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 Animation on how an electropherogram is generated

August 5, 2020
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          Transcript
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.
    
  
Disclaimer:
              Opinions or points of view expressed in these recordings represent those of the speakers and do not necessarily represent the official position or policies of the U.S. Department of Justice. Any commercial products and manufacturers discussed in these recordings are presented for informational purposes only and do not constitute product approval or endorsement by the U.S. Department of Justice.
  
Date Published: August 5, 2020
    
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