DNA gel electrophoresis is a laboratory technique used to separate and analyze DNA fragments. This process relies on fundamental properties of DNA and electricity to sort molecules by size. It enables scientists to visualize DNA, making it a foundational method in molecular biology.
Underlying Scientific Concepts
DNA molecules possess a net negative charge due to the phosphate groups in their sugar-phosphate backbone. Each phosphate group carries a negatively charged oxygen atom at physiological pH, which contributes to the overall negative charge of the entire DNA strand. When an electric current is applied, these negatively charged DNA fragments are repelled by the negative electrode and drawn towards the positive electrode.
The separation of DNA fragments occurs within a gel matrix, which functions much like a sieve. Common gel materials include agarose, a polysaccharide derived from seaweed, or polyacrylamide. As DNA fragments move through the gel, they navigate a network of microscopic pores. Smaller DNA fragments can pass through these pores more easily and quickly, experiencing less resistance. Conversely, larger fragments encounter more friction and resistance from the gel matrix, causing them to move at a slower pace.
A power supply connected to the gel apparatus creates an electric field, with one end of the gel having a negative charge and the other a positive charge. The rate of migration is directly influenced by the strength of the electric field and the concentration of the gel, which determines its pore size.
The Step-by-Step Procedure
The process begins with preparing the gel, typically made from agarose powder dissolved in a buffer solution. The mixture is heated until the agarose fully dissolves, then poured into a casting tray where it solidifies. A comb is inserted into the molten gel, creating small indentations called wells as the gel cools and solidifies. These wells will hold the DNA samples. Gel concentration can be adjusted; higher concentrations create smaller pores, ideal for separating smaller fragments.
Once the gel has set, the comb is removed, and the gel is placed into an electrophoresis chamber. This chamber is filled with a buffer solution that conducts the electric current and maintains a stable pH during the process. DNA samples, often mixed with a loading dye to make them visible and heavier for easy loading, are then carefully pipetted into the wells. A DNA ladder, containing fragments of known sizes, is loaded into one of the wells to serve as a reference for size comparison.
The negative electrode is positioned at the end of the gel where the samples were loaded, while the positive electrode is at the opposite end. The current is allowed to run for a specific duration, ensuring adequate separation of the DNA fragments based on their size.
After the DNA fragments have sufficiently separated, the electric current is turned off. Since DNA is not visible to the naked eye, the gel is stained with a fluorescent dye that binds to DNA. Common stains like ethidium bromide or safer alternatives such as SYBR Gold or SYBR Green are used. When exposed to ultraviolet (UV) light, the bound dye fluoresces, making the DNA fragments visible as distinct bands within the gel.
Practical Uses
In forensic science, DNA gel electrophoresis is a tool for DNA fingerprinting, where unique DNA profiles can be created from crime scene samples. This allows for the comparison of DNA evidence to suspect profiles, aiding in criminal investigations. The distinct banding patterns generated help establish connections or rule out individuals based on their genetic makeup.
The technique is also regularly employed in paternity testing. By comparing the DNA banding patterns of a child with those of the alleged father and the mother, genetic relationships can be determined.
In medical diagnostics, gel electrophoresis helps identify genetic mutations or pathogens. For example, it can detect specific gene deletions or duplications associated with inherited disorders, or identify viral and bacterial DNA in patient samples for infectious disease diagnosis. Researchers use it to analyze gene expression by examining RNA molecules, verify the integrity and size of DNA fragments in cloning experiments, and detect genetic variations or mutations in DNA samples. It is also used to assess the purity and quantity of DNA samples, which is valuable before proceeding with other molecular biology experiments like sequencing.
Understanding the Outcome
When a gel is stained and viewed under UV light, the separated DNA fragments appear as distinct “bands.” Each band represents a collection of DNA fragments of approximately the same size that have migrated to the same position in the gel. A single DNA fragment or a very small group would not be visible, so each band indicates a significant quantity of DNA.
The position of a band within the gel directly indicates the size of the DNA fragment it contains. Therefore, bands closer to the bottom of the gel represent smaller DNA molecules, while those closer to the top are larger ones.
To accurately determine the size of unknown DNA fragments, their bands are compared to the DNA ladder run in an adjacent lane. The DNA ladder consists of a mixture of DNA fragments of known lengths, creating a series of distinct bands with established sizes. By visually aligning the unknown sample’s bands with the nearest bands in the ladder, researchers can estimate the approximate size of their DNA fragments. The intensity or brightness of a band can also provide a rough estimation of the amount of DNA present, with brighter bands indicating more DNA.