How Does Gel Electrophoresis Work in Biology?
Gel electrophoresis is a common technique that is used in different laboratories to properly separate charged molecules such as RNA, DNA, and proteins on the basis of their size. In the process, the charged molecules will be made to pass through a gel-like substance with electric current passing across it. The application of electric current occurs so that the ends of the gel are oppositely charged.
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When the charged molecules move through the gel, the process is known as migration. These molecules properly migrate towards the end that has an opposite charge. Hence, the molecules that have a positive charge will be attracted to the end having the negative charge. With the process, it is easier to separate the smaller charged molecules. However, larger fragments can also be moved, albeit a little slower. In this article, students will learn what is gel electrophoresis and much more.
Agarose Gel Electrophoresis
The use of agarose gel electrophoresis is done in order to separate the fragments of DNA. The process of restriction of endonuclease can be used for cutting the DNA into fragments. In this case, the various samples will create fragment lengths of variable types. The fragments are separated properly because DNA tends to have a negative charge due to the fact that a phosphate group is present. Agarose gel is used for the placement of the DNA samples and the calculation of the size is done for the process to complete. Different sequences with specific patterns can be identified with the help of this process.
Polyacrylamide Gel Electrophoresis
The applications of gel electrophoresis in order to separate the fragments of proteins uses Polyacrylamide Gel Electrophoresis. The proteins are basically folded to create different shapes that have negative as well as positive regions. The proteins should be properly treated using an anionic detergent so that a uniform negative charge can be imparted. The polyacrylamide gel is used for placing the protein samples and there is a comparison of the sizes that takes place. The proteins, once separated, are properly transported to a particular membrane.
How Does the Process of Gel Electrophoresis Work?
There are some important steps and processes that take place during the process of gel electrophoresis. With careful study of the gel electrophoresis principle, students will be able to understand the process better.
Preparation Of The Gel
The agarose gels are the main components of the process. These gels are commonly used in order to visualize all the DNA fragments. The amount of concentrated gel used in the process depends on the size that the DNA fragments have. With more concentration of agarose, the matrix will be denser. The separation of smaller fragments requires the concentration of agarose to be more in the process of agarose gel electrophoresis. In order to create the gel, experts mix the agarose powder with the electrophoresis buffer and melt the powder on high heat.
Preparation of DNA
A particular type of dye is added to the DNA sample before the entire process is begun. This increases the sample’s viscosity and hence it will not float out from the wells. This also helps in the process of proper migration. The marker of DNA, also called the DNA ladder, will be loaded right into the gel’s first well. These markers tend to have a proper length so that they can help in the approximation of the fragment size in the best way. Then the DNA samples will be transferred into the gel wells using a pipette. After that, the lid is properly placed on the tank to start the process.
Fragment Separation
Once the samples of the DNA have been transferred to the tank for the procedure of agarose gel electrophoresis to begin, the electrical current is turned on. After that, the charged particles in the DNA with a negative charge will move without any difficulty towards the gel’s positive side. It is easier to move the shorter DNA fragments and the process doesn’t even take a lot of time. The total distance that the DNA migrates can be easily identified by monitoring the entire process of migration. The buffer dye helps in that process by leaving a mark. The electric current should be kept running for a long enough time to make sure that all DNA fragments have moved far across so that the gel is able to properly separate them.
Visualizing the Results
After the migration process of the DNA fragments is complete, the electric current will be turned off. The gel is efficiently removed from the tank. In order to visualize the fragments of the DNA, the gel will be stained with a dye that is fluorescent in colour. This will bind itself to the DNA fragments. After binding, the fragments are put on the ultraviolet transilluminator that will allow the people to see some bright bands which are the stained DNA.
Conclusion
The article above attempts to provide an explanation for the process of gel electrophoresis. Students can use this article and study the gel electrophoresis diagram to gain more information about the topic.
FAQs on Gel Electrophoresis Made Simple: Types, Process & Applications
1. What is gel electrophoresis, and what is its fundamental principle?
Gel electrophoresis is a laboratory technique used to separate macromolecules like DNA, RNA, and proteins based on their size and charge. The fundamental principle is that charged molecules, when placed in an electric field, will migrate through a gel matrix. This gel acts as a molecular sieve. Smaller molecules navigate through the pores of the gel more easily and travel a longer distance, while larger molecules are impeded and travel shorter distances. This differential movement results in the separation of molecules into distinct bands.
