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To determine whether a DNA type can be detected from a touch sample, we established a scenario imitating a crime in which an assailant attempted to push a hand towel into the victim’s mouth. Skin secretion from the assailant and saliva from the victim were assumed to be attached to the towel. These towels potentially contained a mixture of cells from skin and saliva with a low copy number (LCN). Extracted DNA was used as a template for DNA analysis using AmpFlSTR Identifiler Plus Kit (Thermo Fisher), mtDNA (D-loop) sequencing using Dye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher), and MPS sequencing using the MiSeq FGx System (Verogen). The results showed that a mixed profile of the assailant and the victim was obtained from four samples, while a single profile of the assailant was obtained from six samples out of ten using each of the above three methods. Regarding these three methods, their detection power was approximately equivalent and extremely high; the SNP method by FGx is the most sensitive and reliable for the analysis of mixed samples.
Recent progress in DNA technology has enabled DNA detection from poor-quality samples or trace amounts of cells. Here, we examined the possibility of detecting trace amounts of DNA. Therefore, we established a scenario imitating a crime in which an assailant attempted to push a hand towel into the victim’s mouth and murdered them by suffocation. Skin secretion from the assailant and saliva from the victim were assumed to be attached to the towel. In this case, it is a focus of interest whether we can detect a DNA type of cells derived from the trace amount of skin from the criminal in addition to DNA derived from saliva of the victim. In brief, the evidence for identification of the criminals can be obtained or not.
2. Materials and methods
We obtained part of the towel that the assailant had touched and extracted DNA from it. Various crime scenes were assumed, which were based on imitated scenarios in which a criminal conducted an act of violence using towels on ten victims.
These towels were suspected to contain a mixture of cells from skin and saliva. Because the saliva would be invisible to the naked eye, a specific light device (Polilight-Flare Plus, Hitachi) was used to identify its staining.
Organic DNA extraction was carried out using phenol/chloroform with extraction buffer (Promega) in a centrifuge tube. Extracted DNA was used as a template for DNA analysis using three methods:
1)
STR typing using AmpFlSTR Identifiler Plus Kit (Thermo Fisher);
2)
Mitochondrial DNA (D-loop) sequencing using Dye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher); and
3)
Hyper-sensitive STR and SNP analysis using MiSeq FGx System (Verogen).
3. Results
After comparison with a reference sample, a mixed profile of the assailant and the victim was obtained from four samples, while a single profile of the assailant was obtained from six samples out of ten using each of these three methods.
Among these methods, STR typing using AmpFlSTR Identifiler Plus Kit had a high rate of detection of the assailant–victim mixed profile (Fig. 1). However, there were samples in which the results indicated a slight mixture for a nonspecific band [
]. By the mtDNA analysis, DNA derived from saliva tended to be predominantly detected. Among the samples, three cases out of ten showed a clear mixed base pattern (Fig. 2).
Fig. 1Result of STR typing using AmpFlSTR Identifiler Plus Kit (Thermo Fisher).
The FGx method also had high power to detect mixed alleles from a towel; 17/59 loci could be typed with STR markers, while 70/94 loci were typed with SNP markers. Notably, the detection power was particularly good for SNPs, for which consistent results were obtained. We can obtain further information when we refer to data under the analytical threshold.
4. Discussion
All testing proved effective for examining ten touch-samples containing skin cells in actual forensic cases. Detection of the DNA profile was possible from a towel sample touched by an assailant, using each of these methods. Therefore, we should use these methods appropriately depending on the nature of the criminal case. Among the three studied methods, the detection power was approximately equivalent and extremely high.
The STR method by capillary electrophoresis had high detection power and was effective for a screening test. Meanwhile, the mtDNA testing could precisely reproduce the dominant DNA type, but the detection power for mixed DNA was rather weak.
As such, the SNP method by FGx is the most sensitive and reliable for analyzing trace amounts of mixed DNA.
5. Conclusion
We conducted DNA typing from a touch sample by STR typing, mtDNA sequencing, and using an FGx system with a next-generation sequencer. Of these approaches, the SNP method by FGx was found to be the most sensitive and reliable for analyzing trace amounts of mixed DNA.
Declaration of Competing Interest
None.
Acknowledgments
We thank Edanz (www.edanzediting.co.jp) for editing the English text of a draft of this manuscript.
References
Wickenheiser R.A.
Trace DNA: A review, discussion of theory, and application of the transfer of trace quantities of DNA through skin contact.
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