Toenails as an alternative source material for the extraction of DNA from decomposed human remains
Introduction
In the State of Victoria, Australia, deceased individuals (not suitable for visual identification) are typically identified by comparison of the DNA profile obtained from a sample of the deceased to the DNA profile of a reference sample from a relative or a known sample of the deceased; a process performed by the Victorian Institute of Forensic Medicine (VIFM). The sample routinely obtained for identification in decomposed cases is bone, as blood (suitable for DNA analysis) is rarely obtained from decomposed remains. Bone typically provides a good yield of adequate quality DNA due to its hard structure protecting the DNA from degradation [1] leading to Short Tandem Repeat (STR) DNA profiles suitable for identification purposes.
Despite the success of bone as a primary sample type for decomposed cases, its retrieval and processing for DNA identifications has some associated issues including; the invasiveness of the surgical procedure to remove the bone from the deceased; the occupational health and safety (OH&S) risks to staff when using saws to retrieve and sample the bone; the time taken to prepare and sample the bone; and the requirement for refrigerated storage of the sample.
Many of the forensic studies analysing nail material focus on identifying perpetrators of crimes from fingernail scrapings of the victims (see [2], [3], [4], [5], [6]). There are few studies to date that describe the extraction of DNA from nail material from decomposed remains for identification purposes (some examples are displayed by [7], [8], [9]). Toenails were selected for this study as they are less exposed to exogenous DNA sources due to feet often being protected by shoes or socks. This idea that toenails may display lower numbers of mixed profiles when compared to fingernails has been suggested previously [7].
The theory underpinning the use of nails for DNA extraction is that, analogous to bone, they are composed of a hard biological material which is resistant to environmental damage and the effects of decomposition [10], [11]. In comparison with bone, toenails can be collected easily and non-invasively which results in fewer OH&S risks [7], [12]. Nail material may also require less time to process to obtain a DNA profile [7] leading to a quicker identification of the body which in turn provides faster answers to distressed families waiting for the identification results. In the circumstances of a mass fatality event, nails also have the benefit of being able to be collected by persons with minimal training in sample collection and take up minimal storage space without the requirement for refrigeration [7], [13].
In an effort to overcome the issues associated with bone samples for decomposed cases, a study was commenced to develop, validate and implement a method using toenails as the source material for DNA extraction. Described in this study is the optimisation of two DNA extraction methods for toenail material – one organic and one utilising a silica based column purification. The application of the silica based method for the extraction of DNA from toenail samples for 30 decomposed coronial cases, and the comparison to the conventional sample taken, is also described.
Section snippets
Extraction protocol selection
Two extraction protocols optimised by the Molecular Biology Laboratory (MBL) at the VIFM for hair samples were selected as the basis for optimisation using toenails as the source material. One protocol utilised the Qiagen DNA Investigator Kit and the other comprised of an in-house prepared digestion buffer with phenol chloroform purification and subsequent concentration by filtration.
Toenails and buccal swabs from live donors
Consenting donors were asked to provide clippings of their own toenails for use in the study. Buccal swabs were
Selection, optimisation and verification of methods developed
Methods already in use in the MBL for the extraction of DNA from hair samples were selected for adaption to nail material donated from live volunteers. The use of existing methods allowed for easy implementation without requiring the purchase of additional equipment or reagents. Live donor volunteers were used to prevent the unnecessary removal of material from deceased individuals during protocol optimisation. Both optimised methods were successful in extracting sufficient amounts of DNA from
Conclusions and wider implications
This study has successfully optimised two extraction methods capable of extracting DNA from toenail material. The protocol utilising the QIAGEN DNA Investigator Kit has been shown to successfully recover sufficient DNA from toenail samples from decomposed remains (case work) for STR profiling for identification purposes. Even with toenails mostly displaying an overall poorer profile quality when compared to the conventional sample for that case, the profiles for 28 of the toenails would have
Acknowledgments
The authors would like to sincerely thank the live volunteers for the provision of toenail samples that enabled the methods to be developed. We would also like to acknowledge the input and contributions of staff in the Forensic Technical Services and the Molecular Biology Laboratory at the Victorian Institute of Forensic Medicine for their technical expertise and support. The project would not have occurred without the support of the Coroners Court of Victoria. VIFM Ethics Approval No. EC
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2021, Forensic Science InternationalCitation Excerpt :Slightly less full STR profiles were obtained when fingernails were used for extractions with both kits (75% for DNeasy® and 70% for SwabSolution™). Schlenker et al. [16] reported in their study, that 26 out of the 30 analyzed toenail samples achieved a full STR profile. Piccinini et al. [14] achieved similar results for fingernails collected from deceased exhumed after 2–15 years.