2. What are the key steps involved in performing agarose gel electrophoresis for DNA separation?
The separation of DNA fragments using agarose gel electrophoresis involves several key steps:
- Gel Preparation: An agarose powder is dissolved in a buffer solution, heated, and then poured into a casting tray with a comb to create wells for sample loading.
- Sample Loading: The DNA samples, mixed with a loading dye, are carefully pipetted into the wells of the solidified gel. A DNA ladder (a mixture of DNA fragments of known sizes) is also loaded into one well for reference.
- Electrophoresis: The gel tray is placed in an electrophoresis tank filled with buffer, and an electric current is applied. Since DNA is negatively charged (due to its phosphate backbone), it migrates towards the positive electrode (anode).
- Staining and Visualization: After the run, the gel is stained with a fluorescent dye like ethidium bromide, which binds to the DNA. The separated DNA bands are then visualized under UV light.
3. What are the primary applications of gel electrophoresis in biotechnology and medicine?
Gel electrophoresis is a versatile tool with numerous important applications, including:
- DNA Fingerprinting: Used in forensic science to compare DNA samples from a crime scene with suspects, and in paternity testing.
- Medical Diagnostics: To detect genetic mutations associated with diseases like cystic fibrosis or sickle cell anaemia.
- Recombinant DNA Technology: To verify the successful insertion of a gene into a plasmid by checking the size of the DNA fragments after restriction enzyme digestion.
- Protein Analysis: Techniques like SDS-PAGE are used to separate proteins to study their size, purity, and expression levels.
- Evolutionary Biology: To compare DNA sequences among different species to determine evolutionary relationships.
4. Why do smaller DNA fragments travel further than larger ones in a gel?
Smaller DNA fragments travel further through the gel due to the molecular sieving effect of the gel matrix (e.g., agarose). The gel is a mesh-like network of pores. Smaller DNA fragments can easily navigate through these pores with minimal resistance, allowing them to move quickly towards the positive electrode. In contrast, larger DNA fragments are more cumbersome and get entangled in the gel matrix more frequently. This increased friction and obstruction significantly slows their migration, causing them to travel a shorter distance from the well in the same amount of time.
5. How does SDS-PAGE for separating proteins differ from agarose gel electrophoresis for DNA?
While both techniques separate molecules based on size, they have key differences. Agarose gel electrophoresis separates DNA based on its intrinsic negative charge and size. In contrast, SDS-PAGE (Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis) is used for proteins. Its key differences are:
- Sample Treatment: Proteins are treated with the detergent SDS, which denatures them and imparts a uniform negative charge. This eliminates the influence of the protein's native charge and shape, ensuring separation is based solely on size.
- Gel Matrix: It uses a polyacrylamide gel, which has a smaller pore size than agarose, making it ideal for separating smaller protein molecules.
- Purpose: Agarose gel electrophoresis separates DNA or large RNA. SDS-PAGE specifically separates proteins based on their molecular weight.
6. What is the importance of a 'DNA ladder' in gel electrophoresis?
A DNA ladder, also known as a molecular-weight marker, is a crucial control used in gel electrophoresis. It is a solution containing a mixture of DNA fragments of known, predetermined sizes. The DNA ladder is loaded into one of the wells on the gel alongside the unknown samples. When the gel is run, the ladder separates into a series of distinct bands. By comparing the migration distance of the unknown DNA bands to the bands of the known ladder, one can accurately estimate the size (in base pairs) of the sample DNA fragments.
7. Can gel electrophoresis separate molecules that are not charged?
No, standard gel electrophoresis cannot separate neutral (uncharged) molecules. The technique's driving force is the interaction between the charged molecules and the applied electric field. Molecules must possess a net positive or negative charge to be pulled towards the oppositely charged electrode. Neutral molecules would not experience this electrostatic force and would therefore not migrate through the gel in a directed manner. However, techniques like SDS-PAGE can be used to impart a uniform charge onto molecules like proteins, allowing for their separation.
