What is Electrophoresis?
Because shorter strands of DNA move much faster through the gel than longer strands, the fragments are ordered in size order. An electric current is used to move molecules across a gel for separating. The gel's pores act as a filter, allowing smaller molecules to move more quickly than larger molecules. The electrophoresis conditions can be adjusted to separate molecules in a certain size range.
Let us understand what is electrophoresis and what is electrophoresis from this article.
What is Gel Electrophoresis?
Gel electrophoresis is used for separating the charged molecules, such as DNA, based on their size.
When an electric current is passed through a gel, charged molecules travel across it. An electric current passes through the gel, creating a positive charge on one end and a negative charge on the other. The charged molecules can be separated by an electric field. In gel electrophoresis DNA molecules migrate from the negative to a positively charged electrode.
Migration
The term "migration" refers to the movement of charged molecules. Molecules move in the direction of the opposite charge. As a result, a molecule with a negative charge will be attracted to the positive end (opposites attract!). When an electric current is passed over the gel, it forms a permeable matrix, similar to a sieve, through which molecules can pass.
Smaller molecules migrate faster across the gel, covering a greater distance than larger fragments, which migrate more slowly and cover a shorter distance. As a result, the molecules are separated according to their sizes.
Gel Electrophoresis and DNA
Electrophoresis allows you to distinguish between different lengths of DNA fragments.
Because DNA is negatively charged, it will migrate to the positively charged electrode when an electric current is applied to the gel.
The fragments are arranged in size order because shorter strands of DNA pass through the gel considerably faster than longer strands.
The DNA on the gel can be seen once it has been separated using dyes, fluorescent tags, or radioactive labels. On the gel, they will appear as bands.
At the same time as the samples, a DNA marker with known length fragments is usually passed through the gel.
In gel electrophoresis DNA molecules migrate from the negatively charged electrode to the positively charged electrode.
We can calculate the length of the DNA fragments in the samples by comparing the bands of the DNA samples with those of the DNA marker.
How is Gel Electrophoresis Carried Out?
Preparing the Gel
Agarose gels are commonly used to recognize DNA fragments. The amount of agarose in the gel is determined by the size of the DNA fragments we are working with. The denser the matrix, the higher the agarose content, and vice versa. Smaller DNA fragments are separated using higher agarose concentrations, whereas larger molecules require a lower agarose concentration.
To make a gel, combine agarose powder with an electrophoresis buffer and heat to a high temperature until the agarose powder has completely melted. The molten gel is then placed on a gel casting tray with a "comb" on one end to produce wells for pipetting the sample into.
The comb is removed once the gel has cooled and set (it will now be opaque rather than transparent). Pre-made gels are now widely used. The gel is then placed in an electrophoresis tank, which is subsequently filled with an electrophoresis buffer until the entire surface of the gel is covered. The electric current is carried by the buffer. The type of buffer used is determined by the size of the DNA fragments present in the sample.
The charged molecules can be separated by an electrical field through a gel that contains small pores.
Preparing the DNA for Electrophoresis
Prior to electrophoresis, a dye is added to the DNA sample to increase its viscosity, stopping it from floating out of the wells and allowing the migration of the sample through the gel to be detected.
In the first well of the gel, a DNA marker (also known as a size standard or a DNA ladder) is added. Because the marker's fragments are of a known length, they may be used to calculate the size of the fragments in the samples.
Pipette the prepared DNA samples into the remaining wells of the gel.
The lid is then placed on the electrophoresis tank, ensuring that the gel and positive and negative electrodes are aligned properly (we want the DNA to migrate across the gel to the positive end).
Separating the Fragments
The electrical current is then turned on, which causes the negatively charged DNA to flow through the gel towards the positive side.
Shorter DNA molecules move quicker than longer strands, the ability to travel further in the time the current is running.
The migration of the loading buffer dye can be used to assess how far the DNA has migrated in the gel.
The electrical current is placed on long enough for the DNA fragments to migrate far enough across the gel to be separated, but not so long that they run off the end.
The Gel Electrophoresis Diagram is represented as follows.
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A gel is contained within a buffer tank. An electrical current is conducted across the gel as the DNA samples are deposited in wells at one end. Negatively charged DNA is attracted to the positive electrode.
Visualizing the Results
The electrical current is turned off and the gel is removed from the electrophoresis tank once the DNA has migrated far enough across the gel.
