Fragment patterns and molecular genotypes
One application of gel electrophoresis in DNA analysis is that it can reveal an individual’s genotype at a specific genetic locus. In this case, the DNA segments loaded into a sample well are copies of the DNA from one chromosomal region or locus from a single individual. In most applications, the DNA segment copies are made with the use of the PCR technique. In other applications (i.e. Southern blotting), these segments are detected among thousands of different length DNA segments that have been run through the gel. Molecular genotypes can be inferred from the fragment banding pattern observed if one keeps in mind what is happening at the molecular level.
We will describe the molecular basis of a genotype using a diploid organism as an example. In any tissue source from which we obtain DNA, this organism will have two copies of every chromosome and thus two copies of each gene on those chromosomes. If copies are made of the DNA at a specific locus, half of these copies in our sample will originate from the locus of one chromosome and the other half from the homologous chromosome. We can use electrophoresis to observe these DNA segments and determine if the organism was homozygous or heterozygous with respect to the DNA at that locus. Homozygous individuals will have the same DNA sequence at this locus in both chromosomes. Heterozygous individuals will have two different versions of this DNA. If the DNA sequence differences that occur result in a longer DNA segment from one chromosome (Fig. 21, lane 1) and a shorter segment from the other (Fig 21, lane 2), then we can visualize this difference in the electrophoresis banding pattern observed in the gel. If an individual is homozygous at the locus being analyzed, both chromosomes have the same DNA (Fig 21 lanes 1 and 2). If an individual is heterozygous at the locus, the DNA segment copies from one chromosome will be different than the DNA segment copies from the homologous chromosome (Fig. 21, lane 3). We will therefore see more DNA bands in our gel from a heterozygote.
Fig. 21. The genotype of an individual at a specific locus can be inferred from a fragment banding pattern. (Image by D. Lee)
The molecular genotypes just described are illustrated in the animation. Sample A or sample B would be from individuals that are homozygous and sample A & B would be from a heterozygous individual. In this case, one version of this locus has two smaller segments of DNA and the other version has one longer segment. Therefore, the heterozygous sample contains all three DNA segments. One segment is observed in one of the homozygous types and the other two DNA segments observed in the other homozygous type. The DNA sequence differences that result in the DNA segment lengths are described in another lesson.