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Development of a novel panel for blood identification based on blood-specific CpG-linked SNP markers

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Abstract

Blood-containing mixtures often appear in murder and robbery cases, and their identification plays a significant role in solving crimes. In recent years, the co-detection of DNA methylation markers (CpG) and single nucleotide polymorphism (SNP) markers has been shown to be a promising tool for the identification of semen and its donor. However, similar research on blood stains that are frequently found at crime scenes has not yet been reported. In this study, we employed blood-specific CpG-linked SNP markers (CpG-SNP) for blood-specific genotyping and the linking of blood and its donor. The tissue-specific CpG markers were screened from the literature and further verified by combining bisulfite conversion with amplification-refractory mutation system (ARMS) technology. Meanwhile, adjacent SNP markers with a minor allele frequency (MAF) greater than 0.1 were selected within 400 bp upstream and downstream of the CpG markers. SNP genotyping was performed using SNaPshot technology on a capillary electrophoresis (CE) platform. Finally, a multiplex panel, including 19 blood-specific CpG linked to 23 SNP markers, as well as 1 semen-specific CpG, 1 vaginal secretion-specific CpG, and 1 saliva-specific CpG marker, was constructed successfully. The panel showed good tissue specificity and blood stains stored at room temperature for up to nine months and moderately degraded (4 < DI < 10) could be effectively identified. Moreover, it could also be detected when blood content in the mixed stains was as low as 1%. In addition, 15 ng of DNA used for bisulfite conversion was required for obtaining a complete profile. The cumulative discrimination power of the panel among the Han population of northern China could reach 0.999983. This is the first investigation conducted for the simultaneous identification of blood and its donor regardless of other body fluids included in mixed stains. The successful construction of the panel will play a vital role in the comprehensive analysis of blood-containing mixtures in forensic practice.

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All data generated or analyzed during this study are included in this published article and its supplementary materials.

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Funding

This work was supported by the Basic Research Program of Shanxi Province [No. 20210302123313] and the National Natural Science Foundation of China [No. 81701868, No. 82171876 and No. 82030058].

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Author notes

  1. Zeqin Li and Na Liu contributed equally to this work.

Authors and Affiliations

  1. School of Forensic Medicine, Shanxi Medical University, Jinzhong, 030600, Shanxi, China

    Zeqin Li, Na Liu, Fang Yuan, Jinding Liu, Feng Liu, Jianbo Ren, Jiangwei Yan & Gengqian Zhang

  2. Shanxi Key Laboratory of Forensic Medicine, Jinzhong, 030600, Shanxi, China

    Zeqin Li, Na Liu, Fang Yuan, Jinding Liu, Feng Liu, Jianbo Ren, Jiangwei Yan & Gengqian Zhang

  3. Department of Biotechnology, Biomedical Sciences College, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, 250117, Shandong, China

    Zimeng Guan

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  1. Zeqin Li
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  3. Fang Yuan
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Contributions

The authors, Gengqian Zhang and Zeqin Li, were responsible for experimental conception and design. Zeqin Li, Na Liu, Fang Yuan, and Zimeng Guan carried out experimental preparation, operation, and data processing. The first draft of the manuscript was written by Zeqin Li and Na Liu. Zimeng Guan, Jinding Liu, Feng Liu, and Jianbo Ren gave valuable opinions and suggestions on the writing and revision of the manuscript. The authors Gengqian Zhang and Jiangwei Yan revised it critically for important intellectual content. All authors approved the final manuscript.

Corresponding authors

Correspondence to Jiangwei Yan or Gengqian Zhang.

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Approval was obtained from the ethics committee of Shanxi Medical University (No. 2021GLL052). The procedures used in this study adhere to the tenets of the Declaration of Helsinki.

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The authors affirm that human research participants provided informed consent for the publication of the data in Supplementary Table S7.

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Li, Z., Liu, N., Yuan, F. et al. Development of a novel panel for blood identification based on blood-specific CpG-linked SNP markers. Int J Legal Med 138, 1205–1219 (2024). https://doi.org/10.1007/s00414-023-03105-y

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