Abstract
Short tandem repeat (STR) is regarded as a crucial tool for personal identification as well as parentage testing. Thus, genotyping errors of STRs could have negative effects on the reliability of forensic identification. A null allele at the combined DNA index system (CODIS) core loci D2S1338 was found in a father-daughter pair with the AGCU Expressmarker 22 kit which was a commonly used commercial kit during our daily laboratory work. This null allele caused the father and daughter to not conform to the laws of inheritance, thus potentially generating erroneous conclusions that excluded parentage. To figure out the reason for this phenomenon, re-amplification with new primers and then large fragment Sanger sequencing was conducted. We found a G to G/T variation at the position which is fifty-nine bases away from the 3’ end of the core repeat in both samples. This probably could be considered a novel variant at the primer binding region which had not been reported that resulted in the emergence of the null allele. We also found that there was more than one single-nucleotide polymorphism (SNP) with minor allele frequency (MAF) greater than 0.1 in the upstream and downstream sequences of D2S1338. When designing primers for amplification of D2S1338, the possible adverse results of these SNPs should be taken into account and avoided.
This is a preview of subscription content, log in via an institution to check access.
References
Li K, Shrivastava S, Stockwell TB (2015) Degenerate primer design for highly variable genomes. In: PCR Primer Design. Basu C (ed.) Springer New York, New York, NY, pp 103–115.
Yao Y, Yang Q, Shao C, Liu B, Zhou Y, Xu H, Zhou Y, Tang Q, Xie J (2018) Null alleles and sequence variations at primer binding sites of STR loci within multiplex typing systems. Leg Med (Tokyo) 30:10–13. https://doi.org/10.1016/j.legalmed.2017.10.007
Kline MC, Hill CR, Decker AE, Butler JM (2011) STR sequence analysis for characterizing normal, variant, and null alleles. Forensic Sci Int Genet 5:329–332. https://doi.org/10.1016/j.fsigen.2010.09.005
Takayama T (2022) A novel mutation at the AMEL primer binding region on the Y chromosome in AMELY negative male. INT J LEGAL MED 136:519–526. https://doi.org/10.1007/s00414-022-02781-6
Yao Y, Yang Q, Shao C, Liu B, Zhou Y, Xu H, Zhou Y, Tang Q, Xie J (2018) Null alleles and sequence variations at primer binding sites of STR loci within multiplex typing systems. LEGAL MED-TOKYO 30:10–13. https://doi.org/10.1016/j.legalmed.2017.10.007
Zou Y, Guo JJ, Li QP, Zuo DH, Liu JS, Guo YD, Yan J, Zha L, Cai JF, Lan LM (2016) Genetic polymorphisms of 21 STR loci in Hunan Province-based Han population. Fa Yi Xue Za Zhi 32:356–362. https://doi.org/10.3969/j.issn.1004-5619.2016.05.010
He G, Li Y, Wang Z, Liang W, Luo H, Liao M, Zhang J, Yan J, Li Y, Hou Y, Wu J (2017) Genetic diversity of 21 autosomal STR loci in the Han population from Sichuan province, Southwest China. Forensic Sci Int Genet 31:e33–e35. https://doi.org/10.1016/j.fsigen.2017.07.006
Zou X, Li Y, Wei Z, Wang T, Hu Y, Zhu Y, Li J, Tang R (2018) Population data and forensic efficiency of 21 autosomal STR loci included in AGCU EX22 amplification system in the Wanzhou Han population. INT J LEGAL MED 132:153–155. https://doi.org/10.1007/s00414-017-1680-9
Lorente M, Lorente JA, Wilson MR, Budowle B, Villanueva E (1993) Composite PAGE: an alternate method for increased separation of amplified short tandem repeat alleles. INT J LEGAL MED 106:69–73. https://doi.org/10.1007/BF01225043
Sajantila A, Lukka M (1993) Improved separation of PCR amplified VNTR alleles by a vertical polyacrylamide gel electrophoresis. INT J LEGAL MED 105:355–359. https://doi.org/10.1007/BF01222121
Gjertson DW, Brenner CH, Baur MP, Carracedo A, Guidet F, Luque JA, Lessig R, Mayr WR, Pascali VL, Prinz M, Schneider PM, Morling N (2007) ISFG: recommendations on biostatistics in paternity testing. Forensic Sci Int Genet 1:223–231. https://doi.org/10.1016/j.fsigen.2007.06.006
Morling N, Allen RW, Carracedo A, Geada H, Guidet F, Hallenberg C, Martin W, Mayr WR, Olaisen B, Pascali VL, Schneider PM, Paternity TCOT (2002) Paternity testing commission of the international society of forensic genetics: recommendations on genetic investigations in paternity cases. FORENSIC SCI INT 129:148–157. https://doi.org/10.1016/S0379-0738(02)00289-X
Sanger F, Coulson AR (1975) A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J MOL BIOL 94:441–448. https://doi.org/10.1016/0022-2836(75)90213-2
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. P NATL ACAD SCI USA 74:5463–5467. https://doi.org/10.1073/pnas.74.12.5463
Kannan S, Zacharias M, Pacific Northwest National Laboratory PNNL RWUE (2007) Folding of a DNA hairpin loop structure in explicit solvent using replica-exchange molecular dynamics simulations. BIOPHYS J 93:3218–3228. https://doi.org/10.1529/biophysj.107.108019
Funding
This work was funded by the National Natural Science Foundation of China, grant number 81772030.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Ethics approval
Although ethics approval was not obtained, when collecting materials, the content and purpose of the test were fully explained to the material donor, and written informed consent was obtained. In addition, the samples involved in this study were derived from a case in forensic casework, and the loci information disclosed (total of 7 loci) in the study were not sufficient to identify any individual or case.
Conflict of interest
The authors declare no competing interests.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Jiang, L., Song, M., Wang, Z. et al. Large fragment Sanger sequencing identifies the newly encountered variant that caused null alleles in parentage testing. Int J Legal Med 137, 57–61 (2023). https://doi.org/10.1007/s00414-022-02901-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00414-022-02901-2