Macromolecules (DNA, RNA, protein) play an essential role in biology. They perform a wide variety of functions and are necessary for cell survival. The study of macromolecules is an integral part of studying biology. But to really understand molecules, we have to be able to isolate and study them alone. To do this, we developed many laboratory techniques used to isolate and purify macromolecules for further analysis. One such technique is gel electrophoresis, a way to use electricity to move and separate different size macromolecules through a gel mesh. In this article, we review the steps and types of gel electrophoresis!
Gel electrophoresis definition
Gel electrophoresis has been around for decades, in fact since the 1960s. It is a ubiquitous method used to quantify and purify macromolecules (DNA, RNA, protein).1 It is widely used and very reliable. It is a fundamental part of many other important lab techniques and biotechnologies, like polymerase chain reaction (PCR), genome editing, and gene sequencing.
Gel electrophoresis is a lab technique where charged macromolecules are run through a gel with an electric field to separate them by size.
Types of gel electrophoresis
There are two main types of gel electrophoresis, nucleic acid and protein. Nucleic acid gel electrophoresis is used for DNA and RNA. The type of gel electrophoresis and the length of fragments in the samples used influence the gel used. Choosing a suitable gel is an integral part of gel electrophoresis, and there are multiple options. Gel types include agarose, agar, polyacrylamide, and agarose-acrylamide composites.
Agarose and polyacrylamide gels are the most commonly used; agarose gel is best for the majority of nucleic acids and large proteins while polyacrylamide gel is best for smaller nucleic acids and the majority of proteins.3 The density of the agarose depends on the size of the DNA or RNA fragments that need to be analyzed.
For shorter fragments, a higher concentration of agarose is used when creating the gel.
Gel electrophoresis steps
The process of gel electrophoresis involves many steps. Gel electrophoresis involves the use of a gel. While gels are solid, they are porous matrices, allowing macromolecules to travel through them.2 Gels can be made in the lab or purchased premade. The gels have wells, or indentations, at one end where samples are placed.
When the gel is ready for use, it is placed in an electrophoresis tank and covered with a liquid buffer. It is the buffer that conducts the electric current. The size of the macromolecule fragments to be run determines the type of buffer used. Positive and negative electrodes are placed at opposite ends of the gel, and an electric current is applied to the gel to create an electrical field.
Buffers. You may recall learning about buffers in chemistry class and painstakingly trying to balance buffer equations. One important principle that your teacher may have mentioned is Le Chatelier's Principle. Le Chatelier's Principle states that changes in concentration, pressure, and temperature determine how fast and efficiently a chemical reaction can occur. A buffer is an aqueous solution made of a weak acid and its conjugate base or a weak base and its conjugate acid. Buffers work to maintain a constant pH during chemical reactions and do this using Le Chatelier's Principle.
Before being loaded into the gel, the macromolecule samples, consisting of several fragments of different sizes and concentrations, are dyed. Dying allows for better visualization and make samples heavier, so they do not float out of the wells after they are loaded. Using a pipette, a macromolecule marker, or a ladder and the macromolecule samples are loaded into the gel wells. The macromolecule marker/ladder is always loaded into the first well. The marker/ladder is composed of DNA, RNA, or protein fragments of known lengths.
The marker/ladder serves as a ruler of sorts; the position of samples is compared to that of the fragments in the marker to determine the length of the samples.
Macromolecules migrate or move across the gel, moving towards the opposite charge. For example, DNA and RNA are negatively charged due to the negatively charged phosphate molecules in nucleic acid backbones.1 Therefore, for nucleic acid gel electrophoresis, the negative electrode is placed at the end of the gel with the wells, and the positive electrode is placed at the other end so that the DNA and RNA will migrate away from the wells to the positive end.
Macromolecules move according to size, with smaller molecules migrating farther distances.
Electrode: A small metal device that carries electrical signals to its target.
The electric current is applied long enough to allow the macromolecule fragments to separate. If a gel is not run long enough, it will result in poor or no separation of fragments within each sample. If a gel is run too long, fragments will run right out of the gel and be lost in the buffer.
Once the gel has run long enough to allow for adequate fragment separation, the gel is removed from the electrophoresis tank and stained with a fluorescent dye that binds to macromolecules. The gel is then placed under a light so that the stained macromolecules glow. Macromolecule fragments of the same size will appear as bands, and the thicker the band, the higher the concentration of fragments.
DNA gel electrophoresis
DNA gel electrophoresis is a type of nucleic acid gel electrophoresis used to identify, quantify, and purify DNA fragments. DNA gel electrophoresis can also be used in other contexts, like PCR, DNA cloning, or DNA sequencing. Let's take a look at each of these processes.
PCR stands for polymerase chain reaction and is a laboratory technique used to make copies of a specific DNA sequence from a sample that has very little of that sequence.
PCR is the main test that is used for detecting COVID-19 infection. When a sample is collected from a person with COVID-19 it is sent to a lab for PCR analysis. Since the amount of viral DNA is so low compared to the host DNA.
During a PCR, short synthetic DNA fragments or oligos called primers bind a portion of the questioned genomic DNA to be amplified.4 Once the primer recognizes the piece of DNA that we are interested in, multiple rounds of DNA synthesis occur to amply the segment into many copies. Electrophoresis is a crucial step that needs to be done prior to starting a PCR. As previously mentioned electrophoresis is used to isolate and purify DNA or RNA to ensure that the sample is not contaminated with confounding cellular or tissue matter. If a sample was not properly purified, the primer could have trouble recognizing the DNA sequence of interest and could produce inaccurate results.
Gel electrophoresis is also used in DNA cloning and in DNA sequencing. These laboratory techniques allow us to understand more about the human genome as well as other animal genomes. DNA sequencing is the process of determining the exact position of each of the four bases which allows us to know which codons are present in which genes. This allows us to know what proteins are going to be expressed which can indicate potential mechanisms for rare diseases with no cure.
Gel electrophoresis diagram
To summarize, during gel electrophoresis, charged macromolecules are loaded into a gel with an electric field. One end of the gel has a positive charge, and the other end has a negative charge. The macromolecules move from the wells at one end of the gel to the opposite end, the end with a charge opposite that of the macromolecules. Smaller molecules travel farther.
Macromolecules and the gel are stained with dye to allow for the visualization of macromolecule fragments. A ladder/marker is always used since it serves as the ruler for measuring the length of the fragments. The size of fragments is estimated based on their location relative to fragments in the marker, whose lengths are known.
Gel electrophoresis - Key takeaways
- Gel electrophoresis is a prevalent lab technique used to quantify and purify macromolecules (DNA, RNA, protein).
- Gel electrophoresis involves running charged macromolecules through a gel with an electric field to separate them by size.
- There are two main types of gel electrophoresis, nucleic acid and protein. Nucleic acid gel electrophoresis is used for DNA and RNA.
- The type of gel used is determined by the type of macromolecule and the size of the macromolecule samples being analyzed.
References:
Nucleic Acid Gel Electrophoresis—A Brief Overview and History, n.d. https://www.thermofisher.com/us/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/na-electrophoresis-education/na-separation-overview.html
What is gel electrophoresis?, 21 July 2021. https://www.yourgenome.org/facts/what-is-gel-electrophoresis
Overview of protein electrophoresis, n.d. https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-electrophoresis.html
Polymerase Chain Reaction (PCR), 26 August 2022 https://www.genome.gov/genetics-glossary/Polymerase-Chain-Reaction
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