How Does Gel Electrophoresis Work in Physics?
Gel Electrophoresis is made of two words, viz: Gel and Electrophoresis.
Here, Gel is semi-solid. They are colloids or the aggregates of fine particles dispersed in a continuous medium. A medium in which the liquid remains viscous and behaves more or less like a solid or semi-solid.
However, Electrophoresis is the motion of charged particles in a gel/fluid under the influence of an applied electric field.
Gel Electrophoresis is a method of separating DNA fragments, macromolecules like proteins, and RNA, according to their size and charge.
This page explains to you what gel electrophoresis is. Also, how DNA/RNA fragment separation occurs in detail.
Introduction to Gel Electrophoresis
Assume you have quite recently done a PCR (Polymerase chain reaction), making numerous duplicates of an objective DNA region.
On the other hand, you've done some DNA cloning, attempting to "glue" a quality into a round DNA plasmid.
Presently, you need to check and see whether your PCR worked, or whether your plasmid has the right gene in it. What strategy would you be able to use to picture (straightforwardly notice) the parts of DNA?
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Here, we approach it with a technique called Gel Electrophoresis.
Do you know what gel electrophoresis is? If not, let’s understand it in brief.
What is Gel Electrophoresis?
Electrophoresis involves passing a current through a gel containing the DNA molecules.
Based on their size and charge, the molecules travel through the gel in varying directions or at different speeds. This allows them to separate from one another.
Also, all the DNA fragments carry the same amount of charge per mass.
Therefore, gel electrophoresis of DNA fragments separates them on the basis of their size only.
Using electrophoresis, we can ascertain how many different DNA fragments are present in a sample.
Further, we can see how large they are in comparison with one another.
Furthermore, we can also determine the absolute size of a DNA piece by investigating it next to a standard "yardstick" made up of DNA fragments of known sizes.
How Electrophoresis Works?
In a technique of gel electrophoresis, we load a DNA or macromolecule-like RNA or proteins sample into pocket-sized indentations or wells at one end of a gel. After this, we supply an electric current to pull them through the gel.
Since DNA fragments are negatively charged, they move towards the positive electrode of the gel box.
However, in comparison to the large fragments, the small fragments move faster through the gel.
When a gel completely stains with a DNA-binding dye, the DNA fragments are visible as bands. Here, each band represents a group of DNA fragments of the same size.
Now, let us understand how electrophoresis apparatus works in DNA fragment separation:
Gel Electrophoresis Apparatus
Firstly, we add gel to a gel box.
Secondly, we affix one end of this box to a positive electrode and another to a negative electrode.
Thirdly, we fill the main body of the box (a place filled with a gel) with a current-conducting salt-containing buffer solution.
Additionally, we position the end of the gel with wells towards the negative electrode.
However, we position the ends without wells toward the positive electrode. It is the end where DNA fragments will migrate.
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How Do DNA Fragments Move?
After supplying the power to the gel box, the current begins to flow through the gel.
The fragments of DNA molecules possess a negative charge due to which the phosphate groups in their sugar-phosphate backbone start moving through the matrix of the gel towards the positive pole.
After the power is on, the current passing through the gel, keeps the gel running.
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As the gel keeps on running, smaller pieces of DNA travel faster through the pores of the gel matrix than longer ones.
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The movement of gel continues and the shortest pieces of DNA reach close to the positive end of the gel.
However, the longest pieces of DNA persist near the wells.
Even so very small pieces of DNA run right off the end of the gel if we keep the power on for a long period.
How DNA Fragments Look Like After Separation?
After the DNA fragment separation is complete, we can examine the gel and also see what sizes of bands are found on it.
When a gel stains with a DNA-binding dye, we place it under UV light. Under the UV light, the DNA fragments shine.
These shining DNA fragments are present at different locations along the line of the gel.
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A band in the above diagram (b) is a well-defined “line” of DNA on a gel.
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Here, each band comprises several same-sized DNA fragments that have all traveled as a group to the same position.
A single or a small group of DNA fragments are not easily visible on a gel.
By comparing the bands (from Diagram. b) in a sample to the DNA ladder ( Diagram. a), we can estimate their approximate sizes.
For instance, the bright band of the DNA molecule on the gel is around 700700700 base pairs (bp) in size.
