Gel electrophoresis is a fundamental technique in molecular biology used to separate and analyze DNA fragments by size. Understanding how DNA moves through a gel is important for various applications, including genetic research, forensic analysis, and medical diagnostics. The method provides a visual representation of DNA fragments, making it possible to identify specific genetic patterns.
Setting the Stage for Movement
DNA movement during gel electrophoresis relies on several key components. The gel, typically made from agarose, forms a porous, jelly-like matrix when cooled, acting much like a sieve at a molecular level. The concentration of agarose determines the density of this matrix and, consequently, the size of its pores.
The gel is submerged in a buffer solution, which serves two main purposes: maintaining a stable pH and conducting electricity. Common buffers like Tris-borate-EDTA (TBE) or Tris-acetate-EDTA (TAE) ensure the DNA remains stable and the electrical current flows efficiently. DNA molecules possess an inherent negative charge due to the phosphate groups in their backbone. This negative charge is crucial for their directed movement through the gel.
The Electric Push: Why DNA Travels
An electric field drives DNA migration through the gel. The gel is placed within an electrophoresis chamber, which has electrodes at opposite ends. A power supply then applies an electric current across the chamber, creating a positive electrode (anode) at one end and a negative electrode (cathode) at the other. This setup establishes a directional pull for charged molecules.
Because DNA carries a net negative charge, it is repelled by the negative cathode and attracted to the positive anode. This electrical attraction causes the DNA fragments to migrate from the wells, loaded near the negative electrode, towards the positive end of the gel. The strength of the electric field influences the speed at which the DNA moves, with a stronger field generally leading to faster migration.
Navigating the Gel: How Size Matters
As negatively charged DNA fragments are pulled by the electric field, they navigate the gel’s intricate network of pores. The gel acts as a molecular sieve, impeding larger molecules more than smaller ones. This differential resistance is the basis for separating DNA fragments by size. Smaller DNA fragments weave through the gel’s pores more easily and quickly, traveling farther down the gel.
In contrast, larger DNA fragments encounter more obstacles and resistance. They move slower and therefore travel a shorter distance from the loading wells. This phenomenon, sometimes described by models like “biased reptation” where DNA “snakes” through the pores, results in DNA fragments separating into distinct bands according to their length. Adjusting the agarose concentration in the gel directly impacts the pore size; a higher concentration creates smaller pores, which improves the separation of smaller DNA fragments, while a lower concentration is better for separating larger fragments.
Revealing the Invisible Journey
After the electric current is switched off, separated DNA fragments remain invisible. To visualize these bands, the gel is stained with a DNA-binding dye. Common dyes, such as ethidium bromide or SYBR Safe, bind with the DNA molecules. When exposed to UV light, these bound dyes fluoresce, making the DNA bands glow.
The intensity of fluorescence corresponds to the amount of DNA in each band. To determine fragment sizes, a DNA ladder or marker is run alongside the samples. This ladder consists of DNA fragments of known lengths, providing a reference. By comparing the migration distance of the sample bands to the known bands of the ladder, researchers can estimate the size of their DNA fragments.