What is electrophoresis?
Electrophoresis is a laboratory technique used to separate charged molecules, such as DNA, RNA, or proteins, based on their size and electrical charge, using an electric field and a porous medium (gel) that acts as a sieve.
How does it work?
- The sample is placed in a porous gel (agarose or polyacrylamide).
- An electric current is applied:
- Negative molecules migrate toward the anode (+) and positive ones toward the cathode (-).
- Smaller molecules migrate faster through the gel.
This allows the separation and visualization of DNA, RNA, or protein fragments, which is essential for molecular analysis and diagnostics.
Relevant Types of Electrophoresis
- Agarose gel electrophoresis – Ideal for separating large molecules such as DNA
- Polyacrylamide gel electrophoresis – Higher resolution for smaller molecules (proteins), requires careful handling
- Capillary electrophoresis – Highly efficient, requires less sample and reagents, provides digital results
- Isoelectric focusing and 2D electrophoresis –High-resolution separation, useful in advanced proteomic analysis
Main Applications in Science and Medicine
In Genetics and Molecular Biology
• Genotyping and analysis of genetic variations – Determines variants of specific genes by comparing migration patterns with standard molecular weight markers.
• Diagnosis of genetic diseases – Identification of genetic expansions, for example, trinucleotide repeat disorders.
• Molecular oncology – Monitoring of microsatellite instability in tumors, a key marker in cancer.
In Research and Development
• Verification of PCR and sequencing products – Confirmation of amplification results before sequencing or cloning.
• Proteomics and protein analysis – Characterization of protein expression profiles, purity assessment, and biomarker detection.
Clinical Diagnostics and Health
• Serum protein or hemoglobin profiles for the diagnosis of hematological diseases, such as sickle cell anemia and thalassemias.
• Biomarker evaluation in infections and metabolic conditions.
Forensic Science and Paternity
Electrophoresis is an essential tool for identifying DNA profiles, comparing them with databases, and solving forensic cases or determining parentage.
Practical Example: Analysis of Variants in the PER3 Gene
Step 1: DNA Sample Extraction and Preparation
The analysis of genetic variants by electrophoresis begins with a critical step: DNA extraction and proper sample preparation. In the case of the PER3 gene, a gene associated with circadian rhythms and sleep patterns, the quality of the DNA obtained is essential to ensure reliable results in the later stages of molecular analysis.
Step 2: PCR Amplification and Sample Staining
At this stage, the polymerase chain reaction (PCR) is used to specifically amplify the target fragment of the PER3 gene. This procedure makes it possible to obtain millions of copies of the target DNA from a minimal initial amount, ensuring a detectable and reproducible signal in subsequent analyses.
The correct selection of buffers, dNTPs, primers, enzymes, and reaction conditions is key to achieving efficient and specific amplification, reducing the appearance of nonspecific products.
Step 3: Electrophoresis and Genetic Variant Analysis
Once the DNA has been amplified, the PCR products are analyzed by gel electrophoresis, a technique that separates fragments according to their size and allows differences between samples to be visualized. From the migration patterns obtained, it is possible to identify genetic variants, compare individuals, and validate the amplification results.
The process is carried out as follows:
• Electrophoresis Gel Preparation
An agarose gel is prepared with the appropriate concentration according to the expected size of the PER3 fragment. The correct formulation of the gel is key to obtaining good resolution and separation of DNA bands.
• Use of Running Buffers (TAE / TBE)
Buffers ensure electrical conductivity and pH stability during electrophoresis, preventing distortions in DNA migration and ensuring reproducible results.
• Sample Loading and Electric Field Application
PCR products are loaded into the gel wells together with a molecular weight marker. When the electric field is applied, DNA fragments migrate through the gel according to their size.
• Separation and Resolution of DNA Fragments
Electrophoresis separates the amplified fragments, making it easier to observe the size differences of the DNA between the analyzed samples.
• DNA Band Visualization
Once the run is complete, DNA is visualized using specific stains and appropriate illumination systems, allowing well-defined and clearly contrasted bands to be observed.
• Comparison with the Molecular Weight Marker
The position of the bands is compared with the marker to confirm the size of the amplified PER3 fragment and detect possible variations.
• Identification of Genetic Variants
Differences in the migration pattern allow insertions, deletions, or polymorphisms associated with the PER3 gene to be identified across different samples.
• Comparative Analysis Between Individuals or Groups
The results obtained are compared between samples to evaluate genetic variability, population studies, or associations with specific biological characteristics.
• Process Quality Control
Electrophoresis acts as a verification tool for the complete process, allowing the quality of DNA, PCR efficiency, and reliability of the reagents used to be evaluated.
• Relationship with Laboratory Products and Consumables
This step requires the use of agarose gels, electrophoresis buffers, molecular weight markers, stains, electrophoresis chambers, and documentation systems, all essential to obtain clear and reproducible results.
Step 4: Results Analysis:
Once migration in the gel is complete and the run is stopped, separated bands are observed, representing DNA/RNA fragments or proteins according to the type of electrophoresis performed. These bands are visualized because the molecules have been stained with an agent that allows them to be seen under light, for example, with ethidium bromide or safer alternative stains.
What do the bands mean?
Position in the gel: the distance traveled by each band depends on the size and charge of the molecules. Smaller fragments migrate farther than larger ones.
Comparación con un marcador: Colocar un marcador de peso molecular (o escalera) te permite estimar el tamaño de tus fragmentos comparando la ubicación de tus bandas con la de los estándares conocidos.
Number of bands:
A single band may indicate a single fragment present or no variation in the sample.
Multiple bands suggest that there is more than one fragment or different variants in the sample.
Band intensity: the more intense or thicker the band, the greater the amount of that molecule present.
Source: Genotipia. (s. f.). Electrophoresis: what it is and what it is used for. https://genotipia.com/electroforesis/
