Occasionally new rare alleles may fall outside the allele range spanned by the locus allelic ladder. If these peaks fall between two STR loci in a multiplex set, they can be challenging to assign to a particular locus unless testing is performed with individual locus-specific primer sets or a different multiplex. These extreme 'off-ladder' alleles can be confirmed with singleplex amplification of the two loci in the multiplex bracketing the new allele. Alternatively the sample could be amplified again using a separate multiplex where the loci are present in a different order. For example, if a PCR product was observed between the typical VWA and D16S539 allele ranges when using the SGM Plus kit, then it could be either a large VWA allele or a small D16S539 allele, which is doubtful because allele 5 is the smallest run on the D16S539 ladder. A different kit, such as PowerPlex 16, could be used on this same sample to help address the source of the new allele since the loci are put together in a different combination. With PowerPlex 16 a large VWA allele would appear between the VWA and D8S1179 expected allele ranges, but a small D16S539 allele would fall between D7S820 and D16S539 (see Figure 5.4).
Three-banded or tri-allelic patterns are sometimes observed at a single locus in a multiplex STR profile. These extra peaks are not a result of a mixture but are reproducible artifacts of the sample. Extra chromosomal occurrences or primer point mutations have been known to happen and result in a three-banded pattern (Crouse et al. 1999). For example, three-banded patterns have been observed in the 9948 cell line with CSF1PO and in the K562 cell line with D21S11 (see D.N.A. Box 7.1).
The three peaks or bands seen at a particular locus may or may not be equal in intensity. While the TPOX three-banded patterns reported by Crouse and co-workers (1999) were approximately equal in intensity (similar to Figure 6.7a), there are also occasions when tri-allelic patterns occur with peaks of unequal intensity. In Figure 6.7b, a sum of two of the alleles is approximately equivalent in amount as the third allele as seen in Figure 6.7b for D18S51. The peak heights for alleles 14 and 15 when added together are similar to amount as the D18S51 allele 22. Thus, it is likely that the 14 and 15 alleles came from one parent while the 22 repeat allele came from the other. There is probably some
Tri-allelic patterns observed at (a) TPOX and (b) D18S51 from different samples. Allele calls are listed underneath each peak. (a) The TPOX result was obtained with the Identifiler STR kit and run on the ABI 3100. (b) This DNA sample was amplified with the Profiler Plus™ STR kit, separated on the ABI Prism 310 Genetic Analyzer and viewed with Genotyper software. Only the green dye-labeled PCR products are shown here for simplicity's sake.
kind of chromosome duplication for the region surrounding the D18S51 marker in this individual. Note that in this example the other STR loci besides D18S51 have two peaks of similar intensity, which suggests that a sample mixture is not likely (see Chapter 7).
More than 50 different tri-allelic patterns have been reported at all 13 CODIS STR loci with most of them being seen at TPOX and FGA. A frequently updated listing of tri-allelic patterns may be found on the STRBase web site: http://www.cstl.nist.gov/biotech/strbase/var_tab.htm.
When amplifying DNA fragments that contain STR repeat regions, it is possible to have a phenomenon known as allele dropout. Sequence polymorphisms are known to occur within or around STR repeat regions. These variations can occur in three locations (relative to the primer binding sites): within the repeat region, in the flanking region, or in the primer-binding region (Figure 6.8).
If a base pair change occurs in the DNA template at the PCR primer binding region, the hybridization of the primer can be disrupted resulting in a failure to amplify, and therefore failure to detect an allele that exists in the template DNA. More simply, the DNA template exists for a particular allele but fails to
Possible sequence variation in or around STR repeat regions and the impact on PCR amplification. The asterisk symbolizes a DNA difference (base change, insertion or deletion of a nucleotide) from a typical allele for a STR locus. In situation (a), the variation occurs within the repeat region and should have no impact on the primer binding and the subsequent PCR amplification (although the overall amplicon size may vary slightly). In situation (b), the sequence variation occurs just outside the repeat in the flanking region but interior to the primer annealing sites. Again, PCR should not be affected although the size of the PCR product may vary slightly. However, in situation (c) the PCR can fail due to a disruption in the annealing of a primer because the primer no longer perfectly matches the DNA template sequence.
5'flanking 3'flanking Forward primer region STR region Reverse primer binding region Repeat region binding region /-\!-\/-W-W-\
Example: TH01 9.3 allele (-A in 7th repeat)
D18S51 13.2 allele (+AG in 3'-flanking region)
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