Gel electrophoresis is a widely used technique for separating DNA fragments based on their size and charge. Here’s how it works, broken down into its underlying principles:
1. Gel Medium
The gel typically used in gel electrophoresis is made from agarose or polyacrylamide. Agarose gels are commonly used for separating larger DNA fragments, while polyacrylamide gels can separate smaller fragments with higher resolution.
2. DNA Charge
DNA molecules are negatively charged due to the phosphate backbone. When an electric field is applied across the gel, the negatively charged DNA fragments migrate toward the positive electrode (anode).
3. Electric Field Application
Once the DNA samples are loaded into wells at one end of the gel, an electric current is applied. The electric field causes the DNA fragments to move through the gel matrix.
4. Size-Based Separation
As the DNA fragments move, they encounter resistance from the gel matrix. The gel’s porous nature allows smaller DNA fragments to navigate through it more easily and quickly than larger fragments. Because of this sieving effect:
- Smaller fragments travel faster and farther through the gel.
- Larger fragments move more slowly and thus travel shorter distances.
5. Visualization
After the electrophoresis run is complete, the separated DNA fragments can be visualized. This is often done using a DNA-binding dye (like ethidium bromide or SYBR Green) that fluoresces under UV light, allowing the DNA bands to be seen.
6. Analysis
The pattern of bands on the gel can then be analyzed and compared against a molecular weight marker or ladder, which consists of DNA fragments of known sizes. This allows the determination of the approximate size of the DNA fragments in the samples based on how far they have migrated through the gel.
Summary
In summary, gel electrophoresis utilizes the negative charge of DNA and the sieving properties of a gel matrix to separate DNA fragments based on size. The application of an electric field causes the DNA to migrate through the gel, with smaller fragments moving faster than larger ones, resulting in a distinct pattern of separated bands that can be visualized and analyzed.