desired form (otherwise it will just solidify in the bottom of the flask wasting the expensive agarose).
An electric current is sent through the gel, causing one end to be positively charged and the other to be negatively charged. When an electric current is delivered through a gel, charged molecules travel across it. Migration refers to the movement of charged molecules. Molecules move in the opposite direction of their charge. As a result, a negatively charged molecule will be drawn towards the positive end. The gel is made up of a permeable matrix, similar to a sieve, through which molecules can flow when an electric current is sent across it. Smaller molecules migrate more quickly through the gel and hence travel further than bigger fragments, which migrate more slowly and thus travel a shorter distance. As a result, the molecules are sorted based on their size.
Separation of DNA Fragments by Gel Electrophoresis
A gel is filled with a solution of DNA molecules. Because each DNA molecule is negatively charged, an electric field may drag it through the gel. Smaller DNA molecules travel through the gel faster than bigger DNA molecules. As a result, a series of ‘bands’ form, with each band comprising DNA molecules of varying sizes. The tiniest DNA fragments are found in the bands furthest from the start of the gel. The biggest DNA fragments are seen in the bands closest to the beginning of the gel. To see the DNA, the gel is dyed with a fluorescent dye such as Ethidium Bromide that binds to the DNA and placed in front of a UV transilluminator, which reveals the stained DNA as bright bands.
Also Read: Capillary Electrophoresis