Identification and secondary structure analysis of a region affecting electrophoretic mobility of the STR locus SE33

SE33 is one of the most informative markers in forensic use due to its high power of discrimination. During the course of developing the AmpFℓSTR(®) NGM SElect™ PCR Amplification Kit several SE33 primer designs were screened with one primer pair yielding a high frequency of discordant alleles when compared to the AmpFℓSTR(®) SEfiler Plus™ PCR Amplification Kit. This discordance was mostly specific to samples of African descent with an estimated frequency of 5.1% and was a result of a mobility shift of approximately +0.84nt. The sequence analysis of the affected alleles revealed that the only difference from the wild type sequence was a single nucleotide polymorphism (SNP) outside of the SE33 repeat but within the amplicon of this particular set of experimental primers. In total, we identified three different SNPs all within 9nt of each other, each of which could cause the mobility shift individually. Further characterization of this region via site directed mutagenesis and thermostability measurements strongly suggests that this polymorphic region contains a secondary structure that, when disrupted due to the presence of a variant SNP, results in a mobility shift relative to the wild type sequence. To overcome this problem, the SE33 primers used in the final configuration of the NGM SElect™ Kit avoided the amplification of this polymorphic region yielding in turn results highly concordant with the SEfiler Plus™ Kit.