Mixture Interpretation

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This next section will review the principles described by Gill et al. (1998b) and Clayton et al. (1998) for interpreting mixed forensic stains using STR typing results. Their six primary steps for interpreting mixtures are outlined in Figure 7.4. The interpretation steps will first be discussed and then an example mixture will be reviewed to put these steps into the context of a real sample.

An understanding of how non-mixtures behave is essential to being able to proceed with mixture interpretation. Mixed DNA profiles need to be interpreted against a background of biological and technological artifacts. Chapter 6 reviewed some of the prominent biological artifacts that exist for STR markers.

Figure 7.4

Steps in the interpretation of mixtures (Clayton et al. 1998).

Step #1

Step #2

Step #3

Step #4

Step #5

Step #6

Contoh Flowchart Membuat Kue

These include stutter products and null alleles. In addition, chromosomal abnormalities, such as tri-allelic (three-banded) patterns resulting from trisomy (the presence of three chromosomes instead of the normal two) or duplication of specific chromosomal regions can occur. In addition, non-specific amplification products can occasionally happen and must be considered prior to making an attempt to decipher a mixed profile. Issues surrounding technological artifacts from fluorescence detection will be covered in Chapters 13 and 15.

Stutter products represent the greatest challenge in confidently interpreting a mixture and designating the appropriate alleles. It is not always possible to exclude stutters since they are allelic products and differ from their associated allele by a single repeat unit. The general guideline for stutter identification of one repeat unit less than the corresponding allele and less than 15% of that allele's peak area is typically a useful one and can be used to mark suspected stutter products. The introduction of pentanucleotide repeat markers with stutter products of less than 1-2% has greatly simplified mixture interpretation (Bacher et al. 1999, Krenke et al. 2002).

After a mixture has been identified as such and all of the alleles have been called, the next step (Figure 7.4, step #3) is to identify the number of potential contributors. For a two-person mixture, the maximum number of alleles at any given locus is four if both individuals are heterozygous and there is no allele overlap. Thus, if more than four alleles are observed at a locus then a complex mixture consisting of more than two individuals is possible. Fortunately, the overwhelming majority of mixtures encountered in forensic casework involve two-person mixtures (Clayton et al. 1998).

Mixtures can range from equal proportions of each component to one component being greatly in excess. The varying proportions of a mixture are usually referred to in a ratio format (e.g., 1:1 or 1:5). Mixtures of known quantities of DNA templates have shown that the mixture ratio is approximately preserved during PCR amplification (Gill et al. 1998a, Perlin and Szabady 2001). Thus, the peak areas and heights observed in an electropherogram can in most cases be related back to the amount of DNA template components included in the mixed sample.

An approximate mixture ratio can be best determined by considering the profile as a whole and looking at all of the information from each locus. The ratio of mixture components is most easily determined when there are no shared alleles at a locus. Thus, it is best to first examine loci with four alleles as a starting point for estimating the relative ratio of the two individuals contributing to the mixture. Determining the ratio when there are shared alleles is more complex because there may be more than one possible combination of alleles that could explain the observed peak patterns (Clayton et al. 1998).

Table 7.3

Four Alleles (A,B,C,D) Three Alleles (A,B,C) Two Alleles (A,B)

Pairwise comparisons of

Table 7.3

Four Alleles (A,B,C,D) Three Alleles (A,B,C) Two Alleles (A,B)

Pairwise comparisons of

two, three and four allele

A,B

C,D

A,A

B,C

A,A

A,B

peak patterns. Reciprocal

combinations are not

A,C

B,D

B,B

A,C

A,B

A,B

shown.

A,D

B,C

C,C

A,B

A,A

B,B

A,B

A,C

A,B

The possible combinations of alleles for two, three, and four allele peak patterns are listed in Table 7.3. For four alleles at a locus, there are three possible pairwise comparisons that exist, if one does not worry about the reciprocal cases, i.e., which allele combinations belong to the minor contributor and which belong to the major contributor. For three alleles at a locus, there are six possible pairwise combinations and for two alleles at a locus there are four possible pairwise combinations (Table 7.3).

Amelogenin, the sex-typing marker, is an effective marker for deciphering the contributions of genetically normal male and female individuals. The predicted X and Y allele peak ratios for a number of possible male and female mixture ratios are listed in Table 7.4. The amelogenin X and Y peak areas are especially useful in determining whether the major contributor to the mixture is male or female.

The next step in examining a mixture is to consider all possible genotype combinations at each locus (Figure 7.4, step #5). Peaks representing the allele calls at each locus are labeled with the designations A, B, and so forth. The possible pairwise combinations from Table 7.3 are considered using the peak areas for each called allele. Each particular combination of alleles at the different loci is considered in light of the information determined previously regarding the mixture ratio for the sample under investigation (step #4). By stepping through each STR locus in this manner, the genotypes of the major and minor contributors to the mixture can be deciphered. In the example shown in Figure 7.5 and Table 7.5, some of these calculations are demonstrated.

The final step in the interpretation of a mixture is to compare the resultant genotype profiles for the possible components of the mixture with the genotypes of reference samples (Figure 7.4, step #6). In a sexual assault case, this reference sample could be the suspect and/or the victim. If the DNA profile from the suspect's reference sample matches the major or minor component of the mixture, then that person cannot be eliminated as a possible contributor to the mixed stain (Clayton et al. 1998).

Mixture Ratio

Allele Combination

Ratio of X:Y Peak Areas

Female

Male

Mixture Ratio

Allele Combination

Female

Male

(X,X)

(X,Y)

X

Y

X:Y

20

1

41

1

41:1

10

1

21

1

21:1

5

1

11

1

11:1

4

1

9

1

9:1

3

1

7

1

7:1

2

1

5

1

5:1

1

1

3

1

3:1

1

2

4

2

2:1

1

3

5

3

1.7:1

1

4

6

4

1.5:1

1

5

7

5

1.4:1

1

10

12

10

1.2:1

1

20

22

20

Possible amelogenin X and Y allele peak ratios with varying quantities of DNA. The 2:1 peak area ratio for the X and Y alleles observed in Figure 7.6 and calculated in Table 7.5 matches a mixture ratio of one part female to two parts male as indicated by the numbers underlined. Thus, there is twice as much male DNA compared to female DNA in the mixed sample shown in Figure 7.5.

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