What is Electrophoresis?
The electrokinetic process which separates the charged particles in a fluid using a field of electrical charge is known as Electrophoresis.
This process is more often used in life science to separate the protein molecules called DNA. Depending on the size and the type of molecules, the separation can be achieved through various methods.
The methods differ in a few ways. But some things are common in every procedure like all need a source for the electrical charge, a buffer solution, and a support medium. To separate molecules based on their size, purity, and density, electrophoresis is used in laboratories.
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Define Electrophoresis
A laboratory technique that is used to separate DNA, protein molecules, or RNA based on the size, type, density, and electrical charge is known as electrophoresis. Through a gel and an electric current, move molecules are separated.
The pores in the gel work as a sieve which allows the small molecules to move faster than the large molecules. According to the desired size range, the conditions used during the electrophoresis can be altered to separate the molecules.
How Does The Electrophoresis Process Works?
The molecules are electrically charged themselves. Due to this when an electric field is applied to the molecules, it results in a force acting upon them. The intensity of the force applied by the electric field depends on the intensity of the charge in the molecule. With a greater charge of the molecules, the force applied by the electric field is also greater.
Therefore, the molecule will move according to its mass through the support medium. DNA analysis, RNA analysis as well as protein electrophoresis is some of the examples for the application of the trophoresis. It is a medical method that is used to separate the molecule found in a fluid sample and also analyze the molecule in the fluid sample.
Different Types of Electrophoresis
Routine Electrophoresis
Routine electrophoresis is traditional clinical laboratory electrophoresis. It is performed on a rectangle-shaped slab gel. This process is used to separate proteins like DNA. Some also use in separating the nucleic acids.
High-Resolution Electrophoresis
High-resolution electrophoresis is routine electrophoresis using a high amount of voltage. This process is used in conditions where more resolution of protein is needed. It is used to separate the CSF proteins for the diagnosis of multiple sclerosis. High-resolution electrophoresis is also used in light chains in urine for the early detection of lymphoproliferative disorders like multiple myeloma.
Polyacrylamide
Polyacrylamide which is also known as PAM is an organic polymer. It is formed from the acrylamides subunits. In the initial stage, it is made with a simple, linear structure, and repeating polymer. But this can be modified to form highly structured, crossed linked, and branched variants. It has various uses. The main purpose of this process is to separate the solids or the liquids in municipal as well as in industrial wastewater sectors.
Capillary Electrophoresis
Capillary electrophoresis is not as complicated as it is pronounced. This electrophoresis works much like a magnet. The magnet has two different poles one known as the North Pole and the other known as the South Pole. The same poles repel each other while the opposite poles attract each other.
Exactly like a magnet in this type of electrophoresis, there are two sides. One side is the positive side which is also called the cathode and the other side is the negative side also known as the anode.
This process is used to separate and detect the short tandem repeat alleles in forensic DNA laboratories. It is a primary method.
Isoelectric Focusing
Chemicals are infused in the gel that makes a pH gradient across the surface of the gel. The proteins will move to the place in the gel where there is no net charge which is their isoelectric point by using a very high vol.
This process is used in prenatal screening to separate various hemoglobins. It is also used to detect the oligoclonal bands in the gamma globulin.
Immunofixation Electrophoresis
In immunofixation electrophoresis, agarose gel electrophoresis separates the proteins in the serum sample. Antiserum is spread directly on the gel against the protein of interest. Precipitation of the protein of interest is formed in the gel matrix.
The precipitated protein is stained after a wash to remove the other protein. This process helps in studying the anti-aging of the proteins and also their split products. It also helps in identifying the proteins found in multiple myeloma.
Pulsed Field Electrophoresis
By alternatively applying the power to different pairs of electrodes, fragment separation can be easily achieved.
The positive electrode and the negative electrode get altered with the most common method during the process of electrophoresis. This process helps in separating the large fragments of the DNA.
FAQs on Electrophoresis Technique Used For DNA Analysis
1. What is the basic principle behind using electrophoresis for DNA analysis?
The fundamental principle of DNA electrophoresis lies in the movement of charged particles through a medium under the influence of an electric field. Since DNA molecules have a uniform negative charge due to their phosphate backbone, they migrate towards the positive electrode (anode) when an electric current is applied. The separation is achieved because a gel matrix acts as a molecular sieve, allowing smaller DNA fragments to move more quickly and farther than larger ones.
