Characterization of a novel D1S80 pseudoallele

During a D1S80 population study conducted for databasing purposes in the New York City Ashkenazi Jewish population, eight out of 96 samples were typed with a band corresponding to the position of a #15 allele. In seven of the eight samples, three bands appeared. Further investigation was needed to explain the high frequency of an allele considered so rare that it is not included in the commercially provided allelic ladder. After extraction of the putative D1S80 15-repeat amplicon band from the 6% polyacrylamide genotyping gel, the amplicon bands were reamplified with D1S80 primers. After retyping as putative 15 alleles, these samples underwent Southern hybridization with a D1S80 locus-specific probe followed by DNA sequence analysis. Sequence analysis revealed that these bands did not arise from true D1S80 15 alleles. However, the PCR product was of a size that fell within the allelic ladder region corresponding to the 15 band and contained end sequences with strong homology to the D1S80 primers. An alignment search of the sequenced product revealed that a portion of the amplicons contained 72% identity to a known gene. These results emphasize the importance of sequencing analysis when questions arise about the authenticity of an allele.