An Example Mixture

An example mixture will now be examined to demonstrate how the steps illustrated in Figure 7.4 may be used to interpret a mixture. Figure 7.5 shows a mixed sample that is a combination of male and female DNA, typical of what might be seen in a sexual assault investigation. The STR markers for the mixture are separated into three panels based on their dye label in order to visualize each STR locus more easily.

The first thing that is obvious in this example is the presence of more than two peaks at a majority of the loci. For example, D3S1358 contains four peaks and VWA has three peaks. There is also an imbalance in the X and Y alleles of the amelogenin sex-typing marker.

With the presence of a mixture established, the alleles are determined. Because there are a maximum of four peaks at any one locus, it is unlikely that there are more than two contributors to this example mixture. Each of the called alleles is labeled with a letter: 'A', 'B', 'C', or 'D'. These universal designations are used to track possible allele combinations through the rest of the mixture calculations.

Figure 7.5 Mixture of male and female DNA typical of what might be seen in a forensic case involving mixed samples. Profiler Plus™ multiplex STR data is displayed here with GeneScan® 3.1. Sample mixture indicators include an imbalance in the amelogenin X and Y alleles as well as greater than two peaks at multiple STR markers.

DNASize(bp)

D3S1358

Amel

D8S1179

D5S818

blue panel

D21S11

green panel D18S51

D13S317

yellow panel D7S820

The relative ratio of the individuals contributing to this example mixture is then estimated by examining loci with four peaks. The green panel STR data from Figure 7.5 is shown in Figure 7.6 with labeled peak areas. There are four peaks present at both the D21S11 and D18S51 loci. Since the peak areas of the D21S11 A and the D alleles are similar and the peak areas of the B and the C alleles are similar to each other, we can assume that AD and BC represent the best possible combination of alleles to explain the data. Likewise,

Figure 7.6

Peak areas for green panel data from example mixture in Figure 7.5. Mixture ratio calculations for these STR markers are shown in Table 7.5. RFUs = relative fluorescence units.

■t |—rtt]—i i i | t i—i | i' —|—l—i—i—|—i—i—i—|—i—r—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—r~

100 120 140 160 180 200 220 240 260 280 300 320

15957 7512;

imbalance

■t |—rtt]—i i i | t i—i | i' —|—l—i—i—|—i—i—i—|—i—r—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—r~

100 120 140 160 180 200 220 240 260 280 300 320

15957 7512;

A I 1

_jJ-.IL_

A BCD J___

1971 6427

H611!.1158.

[116^ [2064 i

4264

3122 3193

2613 ¡1366]

3 peaks at D8S1179

4 peaks at D21S11

4 peaks at D18S51

RFUs

the D18S51 A and C alleles have similar peak areas and can be grouped together so that the best possible combination of alleles at this locus is AC and BD (Table 7.5).

The mixture ratio for the D21S11 alleles is calculated to be 2.3, or approximately 2:1, by dividing the sum of the larger alleles (B and C) by the sum of the smaller alleles (A and D). Thus, the major contributor to this mixture has about twice as much DNA present as the minor contributor. Using the same approach, the D18S51 mixture ratio is calculated to be 1.8 or approximately 2:1

Locus

Component Allele

Peak

Possible

Mixture

Call

Area

Combinations

D21S11

D18S51

D8S1179

12 14 16 17

10 12 14

1611 3122 3193 1158

1169 2613 1366 2064

1971 4264 6427

AD BC

Based on peak profile appearance for 2:1 mixture (see Figures 7.6 and 7.7)

Table 7.5

Peak information for four loci from the data displayed in Figure 7.6. The mixture ratio is determined by comparing the peak areas for the appropriate peak combinations. The original source genotypes are also listed for the male and female components of the mixture, along with the expected number of peaks at each locus.

Amel

2X:1Y

2X:1Y

D3

VWA

FGA

Amel

D8

D21

D18

D5

D13

D7

Male

17,18

14,19

24,24

X,Y

12,14

30,32.2

14,17

12,13

12,12

9,12

Female

15,16

16,16

23,24

X,X

10,14

29,33.2

12,16

7,11

12,13

10,12

Mixture

# Peaks

4

3

2

2

3

4

4

4

2

3

Major

CD

AC

BB

XY

BC

BC

BD

CD

AA

AC

Minor

AB

BB

AB

XX

AC

AD

AC

AB

AB

BC

Original source genotypes for Figure 7.5 mixed sample example for the major contributor (Table 7.5). These mixture ratios will not always be exact due to the influence of stutter products and imbalances in heterozygote peak areas.

Calculating mixture ratios is much easier at loci with four observed alleles than at loci where one or more of the alleles are shared. D8S1179 in this example represents such a situation. Three peaks are present at D8S1179 with one of these peaks representing an allele from both the major and the minor contributor. Each possible combination of alleles is therefore carefully considered to determine which one best fits the observed data.

Expected peak patterns for each of the possible 2:1 mixture combinations are displayed in Figure 7.7. The observed data for D8S1179 (Figure 7.6) fits the scenario of BC and AC allele combinations with BC belonging to the major contributor. Thus, in this case allele C, or 14, is shared. The major contributor's genotype is therefore 12,14 at D8S1179 while the minor contributor's genotype is 10,14.

The major contributor in this example mixture was the male individual at a ratio of two times that of the female DNA in the mixture (Table 7.5). This fact was determined by examining the peak areas for the X and Y alleles and comparing them to the information found in Table 7.4. Thus, in this example it was possible to decipher that the major contributor was a male and that the D21S11 alleles 30 and 32.2, D18S51 alleles 14 and 17, and D8S1179 alleles 12 and 14 belonged to him. By continuing through all of the loci in the manner indicated above, the genotype profile of the major and minor contributors can be distinguished.

Mixture ratios cannot always be calculated at every locus with complete confidence especially those with two or three peaks that have shared alleles between the contributors. Note that in Figure 7.7 when a homozygous individual is the minor component of a mixture all three scenarios have indistinguishable profiles of three fairly balanced peak signals (e.g., VWA in Figure 7.5). Of course, stutter products and imbalanced heterozygote peak signals make calculation of mixture ratios less accurate.

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