Examples of Gel Electrolysis
TBE and Denaturing PAGE (polyacrylamide gel electrophoresis) - For RNA separation
TAE Agarose Gel Electrophoresis - For DNA separation
SDS PAGE - Denaturing gel electrophoresis - For protein identification & separation.
FAQs on Gel Electrophoresis: Definition, Steps & Applications
1. What is gel electrophoresis and what is its basic principle?
Gel electrophoresis is a laboratory technique used to separate macromolecules like DNA, RNA, and proteins. Its principle is based on separating charged molecules in an electric field. A gel matrix, typically made of agarose or polyacrylamide, acts as a molecular sieve. When an electric field is applied, molecules move through the gel at different speeds based on their size, shape, and charge. Smaller fragments navigate the pores of the gel more easily and travel farther than larger fragments.
2. What are the key steps involved in performing a gel electrophoresis experiment?
The key steps for performing gel electrophoresis, particularly for DNA fragments, are as follows:
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 to create wells.
Sample Loading: DNA samples, mixed with a loading dye, are carefully pipetted into the wells of the solidified gel. A DNA ladder (a mix of DNA fragments of known sizes) is also loaded into one well for reference.
Electrophoresis: 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) toward the positive electrode (anode).
Staining and Visualisation: After the run, the gel is stained with a fluorescent dye (like Ethidium Bromide) that binds to DNA. The separated DNA bands are then visualised under UV light.
3. How do the principles of physics explain the movement of DNA in gel electrophoresis?
The movement of DNA in gel electrophoresis is governed by fundamental principles of electricity and mechanics. DNA molecules possess a net negative charge due to their phosphate backbone. According to the principles of electrostatics, when an electric field (E) is applied across the gel, a force (F = qE, where 'q' is the charge of the DNA) acts on the molecules. This force drives the negatively charged DNA towards the positive terminal (anode). The gel matrix provides a frictional or resistive force that opposes this movement, with the resistance being greater for larger molecules, causing them to move slower.
4. What are some important applications of gel electrophoresis in science and medicine?
Gel electrophoresis is a cornerstone technique with many vital applications, including:
DNA Fingerprinting: Used in forensic science to compare DNA samples from a crime scene with suspects.
Medical Diagnostics: Helps in detecting genetic disorders by analysing gene mutations or identifying specific DNA patterns associated with diseases.
Verification of PCR Products: After a Polymerase Chain Reaction (PCR), gel electrophoresis is used to check if the desired DNA fragment was successfully amplified and is of the correct size.
Protein Analysis: Techniques like SDS-PAGE are used to separate proteins based on their molecular weight, which is crucial in proteomics and disease research.
Paternity Testing: Comparing DNA banding patterns between a child and a potential father.
5. How does the concentration of the agarose gel affect the separation of DNA fragments?
The concentration of the agarose gel is a critical factor that determines its resolving power. It directly affects the pore size of the gel matrix. A high concentration of agarose (e.g., 2%) creates a gel with smaller pores, which is ideal for separating small DNA fragments effectively. Conversely, a low concentration of agarose (e.g., 0.7%) results in a gel with larger pores, which provides better resolution and separation for large DNA fragments. Therefore, the choice of gel concentration depends on the size range of the DNA fragments being analysed.
6. What is the difference between agarose gel and polyacrylamide gel (PAGE)?
The primary difference between agarose and polyacrylamide gels lies in their resolving power and the types of molecules they are used to separate. Agarose gels have a lower resolving power and are used to separate larger macromolecules, typically DNA fragments ranging from a few hundred to thousands of base pairs. Polyacrylamide gels (PAGE) have a much higher resolving power due to their smaller and more uniform pore sizes. They are ideal for separating smaller molecules like small DNA fragments (under 500 bp) and proteins with high precision.
7. Besides molecular size, what other factors can influence the migration of DNA in a gel?
While molecular size is the primary factor, several other variables can influence DNA migration in a gel:
DNA Conformation: The shape of the DNA molecule matters. For instance, a tightly coiled supercoiled plasmid moves faster than its linear or open-circular forms, even if they have the same molecular weight.
Applied Voltage: Increasing the voltage will speed up the migration of DNA. However, excessively high voltages can generate heat, potentially melting the gel or causing DNA bands to become smeared and distorted.
Buffer Composition: The type and concentration of the electrophoresis buffer (e.g., TAE or TBE) affect the conductivity and pH, which in turn influences the mobility of the DNA.
