DNA gel electrophoresis is a fundamental laboratory technique used to separate and analyze DNA fragments based on their size. The process involves placing a DNA sample into a porous gel matrix and applying an electric current, which causes the negatively charged DNA molecules to move. Interpreting the resulting patterns of bands on the gel is the final, most informative step of the procedure. This reading process allows scientists to determine the length of the DNA, assess the success of molecular biology experiments, and draw biological conclusions.
What the Gel Reveals
The gel itself is a slab of agarose, a purified sugar polymer that acts like a molecular sieve. Samples are loaded into small indentations at one end of the gel called wells. Because DNA has a negative charge due to its phosphate backbone, it migrates toward the positive electrode when the current is applied.
The movement of the DNA fragments through the agarose matrix is inversely proportional to their size. Smaller fragments move quickly and travel farther down the gel, while larger fragments encounter more resistance and remain closer to the wells. After the process is complete, the separated DNA is stained with a fluorescent dye. The final result is a visual pattern of distinct, glowing bands under ultraviolet light, where each band represents a collection of DNA fragments of approximately the same length.
Using the DNA Ladder to Determine Size
Determining the length of the separated DNA fragments is accomplished by comparing them to a reference tool called a DNA ladder, or molecular weight marker. The ladder is a mixture of DNA fragments of known, pre-determined sizes, which is loaded into one of the gel’s lanes alongside the experimental samples. When the gel is run, the ladder separates into a series of distinct bands, each corresponding to a specific length, such as 100, 500, or 1,000 base pairs (bp).
To estimate the size of an unknown fragment, one visually compares its final resting position on the gel to the bands of the ladder in the adjacent lane. If a sample band aligns horizontally with a band in the ladder, its size is considered to be the same as that known marker. For fragments that fall between two ladder bands, their size is estimated by interpolation. This comparison is performed on the same gel because slight variations in running conditions can affect the speed of migration, making the ladder an individualized internal standard for every experiment.
Interpreting Sample Results
Interpreting the overall pattern of the bands reveals the success and quality of the underlying experiment, beyond merely calculating fragment size.
Presence and Absence
The presence or absence of a band at a specific, expected size is often the most direct outcome, signifying whether a targeted genetic sequence was successfully amplified or cut. For example, in a polymerase chain reaction (PCR) experiment, a band at the predicted length confirms that the target DNA sequence was present and multiplied.
Intensity
The intensity of a band, or how brightly it glows, provides an estimate of the quantity of DNA present in that fragment size. Brighter bands indicate a higher concentration of DNA molecules have collected at that position, while faint bands suggest the presence of smaller amounts. This relative intensity is sufficient for drawing conclusions about gene expression levels or the efficiency of a reaction.
Appearance and Quality
The physical appearance of the bands also speaks to the quality of the sample and the procedure. A single, sharp, and well-defined band suggests the sample is homogenous and the DNA is intact. Conversely, a blurred or elongated band, known as a smear, often indicates that the DNA has been degraded into many random sizes, or that the sample was overloaded. The presence of extra, unexpected bands may point to non-specific amplification or contamination, which requires careful comparison against control samples.
Where Gel Electrophoresis Readings Are Used
The results of gel electrophoresis are applied across many scientific disciplines whenever the size or purity of DNA needs to be verified.
In molecular cloning and genetic engineering, the technique is used to confirm that a desired gene fragment has been successfully cut from a source and inserted into a circular DNA carrier, known as a plasmid. Scientists check for bands matching the predicted lengths of the cut vector and the inserted gene to verify the experiment’s success.
In the field of forensics, gel electrophoresis provides the foundational separation step for DNA profiling, such as in paternity testing or crime scene investigation. By comparing the banding patterns of short tandem repeat (STR) fragments from a suspect’s sample to those from the crime scene, analysts can establish a genetic match or exclusion. Although modern forensic labs often use more sensitive capillary electrophoresis, the principle of separating fragments by size to create a unique genetic profile remains the core concept. This separation method is also used in medical diagnostics to detect specific genetic variations or to analyze the products of gene expression studies.