Distinguishing Genotypes In A Mixed Sample

Several clues exist to help determine that a mixture is present. Answers to the following questions can help ascertain the genotypes that make up the composite DNA profile of the mixture:

■ Do any of the loci show more than two peaks in the expected allele size range?

■ Is there a severe peak height imbalance between heterozygous alleles at a locus?

■ Does the stutter product appear abnormally high (e.g., > 15-20%)?

If the answer to any one of these three questions is yes, then the DNA profile may very well have resulted from a mixed sample. Mixture interpretation has been examined extensively by the Forensic Science Service (Clayton et al. 1998, Gill et al. 1998a, 1998b) and many of their strategies have been incorporated into this section's material.

Usually a mixture is first identified by the presence of three or more prominent peaks at one or more loci. At a single locus, a sample containing DNA from two sources can exhibit one, two, three, or four peaks due to the possible genotype combinations listed below.

Four peaks:

■ heterozygote + heterozygote, no overlapping alleles (genotypes are unique).

Three peaks:

■ heterozygote + heterozygote, one overlapping allele.

■ heterozygote + homozygote, no overlapping alleles (genotypes are unique).

Two peaks:

■ heterozygote + heterozygote, two overlapping alleles (genotypes are identical).

■ heterozygote + homozygote, one overlapping allele.

■ homozygote + homozygote, no overlapping alleles (genotypes are unique).

Single peak

■ homozygote + homozygote, overlapping allele (genotypes are identical).

When two contributors to a mixed stain share one or more alleles, the alleles are 'masked' and the contributing genotypes may not be easily decipherable. For example, if two individuals at the FGA locus have genotypes 23,24 and 24,24, then a mixture ratio of 1:1 will produce a ratio of 1:3 for the 23:24 peak areas. In this particular case, the mixture could be interpreted as a homozygous allele with a large stutter product without further information. However, by examining the STR profiles at other loci that have unshared alleles, i.e., three or four peaks per locus, this sample may be able to be dissected properly into its components.

In an effort to see if it was possible for masking to occur at every locus in a multiplex, the Forensic Science Service conducted a simulated mixture study with 120 000 individual STR profiles in their Caucasian database (Gill et al. 1997). They found that the vast majority of these artificial mixtures showed 15-22 peaks across a six-plex STR marker multiplex. The maximum number in a mixture of two heterozygous individuals with no overlapping alleles at six STRs would be 24 peaks. Thus, in this example with unrelated individuals, simple mixtures can be identified by the presence of three or more alleles at several loci. Out of more than 212 000 pairwise comparisons, there were only four examples where one or two alleles were observed at each locus in the six-plex, and these could be designated mixtures because of peak imbalances (Gill et al. 1997).

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