Genotype refers to the specific genetic makeup of an organism, representing the two versions of a gene, or alleles, inherited from its parents. Determining this internal genetic code often requires visualizing the physical differences between those alleles. Gel electrophoresis is a standard laboratory technique used to separate and analyze macromolecules, such as DNA, based on their size and electrical charge. By combining genetic processing steps, such as Polymerase Chain Reaction (PCR), with this separation method, the visible result on the gel allows scientists to directly deduce the underlying genotype. The visual pattern of DNA fragments on the gel serves as a unique fingerprint for an individual’s genetic status at a targeted location in the genome.
Principles of DNA Fragment Separation
The mechanism behind gel electrophoresis relies on the physical properties of DNA and the structure of the gel matrix. The gel itself, often made from a seaweed derivative called agarose, acts like a molecular sieve with a network of pores. These pores allow DNA fragments to navigate through the substance when an electrical field is applied.
DNA molecules possess a negative charge due to the phosphate groups in their sugar-phosphate backbone. When the gel is placed in a chamber with a buffer solution and an electrical current is applied, the negatively charged DNA is pulled toward the positive electrode, or anode, at the opposite end. This movement through the gel matrix is called migration.
The speed and distance a DNA fragment travels are determined primarily by its length. Shorter fragments encounter less resistance from the dense network of the gel and therefore move faster and farther through the matrix. Conversely, longer fragments are hindered by the pores and migrate more slowly, remaining closer to the starting point.
To establish a reference point for size, a DNA ladder or marker is run alongside the samples in one lane of the gel. This ladder consists of a mixture of DNA fragments of known lengths, often measured in base pairs. By comparing the distance traveled by an unknown sample band to the bands of the ladder, researchers can accurately estimate the precise size of the DNA fragments under investigation.
Relating Allele Size to Band Position
The interpretation of genotype begins with understanding how the two alleles an individual possesses are converted into DNA fragments of differing lengths. Before the gel is run, a specific region of the DNA, or locus, is typically targeted and amplified, often using PCR. Genetic variations, such as a small insertion or deletion of base pairs, can exist between the two alleles at this locus.
When the two alleles have different lengths, the PCR process yields two distinct sizes of DNA fragments from that individual. For example, one allele might contain a 100 base pair insertion that the other does not, resulting in fragments of 500 base pairs and 600 base pairs, respectively. The position where a band settles on the gel is thus a direct representation of the specific length of the allele-derived fragment.
Alternatively, some methods use restriction enzymes, which are proteins that cut DNA only at specific recognition sequences. If one allele contains the recognition site and the other has a mutation that eliminates it, the enzyme will cut the two alleles differently. This results in one allele yielding a single, longer fragment, while the other is cut into two or more shorter fragments, with the band position directly proportional to the fragment size.
Decoding Genotype Patterns on the Gel
The most direct way to determine an individual’s genotype from the gel is by counting the number of distinct bands in a sample lane. The pattern of bands reveals whether the individual is homozygous, possessing two identical alleles, or heterozygous, possessing two different alleles. This visual analysis is possible because the DNA fragments of different sizes separate completely during the electrophoretic run.
An individual who is homozygous for a particular genetic marker will produce two copies of a fragment that are the exact same length. Since the two identical fragments migrate at the same speed, they will settle at the same position on the gel. This merging results in the appearance of only a single, often bright, band for that sample.
In contrast, a heterozygous individual has two alleles that differ in length. Consequently, these two groups migrate to two separate positions on the gel, resulting in two distinct bands in the sample lane.
To assign the actual genotype, the position of each band must be precisely compared to the DNA ladder. If a lane shows two bands, for instance, one aligning with the 500 base pair marker and the other with the 600 base pair marker, the genotype is immediately identified as heterozygous for the 500 bp and 600 bp alleles. If only a single band is present at the 500 base pair position, the genotype is homozygous for the 500 bp allele.
Common Applications of Electrophoresis-Based Genotyping
The ability to translate a visual band pattern into a precise genetic code makes gel electrophoresis a versatile tool across many scientific disciplines.
Applications
Forensic science: Used as a foundational component of DNA fingerprinting to establish unique genetic profiles from biological evidence collected at a crime scene by analyzing multiple variable loci.
Medical diagnostics: Routinely used to identify disease-related alleles. For instance, a genetic disorder caused by a small deletion results in a shorter fragment that is easily separated and visualized.
Paternity testing: Genotypes of the child, mother, and potential father are compared to determine genetic inheritance patterns.
Agriculture and animal husbandry: Helps researchers track specific traits by screening large numbers of plants or animals to identify individuals carrying desired alleles, such as those conferring resistance to disease or improved yield.