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.
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This project was supported by the National Natural Science Foundation of China (NSFC, No. 81525015, 81772031), GDUPS (2017).
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Representative electropherograms of the sample #1. The sample #1 was amplified with AGCU 21 + 1 kit on GeneAmp® 9700, and 1 μL of PCR products were electrophoresed on a 3500xl Genetic Analyzer using a 2 kV, 8 s injection (BMP 3248 kb)
The sequences of alleles 12 and 13 at the D10S1435 locus for sample #1. The cloning primers of the D10S1435 locus used in this study are underlined, the core repeat region is shaded in red (BMP 2620 kb)
Stem-loop structure prediction formed by intramolecular base pairing. The labeled single-stranded DNA sequence of the D6S1017 locus was analyzed by secondary structure prediction software RNAstructure v6.1 software (BMP 1406 kb)
Variation of the extra peak heights. When the concentrations of MgCl2 increase, the peak height ratios of the extra peaks to the true allelic peaks also increase. When 1 μL of 5 mM, 10 mM, 25 mM, 50 mM, 100 mM, and 200 mM MgCl2 were added, the corresponding peak height ratio was 0.2653, 0.9553, 2.0323, 1.0330, 2.0758, and 2.5405, respectively (BMP 8789 kb)
Effects of different sample substrates on the detection of extra peaks. As far as samples #1, #4, #5, and 9947A are concerned, the true peaks and the additional peaks could be detected at the same time regardless of the MgCl2 concentration (BMP 4192 kb)
Representative electropherograms of the D14S1434 and D20S482 loci with additional peaks. The D14S1434 and D20S482 loci had the largest and the second largest amplicons in the black dye channel, respectively. When 1 μL of 5 mM was added, the extra peaks were observed at D14S1434 locus. When 1 μL of 200 mM was added, the extra peaks were observed at D20S482 locus (BMP 4235 kb)
Representative electropherograms of the sample #5 using water instead of formamide for sample preparation. No extra peaks were observed at all loci, but lots of peaks were called off-ladder ‘OL’ alleles. Compared to the same sample diluted with formamide, the allele size of sample prepared with water was smaller (BMP 4773 kb)
Representative electropherogram of the internal size stand. The standard 250 bp peak has the same size in samples diluted by water and formamide. The samples prepared by formamide and the samples prepared by water were electrophoresed in different runs on a 3130xl Genetic Analyzer using a 3 kV, 10 s injection (BMP 5035 kb) (BMP 5035 kb)
Allele size data of sample prepared with formamide or water. No additional peaks were observed at any loci either in the sample preparation with formamide or water. However, for the same allele, the size of the sample prepared with water was smaller than that of the sample prepared with formamide. The sample #2 did not obtain allele size data because of failed injection. Minus is the allele size of the sample prepared with formamide subtract the allele size of the sample prepared with water (XLSX 16 kb)
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Zhou, Y., Lan, Q., Fang, Y. et al. Detection and analysis of the cause of false-tetra-allelic patterns of locus D10S1435 at the sequence level. Int J Legal Med 134, 833–843 (2020). https://doi.org/10.1007/s00414-019-02153-7
- D10S1435 locus
- Secondary structure
- Sequence analysis