Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
de Knijff P (2019) From next generation sequencing to now generation sequencing in forensics. Forensic Sci Int Genet 38:175–180. https://doi.org/10.1016/j.fsigen.2018.10.017
Butler JM, Buel E, Crivellente F, McCord BR (2004) Forensic DNA typing by capillary electrophoresis using the ABI prism 310 and 3100 genetic analyzers for STR analysis. Electrophoresis 25:1397–1412. https://doi.org/10.1002/elps.200305822
Butler JM (2015) Chapter 3 - STR alleles and amplification artifacts. In: Butler JM (ed) Advanced topics in forensic DNA typing: interpretation. Academic press, San Diego, pp 47–86
Fernando P, Evans BJ, Morales JC, Melnick DJ (2001) Electrophoresis artefacts — a previously unrecognized cause of error in microsatellite analysis. Mol Ecol Notes 1:325–328. https://doi.org/10.1046/j.1471-8278.2001.00083.x
McLaren RS, Ensenberger MG, Budowle B et al (2008) Post-injection hybridization of complementary DNA strands on capillary electrophoresis platforms: a novel solution for dsDNA artifacts. Forensic Sci Int Genet 2:257–273. https://doi.org/10.1016/j.fsigen.2008.03.005
Rosenblum BB, Oaks F, Menchen S, Johnson B (1997) Improved single-strand DNA sizing accuracy in capillary electrophoresis. Nucleic Acids Res 25:3925–3929
Buel E, LaFountain M, Schwartz M (2003) Using resolution calculations to assess changes in capillary electrophoresis run parameters. J Forensic Sci 48:77–79
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 7:73–80
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
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
Zhu BF, Zhang YD, Shen CM, du WA, Liu WJ, Meng HT, Wang HD, Yang G, Jin R, Yang CH, Yan JW, Bie XH (2015) Developmental validation of the AGCU 21+1 STR kit: a novel multiplex assay for forensic application. Electrophoresis 36:271–276. https://doi.org/10.1002/elps.201400333
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
Butler JM (2015) Chapter 8 - troubleshooting data collection. In: Butler JM (ed) Advanced topics in forensic DNA typing: interpretation. Academic press, San Diego, pp 183–210
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 24:20–29
Calladine CR, Collis CM, Drew HR, Mott MR (1991) A study of electrophoretic mobility of DNA in agarose and polyacrylamide gels. J Mol Biol 221:981–1005
Konrad KD, Pentoney SL Jr (1993) Contribution of secondary structure to DNA mobility in capillary gels. Electrophoresis 14:502–508
Stellwagen E, Abdulla A, Dong Q, Stellwagen NC (2007) Electrophoretic mobility is a reporter of hairpin structure in single-stranded DNA oligomers. Biochemistry 46:10931–10941. https://doi.org/10.1021/bi701058f
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
Wang DY, Green RL, Lagace RE, Oldroyd NJ, Hennessy LK, Mulero JJ (2012) Identification and secondary structure analysis of a region affecting electrophoretic mobility of the STR locus SE33. Forensic Sci Int Genet 6:310–316. https://doi.org/10.1016/j.fsigen.2011.06.008
Britten RJ, Kohne DE (1968) Repeated sequences in DNA. Hundreds of thousands of copies of DNA sequences have been incorporated into the genomes of higher organisms. Science (New York, NY) 161:529–540
Wetmur JG, Davidson N (1968) Kinetics of renaturation of DNA. J Mol Biol 31:349–370
Tabor S, Richardson CC (1987) DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A 84:4767–4771
Bhattacharyya AJ, Feingold M (2001) Single molecule study of the reaction between DNA and formamide. Talanta 55:943–949
Blake RD, Delcourt SG (1996) Thermodynamic effects of formamide on DNA stability. Nucleic Acids Res 24:2095–2103
Cortadas J, Subirana JA (1977) The incomplete denaturation of DNA in N,N-dimethyl formamide. Biochim Biophys Acta 476:203–206
Cavanaugh SE, Bathrick AS (2018) Direct PCR amplification of forensic touch and other challenging DNA samples: a review. Forensic Sci Int Genet 32:40–49. https://doi.org/10.1016/j.fsigen.2017.10.005
Myers BA, King JL, Budowle B (2012) Evaluation and comparative analysis of direct amplification of STRs using PowerPlex® 18D and Identifiler® direct systems. Forensic Sci Int Genet 6:640–645. https://doi.org/10.1016/j.fsigen.2012.02.005
Funding
This project was supported by the National Natural Science Foundation of China (NSFC, No. 81525015, 81772031), GDUPS (2017).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflicts of interest.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Fig. S1
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)
Fig. S2
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)
Fig. S3
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)
Fig. S4
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)
Fig. S5
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)
Fig. S6
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)
Fig. S7
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)
Fig. S8
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)
Table S1
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)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00414-019-02153-7