Gel electrophoresis is a procedure used to separate biological molecules by size. The separation of these molecules is achieved by placing them in a gel with small pores and creating an electric field across the gel. The molecules will move faster or slower based on their size and electric charge. Show The process of gel electrophoresis works because negatively charged molecules move away from the negative pole of the electric current and smaller molecules will move faster than larger molecules. Thus, a size separation is achieved within the pool of molecules running through the gel. The gel works in a similar manner to a sieve separating particles by size. The electrophoresis works to move the particles, using their inherent electric charge, through the sieve. When researchers are trying to distinguish between different segments of DNA, for example, the process is simple. The samples are loaded into channels at the start of the gel. Each DNA molecule has the same charge (-1), because DNA is formed by the same 4 nucleotides and always carries a slightly negative charge regardless of its size. Therefore, each DNA molecule will have the same force pulling it through the gel. However, the size of each molecule hinders its progress through the gel. Large molecules hit parts of the gel matrix, and are slowed down. Small DNA molecules can slip between the various components of the gel matrix, and quickly make their way to the other side of the gel. After a certain amount of time, the dyed DNA molecules can be seen aggregating in different areas of the gel, based on how far they moved during gel electrophoresis. This allows researchers to identify the segments, and compare the DNA of different organisms. The purpose of gel electrophoresis is to visualize, identify and distinguish molecules that have been processed by a previous method such as PCR, enzymatic digestion or an experimental condition. Often, mixtures of nucleic acids or proteins that are collected from a previous experiment/method are run through gel electrophoresis to determine the identity or differentiate between molecules. The broad steps involved in a common DNA gel electrophoresis protocol: The DNA is isolated and preprocessed (e.g. PCR, enzymatic digestion) and made up in solution with some basic blue dye to help visualize the movement of the sample through the gel. TAE buffer provides a source of ions for setting up the electric field during electrophoresis. The weight-to-volume concentration of agarose in TAE buffer is used to prepare the solution. For example, if a 1% agarose gel is required, 1g of agarose is added to 100mL of TAE. The agarose percentage used is determined by how big or small the DNA is expected to be. If one is looking at separating a pool of smaller size DNA bands (<500bp), a higher percentage agarose gel (>1%) is prepared. The higher percentage of agarose creates a denser sieve to increase the separation of small DNA length differences. The agarose-TAE solution is heated to dissolve the agarose. The agarose TAE solution is poured into a casting tray that, once the gel solution has cooled down and solidified, creates a gel slab with a row of wells at the top. The solid gel is placed into a chamber filled with TAE buffer. The gel is positioned so that the chamber wells are closest to the negative electrode of the chamber. The gel chamber wells are loaded with the DNA samples and usually, a DNA ladder is also loaded as reference for sizes. The negative and positive leads are connected to the chamber and to a power supply where the voltage is set. Turning on the power supply sets up the electric field and the negatively charged DNA samples will start to migrate through the gel and away from the negative electrode towards the positive. Once the blue dye in the DNA samples has migrated through the gel far enough, the power supply is turned off and the gel is removed and placed into an ethidium bromide solution. Ethidium bromide intercalates between DNA and is visible in UV light. Sometimes ethidium bromide is added directly to the agarose gel solution in step 2. The ethidium bromide stained gel is then exposed to UV light and a picture is taken. DNA bands are visualized in from each lane corresponding to a chamber well. The DNA ladder that was loaded is also visualized and the length of the DNA bands can be estimated. An example is given in the figure below. Gel electrophoresisThere are two types of gel electrophoresis: native and denaturing. Native gel electrophoresis usually attempts to keep RNA or protein in its native structure while running it through the gel. Denaturing gel electrophoresis attempts to reduce the RNA or protein into its most linear structure before or during gel electrophoresis. The denaturation of the RNA or protein is accomplished by adding a reducing agent to the sample, gel and/or buffer. The reducing agent separates bonds within the RNA or protein molecule and thereby reduces its secondary structure. The secondary structure of a protein or RNA will influence, in a non-linear manner, how fast it migrates through a gel. A denatured, linear form of RNA or protein, however, will migrate proportionally to its linear size (base pairs or kilo Daltons). Denaturing gel electrophoresis is often more accurate for size identification, whereas native gel electrophoresis is usually used to identify larger protein complexes.
Simple laws of physics dictate that when current is applied to a medium containing charged species, those species will migrate towards the opposite charge. Depending on the medium through which they are moving, other characteristics – such as the size of the species present – can impact their movement, leading to separation. This is the basis on which electrophoresis techniques, such as agarose gel electrophoresis, are built – techniques that are widely used across the life sciences. What is electrophoresis? In this article, we will consider how agarose gel electrophoresis works, how it can be interpreted and some of its purposes. Electrophoresis is a technique that uses electrical current to separate DNA, RNA or proteins based on their physical properties such as size and charge. What is agarose gel electrophoresis?Agarose gel electrophoresis is a form of electrophoresis used for the separation of nucleic acid (DNA or RNA) fragments based on their size. Negatively charged DNA/RNA migrates through the pores of an agarose gel towards the positively charged end of the gel when an electrical current is applied, with smaller fragments migrating faster. The resulting bands can then be visualized using ultraviolet (UV) light.
