Electrophoretic separation is a common laboratory technique used to sort large biological molecules such as DNA, RNA, and proteins. This method separates molecules based on their movement through a specific medium when an electric force is applied, allowing scientists to distinguish and isolate them.
The Core Principles of Separation
The separation process in electrophoresis relies on two interconnected forces: an applied electric field and a porous gel, often called a sieving matrix. Biological macromolecules, such as DNA and proteins, naturally carry an electric charge. DNA molecules, for instance, possess a consistent negative charge due to the phosphate groups present along their backbone. When an electric field is established across the gel, charged molecules experience a force. Negatively charged molecules move towards the positively charged electrode (anode), while positively charged molecules migrate toward the negatively charged cathode. This electrical force drives molecular movement.
The rate at which a molecule moves is directly influenced by its net charge and the strength of the electric field. A higher net charge generally results in faster migration towards the oppositely charged pole. The gel itself is immersed in a buffer solution, which conducts the electric current and helps maintain a stable pH, preventing changes that could affect molecular charges.
The second component, the gel matrix, acts as a physical obstacle course for the moving molecules. This gel, typically composed of agarose or polyacrylamide, forms a complex network with microscopic pores. As molecules are propelled by the electric field, they must navigate through these intricate pores. Smaller molecules encounter less frictional resistance, allowing them to traverse the gel matrix more easily and thus migrate faster and farther. Conversely, larger molecules face greater resistance from the gel’s dense structure, causing them to move more slowly and remain closer to their starting point. Ultimately, a molecule’s charge, the electric field strength, and its ability to navigate the gel’s pores determine its speed and travel distance, leading to separation by size and charge.
Common Types of Electrophoresis
Agarose gel electrophoresis is a widely used method, primarily for separating larger nucleic acid molecules like DNA and RNA fragments. This technique utilizes a gel made from agarose, a polysaccharide derived from seaweed. Agarose gels have relatively large pore sizes, well-suited for separating DNA fragments ranging from approximately 50 to 20,000 base pairs. The concentration of agarose in the gel can be adjusted; lower concentrations create larger pores for bigger molecules, while higher concentrations create smaller pores for better separation of smaller fragments.
Polyacrylamide gel electrophoresis, or PAGE, is another common method, particularly suited for separating proteins. Polyacrylamide gels are formed by the polymerization of acrylamide and a cross-linking agent, creating a tighter and more uniform pore structure compared to agarose. This finer mesh is effective for resolving smaller protein molecules, ranging from a few kilodaltons to hundreds of kilodaltons. Proteins can be separated based on their native charge and shape in a technique called native PAGE.
A common variation of PAGE is Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, or SDS-PAGE. In SDS-PAGE, protein samples are treated with sodium dodecyl sulfate (SDS), a detergent that denatures proteins, causing them to unfold into linear chains. SDS also coats the proteins with a uniform negative charge, ensuring that their migration rate through the polyacrylamide gel is almost exclusively determined by their molecular size, rather than their original charge or complex shape. This allows for accurate estimation of protein molecular weight.
Capillary electrophoresis (CE) is an automated approach to molecular separation. Unlike slab gels, CE is performed within a narrow, fused-silica capillary tube filled with a buffer solution. This method offers advantages such as higher speed, improved resolution, and reduced sample and reagent consumption. CE can separate a wide range of charged molecules, including DNA, RNA, proteins, and even small ions, based on their electrophoretic mobility, which is influenced by both charge and size.
Visualizing and Interpreting Results
Once the electrophoretic separation is complete, the separated molecules within the gel are typically invisible to the naked eye. To make them apparent, scientists use specific staining techniques. For DNA, common dyes like ethidium bromide or SYBR Green are applied, which bind to the nucleic acid molecules. When exposed to ultraviolet (UV) light, these bound dyes fluoresce, revealing the DNA as distinct bands on the gel.
Similarly, proteins are often stained with dyes such as Coomassie Brilliant Blue or silver stain, which bind to the protein molecules and become visible as colored bands. To interpret the results and determine the approximate size of the separated molecules, a molecular weight marker is run alongside the samples in a separate lane. This marker, often called a “ladder” (e.g., DNA ladder or protein ladder), consists of a mixture of molecules with precisely known sizes.
By comparing the migration distance of the unknown sample bands to the corresponding bands in the ladder, researchers can accurately estimate the size of their target molecules. The final result of an electrophoresis experiment is typically a gel displaying a series of bands. Each band represents a collection of molecules of a similar size that have migrated to the same position. The molecules that traveled furthest from the starting wells are the smallest, while those that remained closer to the origin are the largest. The intensity of a band can also provide an indication of the relative amount of molecules present in that specific size range.
Real-World Applications
In forensics, electrophoretic separation plays a prominent role in DNA fingerprinting. DNA samples collected from crime scenes, such as blood or hair, can be amplified using polymerase chain reaction (PCR) and then separated by gel electrophoresis. The resulting unique banding patterns of DNA fragments can then be compared to those from suspects, providing strong evidence for identification or exclusion in criminal investigations. This method is also employed in paternity testing, where genetic profiles are compared to establish biological relationships.
In medical diagnostics, electrophoresis assists in identifying various diseases and conditions. For example, it is used to detect abnormal proteins in blood or urine, which can indicate conditions like multiple myeloma or other protein-related disorders. The technique can also help diagnose genetic disorders by revealing specific DNA mutations or abnormal protein variants, such as in the case of sickle cell anemia where an altered hemoglobin protein can be identified. Capillary electrophoresis, with its high resolution, is increasingly used for rapid detection of illicit substances in toxicology and for characterizing microorganisms in infectious disease diagnosis.
Beyond diagnostics, electrophoretic separation is a key tool in scientific research, particularly in molecular biology. Researchers routinely use it to verify the success of molecular cloning experiments by confirming the size of inserted DNA fragments. It is also used to analyze PCR products, ensuring the correct DNA regions have been amplified. Electrophoresis was also used in the early stages of large-scale projects like DNA sequencing, providing a means to separate and analyze DNA fragments.