Occasionally a sample may contain an allele that does not fall within 0.5 bp of an allele from the corresponding locus-specific allelic ladder (Gill et al. 1996). These alleles are designed as 'off-ladder' alleles or microvariants (see Chapter 6). The off-ladder allele peak may be larger or smaller than the alleles spanning the allelic ladder range or it may fall in between the rungs on the allelic ladder.
If the allele is sized to be less than the ladder, it may be designated as smaller than the smallest allele in the ladder used for genotyping purposes. For example, a CSF1PO allele sized below allele 6, which is the smallest in the ladder, would be designated CSF1PO < 6. Likewise an allele sized above allele 15, the largest in the ladder, would be designated CSF1PO > 15. Because the alleles in allelic ladders differ between manufacturers, an allele designation of '> 15' from an amplification using one STR kit could be equivalent to an allele designation '16' from another.
Alleles that are sized between the rungs on an allelic ladder are usually designated by the number of bases beyond the allele just smaller than it. For example, a TH01 allele sized three bases larger than allele 8 would be designated TH01 8.3. It could also be referred to less specifically as TH01 8.x.
Off-ladder alleles can be verified by re-running the amplified product, re-amplifying the sample, or by amplifying the sample with single-locus primers. Heterozygous samples with one 'normal' allele and one microvariant allele make it easy to confirm the microvariant. In this particular case, the normal allele with a full length repeat sequence will fall in an allele bin from the allelic ladder while the microvariant allele possessing a partial repeat sequence will fall between the allele bins created by the allelic ladder (see Figure 6.6). New microvariants are constantly being discovered as more samples are being analyzed around the world at various STR loci. As of April 2004, more than 220 variant 'off-ladder' alleles have been reported for all 13 of the CODIS core STR loci (see http://www.cstl.nist.gov/biotech/strbase/var_tab.htm).
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