Gel electrophoresis is a widely used laboratory method that separates deoxyribonucleic acid (DNA) fragments. This technique isolates DNA molecules based on their length, providing a way to analyze them. It serves as a fundamental tool in various scientific disciplines, allowing researchers to examine DNA for different purposes, such as understanding genetic variations or purifying specific fragments.
DNA’s Electrical Journey
DNA molecules possess an inherent negative electrical charge, originating from phosphate groups in their sugar-phosphate backbone. DNA samples are loaded into wells at one end of a gel matrix. The gel is then placed within an electrophoresis chamber, which is connected to a power supply.
An electric current is subsequently applied across the gel. This creates an electric field, with a negative electrode positioned near the wells where the DNA samples were loaded and a positive electrode at the opposite end of the gel. Due to its negative charge, the DNA is naturally repelled by the negative electrode and simultaneously drawn towards the positive electrode. This electrical force causes the DNA fragments to migrate through the gel.
The Gel’s Filtering Effect
As DNA fragments move through the gel, they encounter a resistance from the gel’s structure. The gel, typically made of agarose, functions like a microscopic sieve with interconnected pores. These pores act as obstacles, impeding the movement of the DNA molecules. The extent of this hindrance depends on the size of the DNA fragments.
Smaller DNA fragments can navigate through the gel’s pores more easily and thus move faster. Conversely, larger DNA fragments encounter more resistance and are slowed down. This differential movement causes the DNA fragments to separate by size, with smaller fragments traveling further down the gel than larger ones. The agarose gel’s concentration can be adjusted to change pore size, allowing for optimal separation of DNA sizes.
Making DNA Visible
DNA is colorless and cannot be seen with the naked eye after migration. To visualize the separated DNA fragments, the gel is stained with special DNA-binding dyes. A common example is ethidium bromide, which intercalates, or inserts itself, between the base pairs of the DNA. Other safer alternatives, such as SYBR Safe or Gelite Safe, are also widely used.
When the stained gel is exposed to ultraviolet (UV) light, these dyes absorb UV energy and re-emit it as visible light, a process called fluorescence. This makes the DNA fragments appear as distinct bands or lines on the gel. The position of these glowing bands indicates the size of the DNA fragments, with smaller fragments located closer to the positive end of the gel.