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.
