Gel electrophoresis

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.

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.

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.

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.

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.

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 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.

References:

1. 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

2. What is gel electrophoresis?, 21 July 2021. https://www.yourgenome.org/facts/what-is-gel-electrophoresis

3. 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

4. Polymerase Chain Reaction (PCR), 26 August 2022 https://www.genome.gov/genetics-glossary/Polymerase-Chain-Reaction