Electrophoresis

Electrophoresis is a widely used technique that passes an electric current through biological molecules (typically DNA, but it can also be protein or RNA) and separates them into larger or smaller bits. It’s utilised in a wide range of applications, from forensics to detecting the identity of people who may have been engaged in a crime by comparing their Genetic sequence and electrophoresis template to one in a database. An electric current moves them over a gel to separate molecules. The pores in the gel can function as a filter, allowing smaller molecules to move through faster than larger molecules. The electrophoresis settings can be tweaked to separate materials in a particular size range.

Principle 

 The mobility and splitting of energetic particles (ions) under the applied electrical field are referred to as electrophoresis. An electrophoretic system consists of two electrodes (anode and cathode) with opposite charges linked by an electrolyte. The diameter, shape, and charge of an ionic particle and the temperature during separation dictate its mobility (m), constant under established electrophoretic circumstances.

Types of Electrophoresis

Electrophoresis is a term that refers to a group of similar analytical procedures. Here are several examples: 

• Affinity electrophoresis is a kind of electrophoresis wherein particulates are separated based on the development of complexes or biospecific interactions.

• A capillary electrophoresis is a form of electrophoresis that separates ions based on their atomic radius, charge, and viscosity. As the name suggests, this procedure is usually done in a glass tube, and it provides fast results and a high-resolution separation.

• Gel electrophoresis is a common type of electrophoresis wherein particles are separated by moving across a porous gel while being influenced by an electrical field. Agarose and polyacrylamide are the two most used gel ingredients. Nucleic acids (DNA and RNA) and nucleic acid fragments are separated using gel electrophoresis.

Working

An electric field is supplied to molecules, which results in a force acting on them because they are electrically charged. The larger the molecule’s charge, the greater the energy produced by the electrical field. Hence the further the compound will change based on its mass through the support medium. DNA and RNA analysis and protein electrophoresis,  medical methods for analysing and separating molecules found in a fluid sample, are electrophoresis applications (most commonly blood and urine samples).

There are two basic parameters in electrophoresis that affect how rapidly and in which direction a particle can migrate. First and foremost, the charge on the sample is significant. Positively charged organisms are drawn to the positive pole of an electromagnetic current, whereas negatively charged species are drawn to the negative pole. If the field is strong enough, a neutral species can be ionised and is not changed otherwise. The particle size has an impact as well. Small ions and molecules can flow considerably faster through a gel or liquid than larger ones.

A charged atom is pulled to an oppositely charged particle in an electric field, but additional forces at work influence how a molecule moves.

Gel electrophoresis and DNA

Electrophoresis allows you to discriminate between different lengths of DNA fragments because DNA is negatively charged. It will migrate to the positive electrode when an electric current is supplied to the gel. Because shorter lengths of DNA move faster across the gel than longer strands, the fragments are ordered in size order.

The DNA on the gel can be observed once it has been separated using dyes, fluorescent tags, or radioactive markers. On the gel, it will appear as bands. At the same time as the samples, a DNA marker with known length fragments is routinely passed through the gel.

Preparation of Gel

The amount of agarose in the gel is influenced by the magnitude of the DNA fragments; the denser the matrix, the greater the agarose content, and vice versa. Smaller DNA fragments are separated using higher agarose concentrations, whereas larger molecules need a lower agarose concentration.

• To form a gel, combine agarose powder with an electrophoresis buffer and boil to a high temperature till the agarose powder has completely melted.

• The melted gel is then poured into a gel chamber with a “comb” at one end to create wells for pipetting the sample.

• Once the gel has chilled and set, the comb is removed (it will then be opaque rather than transparent). Pre-made gels are now widely used.

• The gel is then placed in an electrophoresis tank, which is subsequently filled with an electrophoresis buffer until the surface area of the gel is covered. The buffer carries the electric current.

Conclusion

The migration of molecules in a gel or fluid within a somewhat homogeneous electric field is called electrophoresis. Charge, size, and binding affinity can all be utilised to separate molecules using electrophoresis. Biomolecules such as DNA, RNA, proteins, nucleic acids, plasmids, and fragments of these macromolecules are separated and analysed using this approach. In paternity testing and forensic science, electrophoresis is one of the procedures used to determine source DNA.

In most types of electrophoresis, one of the critical issues is the removal of heat produced by the passage of electric current. Any temperature difference affects migration rates through the medium, distorting the separated molecules’ bands. Conducting electrophoretic studies at a specific temperature would be optimal.