0:00 Once DNA has been extracted amplified and then separated according to allele,
0:05 it must be detected in order to yield useful information to the forensic scientist.
0:10 Before separation the mixture of DNA molecules was treated with a mixture of colored fluorescent
0:16 dyes. These dyes are specially designed each to attach to the primers that amplify
0:22 DNA fragments from particular loci each dye is colored differently and in this
0:27 way the DNA fragments from one locus can be distinguished from another locus's DNA fragments,
0:32 even if the two sets of fragments are otherwise similar. Note that when a dye's color is mentioned
0:38 it is not actually the visual color of the dye that is being referred to but rather the
0:43 color of light that the dye will give off when hit with a laser of a particular wavelength.
1:04 Although there are a couple of different methods of separating detecting and analyzing
1:08 a DNA sample, both the underlying principle and the resultant output remain the same.
1:15 This example will reference an ABI PRISM 310 Genetic Analyzer,
1:19 which uses capillary electrophoresis to separate the DNA move the mouse
1:26 cursor over different components of the analyzer to read about their functions.
1:34 After being extracted amplified and died the DNA sample is injected into
1:39 what is referred to as the top end of the capillary at this point the various alleles
1:45 in the sample are jumbled and cannot be distinguished from one another.
1:51 The capillary is a thin metal tube filled with a chemical matrix to which an electric
1:56 current is applied. This current pulls the DNA fragments through from one end to the other
2:01 but the capillary is designed so that larger fragments encounter more resistance
2:05 and take longer to pass through than do the smaller fragments.
2:12 Once the DNA fragments become separated, the dyes attached to them must be detected which requires
2:17 that they be caused to fluoresce; this is done by shining a laser through the fragments as they
2:22 arrive at the bottom end of the capillary. In the case of the ABI 310 an argon ar plus laser
2:30 is used with a wavelength of 488 nanometers a CCD or charge coupled device panel will electronically
2:40 detect the intensity of any light given off by the fluorescent dyes and is arranged so as to
2:46 differentiate one color from another depending on the angle at which the light exits the prism.
2:55 The CCD panel does not actually tell what color a light is per se,
3:00 instead any light given off by the fluorescent dyes will pass through a prism and will exit
3:04 the prism at a particular angle depending on its color. This way the CCD panel can
3:10 determine the color of the light based on where it lands after exiting the prism.
3:17 The data from the CCD panel is interpreted and displayed by a computer
3:21 in the form of a four-color electropherogram.
3:28 After the mixture of DNA fragments is injected into the capillary and the electric current is
3:33 applied they will start to separate with the shortest ones traveling fastest and most freely.
3:44 The shortest DNA fragment exits the capillary first and passes through the laser beam;
3:49 this causes the dye to give off light, in this case blue,
3:54 that light is angled by the prism toward the blue detector on the CCD panel which registers the
3:59 fluorescence and sends data to the computer where it is represented as a peak on an electropherogram
4:06 since there was only a small amount of DNA of this fragment length
4:10 a weak fluorescence was created and only a small peak is drawn.
4:18 The second shortest fragments exit the capillary next. A similar process occurs
4:24 with these fragments except that these are bonded with a dye that fluoresces red so the prism angles
4:29 the light toward the red detector. Also there is much more genetic material of this length
4:36 so the light is much stronger and a much taller peak is created on the electropherogram.
4:44 The process continues similarly as progressively larger DNA fragments
4:48 proceed out of the bottom end of the capillary
4:51 each time the prism angles the fluoresced light toward the appropriate detector on the CCD panel
4:57 and a peak is created on the electropherogram based on the brightness and color of the light.
5:09 Once all of the fragments have exited the capillary the analyst has an electropherogram
5:14 from which information about the DNA samples profile can be determined.
