Gel Electrophoresis of Nucleic Acids

Gel electrophoresis is a method to separate nucleic acid molecules (DNA or RNA) depending on their size. This approach is capable of efficiently separating molecules as large as 20kb. Following PCR analysis or before cloning, sequencing and Northern or Southern blotting, nucleic acid separation by gel electrophoresis has several uses in molecular biology.

The nucleic acids to be examined are distributed into the wells of an agarose gel. The agarose gel serves as a matrix, containing and separating the target molecules. The gel is immersed in an electrophoresis chamber containing a buffer that permits an electric current to pass. Across the chamber, an electromotive force is applied. Buffer solutions work to mitigate pH variations induced by the electric field and prevent the gel from overheating due to the electric current.

To accurately estimate fragment size, nucleic acid standards with established size bands are put on the gel alongside genuine samples. These commercially accessible goods have uniformly spaced banding patterns and come in various sizes.

Nucleic acid molecules are colourless and must be stained to be seen. For convenient monitoring of their mobility in the gel, a tracking dye such as bromophenol blue is frequently used. Ethidium bromide is a fluorescent dye that may be used to see DNA in agarose and polyacrylamide gels. It is commercially accessible as a ready-to-use solution. A gel imaging system, such as the Gel Doc EZ System, is used to see stained gels.

A historical overview of nucleic acid gel electrophoresis

Electrophoresis was first used to divide nucleic acids in the early 1960s. Nucleic acids were frequently segregated using the density of gradient and centrifugation that depended upon the sedimentation and velocities, which in turn was governed by nucleic acid amount and state of confirmation. The density of gradient and centrifugation managed to take a long period to give results, along with heavy equipment and a significant amount of samples to test. Researchers began to investigate features of DNA mobility in the electrolytic or ionic solutions upon which an electrical field was supposed to be applied, in a procedure known as electrophoresis.

After a few decades, nucleic acid electrophoresis evolved to employ a gel matrix as a separating medium, much like protein electrophoresis. Agar (a naturally occurring carbohydrate), agarose (a component of agar), polyacrylamide (a synthetic gel) and agarose-acrylamide composite gels turned out to be successful in aiding RNA and DNA electrophoresis in the middle or late 1960s. 

These early gel electrophoresis investigations produced fractionation findings that correlated with sedimentation coefficients, or S values, obtained by density gradient centrifugation, a well-developed nucleic acid separation technique at the time. In the late 1960s, with improved knowledge and innovations in agarose manufacture, agarose progressively displaced agar as the favoured gel electrophoresis medium.

Functionalities of gel electrophoresis of nucleic acids

A backbone of sugar, deoxyribose, and phosphate groups in DNA connect the nucleobases, or letters of the DNA code. The backbone of DNA has a negative charge for each nucleobase present, resulting in the same mass-to-charge ratio of DNA across different fragment sizes. When we apply an electrical field to a DNA solution, the DNA molecules migrate towards the positively charged electrode due to the negative charge.

Application of gel electrophoresis of nucleic acids

Agarose gel electrophoresis is extensively used to assess the size of DNA fragments following restriction enzyme digestion, such as in restriction mapping of cloned DNA. It has also become a standard technique in molecular genetics diagnosis or genetic fingerprinting via PCR product analysis.

Agarose gel electrophoresis is also required to separate limited genomic DNA before Southern blot and RNA before Northern blot. Agarose gel electrophoresis is often used to separate circular DNA with distinct supercoiling topologies and fragments that differ owing to DNA synthesis.

DNA damage from increased cross-linking lowers electrophoretic DNA movement proportionately. In addition to being an effective medium for analysing fragment size, agarose gels enable the purification of DNA fragments. Because purification of DNA fragments separated in an agarose gel is required for various molecular procedures such as cloning, the ability to filter segments of interest from the gel is critical.

Increasing the agarose content in a gel slows migration and allows for the easier separation of smaller DNA molecules. On the other hand, increasing the voltage speeds up the movement of DNA molecules. However, increasing the currency voltage is accompanied by decreased band resolution and an increased likelihood of melting the gel (over around 5 to 8 V/cm).

Conclusion

In general, gel electrophoresis is now proven to be a common method to divide nucleic acids in molecular biology. The ‘particular preparative and analytical method’ is critical in standard processes like molecular cloning and PCR, nucleic acid separation and the analysis of highly growing technologies such as genome editing and next-generation sequencing.