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FacebookXLinkedInRedditPinterestEmail Home
 NIJ Training Course for Forensic Analysts: Basic Components of an Electropherogram

September 9, 2020
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An interactive tool used to supplement the NIJ online training course entitled 'STR Data Analysis and Interpretation for Forensic Analysts', as outlined in the first learning module, 'STR Data Analysis & Interpretation Software'. This brief animation shows basic components that appear on an electrophoregram.
  
  
          Transcript
0:08 Click anywhere on this panel to hide it;  then move the mouse cursor over each of the

0:13 yellow arrows to read the explanation  of that feature of electropherograms.

0:21 The numbers along the y-axis of the  electropherogram represent relative

0:25 fluorescent units, or RFUs, which  are used to measure threshold values.

0:31 The taller a peak is, the stronger the  fluorescence was that was created by

0:35 that allele during electrophoresis .The  strength of the fluorescence, in turn,

0:41 is based on the number of copies of that  allele's STR that exist in the sample.

0:50 Furthest to the left of the electropherogram are  found the green peak or peaks that indicate the

0:55 subject's gender. Two peaks as shown here  in the top electropherogram represent an

1:01 x allele and a y allele indicating a male. A  female will display a single tall green peak

1:08 representing two x alleles as shown  here in the bottom electropherogram.

1:16 The extra tall red peaks on the  electropherogram represent the size standard;

1:20 a strong mixture of DNA fragments of  known lengths that is added to the sample.

1:25 Because a certain amount of drift can occur  during electrophoresis the detection equipment

1:30 uses the standard as a reference against which  it maintains calibration throughout the process;

1:36 somewhat like the synchronization track on a  videotape. The red peaks therefore are disregarded

1:42 when analyzing a DNA profile and sometimes an  analyst will not even have the analysis software

1:48 include them on the electropherogram, even though  the size standard is still included in the sample.

1:57 The different colors of the peaks represent  the different colors of dyes used to detect

2:01 the alleles. The dyes are specially designed to  attach to different primers that amplify alleles

2:08 at different loci. As a result, different alleles  can be distinguished from each other even when

2:14 they are of similar length, as with this green  peak, blue peak and two black peaks. Black peaks

2:20 are used to represent yellow dyed alleles for  ease of visibility. If it were not for different

2:26 dyes it would be impossible to tell which of  these four peaks corresponds to which allele.

2:34 In general, if a peak stands alone, as in the  top example, it indicates a homozygous genotype

2:40 at that locus; both alleles are the same. If two  peaks of the same color appear right next to each

2:47 other, as in the bottom example, they indicate  a heterozygous genotype; that locus contains two

2:53 different alleles. Note: homozygous peaks are  generally noticeably taller than corresponding

3:00 heterozygous peaks. This is because in the  homozygous case all of the genetic material

3:06 at that locus is forming a single peak, whereas  in the heterozygous case only half of the genetic

3:12 material at that locus is making up either one  of the peaks. It is split evenly between the two.

3:22 Sometimes strong peaks will have very small  peaks of the same color just to the left of them

3:27 these are called stutters; they  do not actually represent alleles,

3:31 but are essentially just echoes of the  larger peaks. As such they are disregarded.

3:40 The readout in areas of the electropherogram where  no genetic material was detected in the sample

3:46 (zero RFUs) is referred to as the baseline.  This name however is slightly misleading;

3:54 the baseline is not truly aligned. A certain  amount of baseline noise occurs causing an erratic

4:01 baseline in all four colors, which is visible when  the electropherogram is viewed at large scales.

4:08 It is because of this that peaks must be of a  particular height, called the threshold value, to

4:13 be considered valid in order to distinguish them  from meaningless irregularities in the baseline.

4:21 The numbers along the x-axis of  the electropherogram represent DNA

4:25 fragment length in number of base pairs.  The longer the DNA fragment is that forms

4:31 one of the alleles in the sample, the  farther to the right its peak appears.
    
  
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: September 9, 2020
    
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