Once DNA has been extracted amplified and then separated according to allele, it must be detected in order to yield useful information to the forensic scientist. Before separation the mixture of DNA molecules was treated with a mixture of colored fluorescent dyes. These dyes are specially designed each to attach to the primers that amplify DNA fragments from particular loci each dye is colored differently and in this way the DNA fragments from one locus can be distinguished from another locus's DNA fragments, even if the two sets of fragments are otherwise similar. Note that when a dye's color is mentioned it is not actually the visual color of the dye that is being referred to but rather the color of light that the dye will give off when hit with a laser of a particular wavelength.
Although there are a couple of different methods of separating detecting and analyzing a DNA sample, both the underlying principle and the resultant output remain the same. This example will reference an ABI PRISM 310 Genetic Analyzer, which uses capillary electrophoresis to separate the DNA move the mouse cursor over different components of the analyzer to read about their functions.
After being extracted amplified and died the DNA sample is injected into what is referred to as the top end of the capillary at this point the various alleles in the sample are jumbled and cannot be distinguished from one another.
The capillary is a thin metal tube filled with a chemical matrix to which an electric current is applied. This current pulls the DNA fragments through from one end to the other but the capillary is designed so that larger fragments encounter more resistance and take longer to pass through than do the smaller fragments.
Once the DNA fragments become separated, the dyes attached to them must be detected which requires that they be caused to fluoresce; this is done by shining a laser through the fragments as they arrive at the bottom end of the capillary. In the case of the ABI 310 an argon ar plus laser is used with a wavelength of 488 nanometers a CCD or charge coupled device panel will electronically detect the intensity of any light given off by the fluorescent dyes and is arranged so as to differentiate one color from another depending on the angle at which the light exits the prism.
The CCD panel does not actually tell what color a light is per se, instead any light given off by the fluorescent dyes will pass through a prism and will exit the prism at a particular angle depending on its color. This way the CCD panel can determine the color of the light based on where it lands after exiting the prism. The data from the CCD panel is interpreted and displayed by a computer in the form of a four-color electropherogram.
After the mixture of DNA fragments is injected into the capillary and the electric current is applied they will start to separate with the shortest ones traveling fastest and most freely. The shortest DNA fragment exits the capillary first and passes through the laser beam; this causes the dye to give off light, in this case blue, that light is angled by the prism toward the blue detector on the CCD panel which registers the fluorescence and sends data to the computer where it is represented as a peak on an electropherogram since there was only a small amount of DNA of this fragment length a weak fluorescence was created and only a small peak is drawn.
The second shortest fragments exit the capillary next. A similar process occurs with these fragments except that these are bonded with a dye that fluoresces red so the prism angles the light toward the red detector. Also there is much more genetic material of this length so the light is much stronger and a much taller peak is created on the electropherogram.
The process continues similarly as progressively larger DNA fragments proceed out of the bottom end of the capillary each time the prism angles the fluoresced light toward the appropriate detector on the CCD panel and a peak is created on the electropherogram based on the brightness and color of the light.
Once all of the fragments have exited the capillary the analyst has an electropherogram from which information about the DNA samples profile can be determined.
