Gel electrophoresis is a fundamental laboratory technique used to separate biological molecules, particularly DNA fragments, based on their size. When a mixture of DNA fragments is placed into a gel and an electrical current is applied, the fragments separate. The core principle of the technique is that shorter DNA strands consistently travel faster and farther through the gel matrix than longer strands.
The Driving Force Behind DNA Movement
The movement of DNA through the gel is driven by an electrical force, which exploits the molecule’s natural negative charge. Deoxyribonucleic acid (DNA) possesses a strong uniform negative charge along its entire length due to the phosphate groups in its sugar-phosphate backbone.
When the DNA samples are loaded into the gel and an electric field is activated, the negatively charged DNA is repelled by the negative electrode (cathode) and attracted toward the positive electrode (anode). This creates a constant, directed force that pulls all the DNA fragments through the gel, regardless of their size. All linear DNA fragments have a nearly identical ratio of charge to mass, meaning the electrical field pushes every fragment with the same relative force.
The Gel Matrix Acts as a Molecular Sieve
The size-based separation occurs because the gel acts like a microscopic sieve or mesh. The gel, typically made of agarose or polyacrylamide, is a porous network of tangled polymer fibers that create tiny, interconnected channels. The concentration of the gel material determines the average size of these pores, which directly impacts DNA migration.
As the DNA molecules are pulled by the electric field, they must navigate through this dense network of pores. Smaller DNA fragments encounter less physical resistance from the gel fibers. They can easily slip through the pores and channels, resulting in rapid movement through the matrix.
The movement of DNA is described by the “biased reptation” model, which explains the molecule’s snake-like motion. The electric field pulls the leading edge of the DNA through a pore, and the rest of the molecule follows that path. Shorter fragments complete this snaking process quickly and repeatedly, allowing them to cover more distance in a given time.
Longer DNA fragments face significantly more resistance because their bulk makes threading through the small pores difficult. These larger molecules frequently become entangled in the polymer fibers, forcing them to reorient themselves before finding a path forward. This constant need to untangle and reorient causes a substantial delay, slowing their net migration speed.
Practical Uses of Size Separation
Forensic Science
In forensic science, this technique is employed for DNA fingerprinting. Specific regions of DNA are amplified and then separated to create a unique size pattern that can be matched to evidence from a crime scene. This size separation is used for identifying individuals.
Medical Diagnostics
Size separation is used to identify specific genetic mutations or variations associated with disease. Researchers can use restriction enzymes to cut a gene of interest; if a mutation is present, the resulting DNA fragment sizes will differ when analyzed on the gel. This difference in fragment length indicates the presence of an abnormal gene sequence.
Basic Research
Basic research laboratories use this method to analyze experimental products. This includes determining the size of DNA pieces after cutting them with restriction enzymes, known as restriction mapping. It is also used to verify that the Polymerase Chain Reaction (PCR) successfully produced the target DNA fragment of the correct size. By running a DNA ladder, which contains fragments of known sizes, alongside the unknown samples, scientists can precisely estimate the size of every separated fragment.