The DNA is recognized by staining the gel with a fluorescent dye that binds to the DNA and exposing it to a UV transilluminator, which exposes the stained DNA as bright bands.
The colour can also be added to the gel before it is poured.
The banding pattern of the DNA marker/size standard will be visible if the gel has run standard.
The size of the DNA in your sample can then be estimated by imagining a horizontal line running across the bands of the DNA marker. The size of the DNA in the sample can then be estimated by comparing it to the marker's nearest band.
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The lengths of the DNA fragments are compared to a marker that contains known-length fragments.
Did You Know?
Did you know the process of Electrophoresis, which is useful to the students? Let us know about it.
Under the electric fields, it is used to separate charged molecules. This method is most commonly used in molecular biology to separate DNA, RNA, and proteins based on their mass and shape. When we run DNA through electrophoresis, for example, we can differentiate between DNA molecules of various shapes and sizes.
FAQs on Electrophoresis
1. What is electrophoresis and what is its fundamental principle?
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 will migrate through a medium when subjected to an electric field. The medium, typically a gel, acts as a sieve, allowing smaller molecules to move more quickly than larger ones, leading to their separation.
2. Why does DNA move towards the positive electrode during gel electrophoresis?
DNA molecules have a net negative charge. This is due to the phosphate groups that make up the sugar-phosphate backbone of the DNA strand. In an electrophoresis setup, electrodes create a positive and a negative pole. Since opposite charges attract, the negatively charged DNA is pulled through the gel matrix towards the positive electrode (the anode).
3. What are the key components required for a typical gel electrophoresis experiment?
A typical gel electrophoresis setup requires several key components:
- Electrophoresis Chamber (Tank): A plastic tank to hold the gel and buffer solution, with a positive and a negative electrode.
- Gel: A porous matrix, commonly made from agarose or polyacrylamide, that separates the molecules.
- Buffer Solution: A liquid that conducts the electric current and maintains a stable pH.
- Power Supply: Provides the electric current needed to move the molecules.
- DNA Samples with Loading Dye: The samples to be separated, mixed with a dye to make them visible and help them sink into the wells.
- DNA Ladder/Marker: A sample containing DNA fragments of known sizes, used for comparison.
4. How does the concentration of the agarose gel impact the separation of molecules?
The concentration of the agarose gel is crucial because it determines the size of the pores within the gel matrix.
- A high-concentration gel (e.g., 2% agarose) has smaller pores and is better for separating small DNA fragments effectively, as it provides more resistance to their movement.
- A low-concentration gel (e.g., 0.7% agarose) has larger pores, which allows for the efficient separation of large DNA fragments that would struggle to move through a denser gel.
Therefore, the gel concentration must be chosen based on the expected size of the molecules being analysed.
5. What are some important real-world applications of electrophoresis?
Electrophoresis is a vital technique with many important applications in science and medicine. Some examples include:
- DNA Fingerprinting: Used in forensic science to compare DNA samples from crime scenes with suspects.
- Medical Diagnostics: To detect genetic disorders (like sickle cell anaemia) by analysing DNA mutations or to separate proteins in blood samples.
- Biotechnology: To verify the size of DNA fragments after PCR amplification or restriction enzyme digestion in genetic engineering.
- Paternity Testing: To compare the DNA patterns of a child with a potential father.
- Protein Analysis: To separate proteins for study in proteomics research.
6. What is the main difference between gel electrophoresis and capillary electrophoresis (CE)?
The main difference lies in the medium and format of separation. In gel electrophoresis, separation occurs in a slab of gel (like agarose) submerged in a buffer tank. In capillary electrophoresis (CE), separation takes place inside a very thin, buffer-filled silica capillary tube. CE is generally much faster, requires smaller sample volumes, and offers higher resolution, making it a more automated and sensitive technique compared to traditional slab gel electrophoresis.
7. How is the concept of electrophoresis relevant to the study of colloidal solutions?
In the context of surface chemistry, electrophoresis is a key property of colloidal solutions. Colloidal particles acquire an electric charge (either positive or negative) on their surface. When an electric field is applied across a colloidal solution, these charged particles migrate towards the oppositely charged electrode. This movement of colloidal particles under an applied electric potential is defined as electrophoresis, and it serves as definitive proof that colloidal particles are electrically charged.
