Gel electrophoresis is a fundamental laboratory technique used to sort and analyze DNA samples. It relies on applying an electric current to a sample, which forces the molecules to move through a porous gel. Understanding why DNA consistently migrates toward the positive electrode is central to this process.
The Phosphate Backbone: Why DNA Carries a Negative Charge
The directional movement of DNA depends entirely on its inherent electrical charge, which is an intrinsic feature of its molecular construction. DNA is built from repeating units called nucleotides, each containing a sugar, a nitrogenous base, and a phosphate group.
These phosphate groups form the structural backbone of the DNA molecule. The negative charge originates within the phosphate group, where phosphorus atoms are bonded to oxygen atoms. Under the neutral or slightly alkaline pH conditions maintained in a laboratory, the phosphate groups are deprotonated.
Deprotonation means the phosphate group loses a hydrogen ion, leaving a localized negative charge on one of its oxygen atoms. Since this group repeats along the entire length of the DNA strand, the cumulative result is a molecule with a substantial, uniform negative charge, regardless of the fragment’s size or sequence.
Creating the Environment for Electrical Movement
Scientists exploit this fixed negative charge by creating an electrical circuit. The technique requires a power supply to generate an electric field and a buffer solution to conduct the current. This buffer surrounds the gel and the DNA samples, maintaining a stable pH that ensures the phosphate backbone remains negatively charged throughout separation.
When the power is turned on, the current establishes two poles: the negative cathode and the positive anode. DNA samples are placed near the cathode. The fundamental law of electromagnetism—that opposite charges attract and like charges repel—is then put into action.
The negatively charged DNA molecules are repelled by the negative cathode and simultaneously pulled toward the positive anode. This directional force, known as electrophoretic mobility, drives the DNA through the gel. The electric field must be applied at a controlled voltage to ensure a consistent flow.
Utilizing Directed Movement for Analysis
The directed, systematic movement of DNA toward the positive electrode is the foundation for analyzing complex genetic mixtures. Since every DNA fragment has the same charge-to-mass ratio due to the repeating phosphate groups, the electrical force applied to each molecule is proportional to its size. This means the force is applied uniformly per unit of length.
The gel matrix acts as a microscopic, porous network, similar to a sieve. As the DNA is pulled through this mesh by the electric field, the gel imposes resistance based on the fragment’s physical size. Smaller DNA fragments navigate the pores more easily and quickly, experiencing less drag.
Conversely, larger DNA fragments encounter more resistance and are slowed down significantly. This difference in movement rate causes the mixed population of DNA fragments to separate. After the electric current runs for a set period, the molecules are sorted by length, with the shortest fragments traveling the furthest toward the positive electrode. This size-based separation allows researchers to visualize, isolate, and accurately determine the size of specific DNA pieces.