Electrophoresis

Definition: Electrophoresis is a laboratory technique used to separate and analyze charged molecules, such as DNA, RNA, and proteins, based on their size and charge. It works on the principle that charged molecules will migrate in an electric field towards the opposite charge.

Classification:

Gel electrophoresis can be classified in two types based on the criteria of "the type of gel used".

a. Agarose Gel Electrophoresis: 🙋

Purpose: Typically used for separating larger molecules such as DNA fragments and RNA.
Gel Composition: Agarose gels are composed of a matrix made from agarose, a polysaccharide derived from seaweed. The pore size of agarose gels can be adjusted by changing the agarose concentration to accommodate different sizes of molecules.

b. Polyacrylamide Gel Electrophoresis (PAGE): 🙋

Principle

The principle of agarose gel electrophoresis is grounded in the molecular properties of nucleic acids (DNA and RNA) and their interaction with an electric field within a gel matrix. Here’s a deeper look into the working principle:

1. Molecular Movement in Electric Field:

Charge: DNA and RNA molecules are negatively charged due to the phosphate groups in their backbone.
Electric Field Application: When an electric current is applied across the agarose gel (from negative to positive electrodes), the negatively charged nucleic acid molecules migrate towards the positive electrode (anode).
Migration Rate: The rate of migration depends on the size and shape of the molecule. Smaller molecules move faster through the gel matrix, while larger molecules move more slowly.

2. Sieving Effect of the Gel Matrix:

Gel Structure: Agarose gel is a three-dimensional matrix of long-chain polysaccharides derived from seaweed.
Pore Size: The agarose gel matrix contains pores of varying sizes, depending on the concentration of agarose used. Higher agarose concentrations create smaller pores, restricting the movement of larger molecules more effectively.
Size-Based Separation: During electrophoresis, smaller DNA or RNA fragments can more easily navigate through the gel’s pores, traveling farther from the point of origin (loading well), whereas larger fragments are hindered and remain closer to the origin.

3. Visualization and Analysis:

Staining: After electrophoresis, the DNA or RNA bands within the agarose gel are made visible by staining with a fluorescent dye (e.g., ethidium bromide). This dye intercalates between the base pairs of nucleic acids.
UV Light Exposure: The stained DNA or RNA bands fluoresce under UV light, allowing for visualization and photography.
Band Analysis: The position of each band on the gel correlates with the size of the nucleic acid molecules. Comparing the bands against size markers (DNA ladders) allows researchers to estimate the sizes of unknown DNA or RNA fragments.

Application of Agarose Gel Electrophoresis:

Agarose gel electrophoresis is a versatile and widely used technique in molecular biology and biochemistry. Its applications span various fields, each leveraging its ability to separate nucleic acid molecules based on size. Here are some key applications:

1. DNA Fragment Analysis:

PCR Product Analysis: After polymerase chain reaction (PCR), agarose gel electrophoresis is used to verify the success of amplification, confirming the presence and size of the PCR products.
Restriction Digestion Analysis: It helps analyze DNA fragments generated by restriction enzyme digestion, verifying the efficiency of enzymatic cleavage and ensuring the correct sizes of digested fragments.
Genotyping: Used in genetic studies to identify genetic variations (polymorphisms) by separating DNA fragments that differ in size due to sequence differences.
DNA Cloning: Essential for verifying the presence and size of cloned DNA fragments during recombinant DNA technology.

2. RNA Analysis:

RNA Integrity Assessment: Agarose gel electrophoresis is employed to assess the integrity of RNA samples, ensuring that they are intact and suitable for downstream applications such as gene expression studies (e.g., RT-PCR).
Northern Blot Analysis: Used to detect and analyze specific RNA molecules by transferring RNA from the agarose gel to a membrane for hybridization with labeled probes.

3. DNA Purification and Size Fractionation:

DNA Size Selection: In molecular biology experiments, researchers use agarose gel electrophoresis to select specific size ranges of DNA fragments for subsequent applications, such as sequencing or cloning.
Plasmid DNA Purification: Separates plasmid DNA from genomic DNA or RNA contaminants in plasmid purification protocols.

4. Forensic and Diagnostic Applications:

DNA Fingerprinting: Agarose gel electrophoresis is crucial in forensic science for DNA profiling and identifying individuals based on unique patterns of DNA fragments.
Genetic Disease Diagnosis: Used in clinical diagnostics to detect genetic mutations or variants by analyzing DNA fragments associated with specific genetic disorders.

5. Environmental and Microbiological Studies:

Microbial Community Analysis: Helps characterize microbial communities by separating and analyzing DNA fragments (e.g., 16S rRNA gene amplicons) from environmental samples.
Viral DNA/RNA Detection: Used in virology to detect and analyze viral genomes or RNA fragments in clinical or environmental samples.