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.
KeywordsD10S1435 locus Artifacts Secondary structure Sequence analysis Electrophoresis
This project was supported by the National Natural Science Foundation of China (NSFC, No. 81525015, 81772031), GDUPS (2017).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest.
- 8.Crivellente F, McCord BR (2002) Effect of sample preparation and pH-mediated sample stacking on the analysis of multiplexed short tandem repeats by capillary electrophoresis. J Capill Electrophor Microchip Technol 7:73–80Google Scholar
- 9.Lan Q, Wang H, Shen C et al (2018) Mutability analysis towards 21 STR loci included in the AGCU 21 + 1 kit in Chinese Han population. 132: 1287–91. doi: https://doi.org/10.1007/s00414-018-1873-x
- 10.Budowle B, Onorato AJ, Callaghan TF, Manna AD, Gross AM, Guerrieri RA, Luttman JC, McClure DL (2009) Mixture interpretation: defining the relevant features for guidelines for the assessment of mixed DNA profiles in forensic casework. J Forensic Sci 54:810–821. https://doi.org/10.1111/j.1556-4029.2009.01046.x CrossRefGoogle Scholar
- 12.Froger A, Hall JE (2007) Transformation of plasmid DNA into E. coli using the heat shock method. J Vis Exp : JoVE:253. https://doi.org/10.3791/253
- 14.Biega LA, Duceman BW (1999) Substitution of H2O for formamide in the sample preparation protocol for STR analysis using the capillary electrophoresis system: the effects on precision, resolution, and capillary life. Kumamoto Med J 24:20–29Google Scholar
- 18.Meldgaard M, Morling N (1997) Detection and quantitative characterization of artificial extra peaks following polymerase chain reaction amplification of 14 short tandem repeat systems used in forensic investigations. Electrophoresis 18:1928–1935. https://doi.org/10.1002/elps.1150181107 CrossRefGoogle Scholar