Principle Procedure Types and Applications of DNA 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 for DNA Analysis in Chemistry
1. What is electrophoresis technique used for DNA analysis?
Electrophoresis is a laboratory technique used to separate DNA fragments based on their size by applying an electric field through a gel medium. In DNA analysis, the most common method is agarose gel electrophoresis.
- DNA samples are loaded into wells in a gel.
- An electric current is applied across the gel.
- DNA fragments move toward the positive electrode due to their negative charge.
- Smaller fragments travel faster and farther than larger ones.
2. How does gel electrophoresis separate DNA fragments?
Gel electrophoresis separates DNA fragments because DNA is negatively charged and moves through a porous gel matrix at different speeds depending on fragment size.
- The phosphate backbone of DNA gives it a uniform negative charge.
- When voltage is applied, DNA migrates toward the positive electrode (anode).
- The gel acts as a molecular sieve.
- Smaller DNA fragments pass through pores more easily and travel farther.
3. Why is DNA negatively charged in electrophoresis?
DNA is negatively charged because its backbone contains phosphate groups (PO43−) that carry negative charges at physiological pH.
- Each nucleotide in DNA has a phosphate group.
- These phosphate groups ionize and release H+ ions.
- The remaining phosphate groups carry negative charges.
4. What are the main types of electrophoresis used in DNA analysis?
The main types of electrophoresis used in DNA analysis are agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), and capillary electrophoresis.
- Agarose gel electrophoresis: Used for separating larger DNA fragments.
- PAGE: Provides higher resolution for small DNA fragments.
- Capillary electrophoresis: Automated technique used in DNA sequencing and forensic analysis.
5. What is the role of agarose gel in DNA electrophoresis?
The agarose gel acts as a porous matrix that separates DNA fragments according to size during electrophoresis.
- Agarose forms a three-dimensional network with microscopic pores.
- DNA molecules migrate through these pores when voltage is applied.
- Higher agarose concentration creates smaller pores for better separation of small fragments.
6. What is the function of buffer solution in electrophoresis?
The buffer solution in electrophoresis maintains a stable pH and provides ions to conduct electricity.
- Common buffers include TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA).
- Buffers prevent drastic pH changes during the run.
- They supply ions that carry current through the gel.
7. How do you visualize DNA after gel electrophoresis?
DNA is visualized after gel electrophoresis by staining it with a fluorescent dye and observing it under UV light.
- Common dyes include ethidium bromide and safer alternatives like SYBR Green.
- The dye intercalates between DNA base pairs.
- Under UV light, DNA bands fluoresce and become visible.
8. What factors affect the movement of DNA in electrophoresis?
The movement of DNA in electrophoresis is mainly affected by fragment size, gel concentration, applied voltage, and buffer composition.
- Smaller fragments move faster than larger ones.
- Higher gel concentration slows migration.
- Higher voltage increases speed but may reduce resolution.
- Improper buffer can alter conductivity and pH.
9. What is a DNA ladder in gel electrophoresis?
A DNA ladder is a mixture of DNA fragments of known sizes used as a reference to estimate the length of unknown DNA samples.
- It is loaded into one well alongside test samples.
- Each band corresponds to a fragment of known base pair length.
- Unknown fragment sizes are determined by comparing band positions.
10. What are the applications of electrophoresis in DNA analysis?
Electrophoresis is used in DNA analysis for DNA fingerprinting, genetic testing, PCR product analysis, and DNA sequencing.
- In forensic science, it compares DNA profiles.
- In medical genetics, it detects mutations or inherited disorders.
- In molecular biology, it verifies PCR amplification results.
- In biotechnology, it supports gene cloning and research.
