Biology Related Artifact Peaks

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Stutter products are the most common source of additional peaks in an electro-pherogram of an STR sample. When STR loci are PCR-amplified a minor product peak four bases (n — 4) shorter than the corresponding main allele peak is commonly observed (see Chapter 6). Validation studies conducted in a laboratory help define maximum percent stutter for each locus. However, if the target allele peak is off-scale then the stutter product can appear larger than it really is in relationship to the corresponding allele peak (see Moretti et al. 2001). For data interpretation, an upper-limit stutter percentage interpretational threshold can be set for each locus as three standard deviations above the highest stutter percentage observed at that locus (Applied Biosystems 1998).

Incomplete 3'(A) nucleotide addition results with amplifications containing too much DNA template or thermal cycling conditions that affect the optimization of the PCR reaction. The Taq DNA polymerase used for amplifying STR loci will catalyze the addition of an extra nucleotide, usually an 'A', on the 3'-end of double-stranded PCR products (see Chapter 6). The commercially available multiplex STR kits have been optimized to favor complete adenylation. However, when incomplete 3' nucleotide addition occurs 'split peaks' will result, sometimes referred to as +/-A, or N and N + 1 peaks, and the allele of interest will be represented by two peaks one base pair apart. Genotyping software may inadvertently call one of these peaks an 'off-ladder' (microvariant) allele.

Tri-allelic (three banded) patterns result from extra chromosomal fragments being present in a sample or the DNA sequence where the primers anneal being duplicated on one of the chromosomes. These rare anomalies are detected by an extra peak at a single locus, as opposed to multiple loci as would likely be seen in a mixture (see Chapter 7). The three peaks will commonly all be of equal intensity but do not have to be (Crouse et al. 1999).

Mixed sample results are observed if more than one individual contributed to the DNA profile. Mixtures are readily apparent when multiple loci are examined. An analyst looks for higher than expected stutter levels, more than two peaks at a locus of equivalent intensity, or a severe imbalance in heterozygote peak intensities of greater than 30% (see Chapter 7). It is usually difficult to detect the minor contributor below a level of 1:20 compared to the major donor in the DNA profile.

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