2. What are the key steps involved in separating DNA fragments using agarose gel electrophoresis?
The process of separating DNA using agarose gel electrophoresis involves several critical steps:
- Gel Preparation: An agarose gel is prepared by dissolving agarose powder in a buffer solution and pouring it into a casting tray with a comb, which creates wells for the samples.
- Sample Loading: The DNA samples, mixed with a loading dye, are carefully pipetted into the wells of the solidified gel.
- Electrophoresis Run: The gel is submerged in a buffer-filled tank, and an electric current is applied. The negatively charged DNA moves from the negative electrode (cathode) towards the positive electrode (anode).
- Staining: After the run, the gel is stained with a chemical like ethidium bromide (EtBr) or SYBR Green, which binds to DNA.
- Visualisation: The stained DNA bands are visualised under UV light, where they appear as fluorescent bands. The distance travelled indicates the size of the fragment.
3. Why is an agarose gel the preferred medium for DNA electrophoresis in most labs?
Agarose gel is widely preferred for separating DNA fragments for several reasons. Its pore size is ideal for separating a broad range of DNA fragments, from a few hundred to over 20,000 base pairs. It is also non-toxic, easy to prepare, and forms a sturdy gel at low concentrations. This sieving effect, combined with its relative safety and simplicity, makes it more suitable for routine DNA analysis than alternatives like polyacrylamide gel, which is typically used for separating smaller molecules like proteins or very small DNA fragments.
4. How does the uniform negative charge of DNA molecules facilitate their separation by size, not by charge?
The separation of DNA during electrophoresis is based almost entirely on size because of its uniform charge-to-mass ratio. Every nucleotide in a DNA strand carries a negative charge from its phosphate group. This means that a longer DNA fragment has more negative charge, but it also has proportionally more mass. The two factors cancel each other out, resulting in a constant charge-to-mass ratio for any piece of DNA. Therefore, the electric field pulls on all DNA fragments with a similar force relative to their mass, making molecular size the primary factor determining their speed through the gel's pores.
5. What is the difference in movement between small and large DNA fragments through the agarose gel?
The difference in movement is due to the gel's molecular sieving properties. Small DNA fragments can easily navigate the pores of the agarose matrix, facing little resistance. As a result, they travel quickly and move a greater distance from the starting wells. In contrast, large DNA fragments are significantly impeded by the gel matrix. They struggle to snake their way through the pores, causing them to move much more slowly and cover a shorter distance in the same amount of time.
6. What are some important real-world applications of DNA electrophoresis?
DNA electrophoresis is a cornerstone technique with many vital applications in science and medicine, including:
- Forensic Science: Used in DNA fingerprinting to compare DNA samples from a crime scene with those of suspects.
- Medical Diagnostics: Helps in identifying genetic mutations responsible for inherited diseases like cystic fibrosis or sickle cell anaemia.
- Paternity Testing: Compares the DNA patterns of a child with a potential father to establish biological relationships.
- Molecular Biology Research: Essential for checking the results of procedures like PCR (Polymerase Chain Reaction) and for isolating DNA fragments for genetic engineering.
7. Besides fragment size, what other factors can affect how DNA moves during gel electrophoresis?
While size is the primary factor, other variables can influence DNA migration. The concentration of the agarose gel is crucial; a higher concentration creates smaller pores and is better for separating small DNA fragments, while a lower concentration is used for large fragments. The applied voltage also matters; a very high voltage can generate heat, potentially denaturing the DNA or distorting the bands. Lastly, the conformation of the DNA (e.g., supercoiled, linear, or nicked circular) can cause it to move at different rates even if the base pair count is the same.
8. How are the invisible DNA bands made visible for analysis after an electrophoresis run?
Since DNA is invisible to the naked eye, a staining process is required after electrophoresis. The most common method involves soaking the gel in a solution containing an intercalating agent like ethidium bromide (EtBr). This molecule inserts itself between the base pairs of the DNA helix. When the stained gel is exposed to ultraviolet (UV) light, the EtBr fluoresces, revealing the separated DNA fragments as distinct, glowing bands. The position and intensity of these bands can then be analysed.