How does gel electrophoresis work?Agarose is a component of agar. It forms a 3D gel matrix of helical agarose molecules in supercoiled bundles held by hydrogen bonds, with channels and pores through which molecules are able to pass. When heated, these hydrogen bonds break, turning the agarose to liquid and allowing it to be poured into a mold before it resets (Figure 1).
DNA gel electrophoresis steps, the gel electrophoresis machine, electrophoresis buffer and electrical separationThere are a number of key steps4 involved in choosing, setting up, running and analyzing agarose gels that we will now consider. 1. Determine the required gel percentage – 0.7–1% agarose gel is typically adequate for most applications, but it is important to choose a percentage appropriate for your samples and expected fragment sizes. Combine the agarose powder with the same buffer type to be used to run the gel and heat to melt the mixture, avoiding boiling. Tris-acetate-ethylenediaminetetraacetic acid (EDTA) (TAE) or tris-borate-EDTA (TBE)5 are often the buffers of choice, as tris-acid solutions are effective buffers for slightly basic conditions, keeping DNA deprotonated and soluble in water. The EDTA, a chelating agent, inactivates nucleases that may damage the DNA being analyzed.
How to read gel electrophoresisAgarose gels may be visualized on a UV light box in a dark room or using a self-contained light box linked to a camera. Whichever system is utilized, UV light is shone through the gel from below and bands of DNA fluoresce thanks to the intercalating dye bound to them. This may be captured using a camera with a specialized UV filter for your records. Marker ladders come with a guide to indicate the size of each band they include. By comparing this to bands in sample lanes, the sizes of the bands can therefore be determined. The relative amount of DNA between samples may also be compared, as higher DNA concentrations will produce brighter bands. An example is shown in Figure 4.
What is the purpose of gel electrophoresis?There are a number of reasons why the separation of DNA fragments may be desirable, many of which are widely applicable across the life science disciplines. Let’s consider some common purposes.
DNA agarose gel electrophoresis glossary
2. Masek T, Vopalensky V, Suchomelova P, Pospisek M. Denaturing RNA electrophoresis in TAE agarose gels. Anal Biochem. 2005;336(1):46-50. doi:10.1016/j.ab.2004.09.010 3. Krizek DM, Rick ME. Agarose gel electrophoresis of proteins. Curr Protoc Cell Biol. 2002;15(1):6.7.1-6.7.13. doi:10.1002/0471143030.cb0607s15 4. Lee PY, Costumbrado J, Hsu CY, Kim YH. Agarose gel electrophoresis for the separation of DNA fragments. J Vis Exp. 2012;(62):3923. doi:10.3791/3923 5. Sanderson BA, Araki N, Lilley JL, Guerrero G, Lewis LK. Modification of gel architecture and TBE/TAE buffer composition to minimize heating during agarose gel electrophoresis. Anal Biochem. 2014;454:44-52. doi:10.1016/j.ab.2014.03.003 6. Hall AC. A comparison of DNA stains and staining methods for agarose gel electrophoresis. bioRxiv. 2019. doi:10.1101/568253 7. DNA gel-loading dye (10X). Cold Spring Harb Protoc. 2008;2008(8):pdb.rec11373. doi:10.1101/pdb.rec11373 8. Schwarz MJ. DNA diagnosis of cystic fibrosis. Ann Clin Biochem. 1998;35(5):584-610. doi:10.1177/000456329803500502 9. Marwal A, Sahu AK, Gaur RK. Chapter 16 - Molecular Markers: Tool for Genetic Analysis. In: Verma AS, Singh A, eds. Animal Biotechnology. Academic Press; 2014:289-305. doi:10.1016/B978-0-12-416002-6.00016-X 10. Tweedie JW, Stowell KM. Quantification of DNA by agarose gel electrophoresis and analysis of the topoisomers of plasmid and M13 DNA following treatment with a restriction endonuclease or DNA topoisomerase I. Biochem Mol Biol Educ. 2005;33(1):28-33. doi:10.1002/bmb.2005.494033010410 11. Molnar C, Gair J. Chapter 10.1 Cloning and Genetic Engineering. In: Concepts of Biology. Published online May 14, 2015. Accessed February 2, 2022. https://opentextbc.ca/biology/chapter/10-1-cloning-and-genetic-engineering/ 12. Balletbó A. DNA purification from an agarose gel (protocol for NucleoSpin® pCR clean-up gel extraction kit). protocols.io. Published September 22, 2019. Accessed February 2, 2022. doi:10.17504/protocols.io.7hrhj56 13. Downey N. Extraction of DNA from Agarose Gels. In: Casali N, Preston A, eds. E. coli Plasmid Vectors: Methods and Applications. Methods in Molecular BiologyTM. Humana Press; 2003:137-139. doi:10.1385/1-59259-409-3:137 14. Hellman LM, Fried MG. Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions. Nat Protoc. 2007;2(8):1849-1861. doi:10.1038/nprot.2007.249 15. Yousaf N, Gould D. Demonstrating Interactions of Transcription Factors with DNA by Electrophoretic Mobility Shift Assay. In: Gould D, ed. Mammalian Synthetic Promoters. Methods in Molecular Biology. Springer; 2017:11-21. doi:10.1007/978-1-4939-7223-4_2 |