Detection and analysis of the cause of false-tetra-allelic patterns of locus D10S1435 at the sequence level.

A number of artifacts produced in forensic DNA typing make the interpretation more complicated and even lead to typing errors. Here, we reported the cause of false-tetra-allelic patterns of STR locus D10S1435 at the sequence level. To confirm the true genotyping, the sample with four allelic peaks was re-amplified and sequenced. The amplicon sequences of D10S1435, D20S482, D6S1017, and D10S1248 loci were analyzed by software BioXM and RNAstructure. We successfully reproduced the four-peak phenomenon by adding various concentration of magnesium chloride into the loading mixtures to simulate the suboptimal electrophoresis conditions. The false four allelic peaks may be caused by the specific nucleotide sequence of locus D10S1435 which tends to form secondary structures under the suboptimal electrophoresis conditions. The relatively high GC content and extremely uneven distribution give the amplicon a potency to resist complete denaturation at the phase of sample preparation and a tendency to form intra- and intermolecular secondary structures during post-injection.