Glenn M.G. Theunissen
DNA profiling of single sperm cells and single skin flakes in forensics using micromanipulation and whole genome amplification.
Theunissen, Glenn M.G.
Paul Kong Thoo Lin
In forensic DNA profiling, the occurrence of complex mixed profiles is currently a common issue. Cases involving intimate swabs or skin flake tape liftings are prone to mixed profiles, because of more than one donor contributing to a DNA sample. By DNA profiling of single spermatozoa and skin flakes, problems associated with mixed profile could ideally be overcome. However, PCR is not a sensitive enough method to generate DNA profiles by STRs on single cells. Moreover, high quality intact DNA is required, but is not always available in skin flakes due to degradation. Additionally, single skin flakes are difficult to discriminate from other similar looking particles on tape liftings used to secure DNA samples from evidence. The main purpose of this study was to develop a method that enables DNA profiling of single sperm cells and skin flakes. After studying multiple whole genome amplification (WGA) protocols, REPLI-g Single Cell WGA was selected due to its suitability in the pre-amplification step of template DNA. Micromanipulation was used to isolate single spermatozoa. Furthermore, micromanipulation in combination with REPLI-g Single Cell WGA resulted in successful DNA profiling of single spermatozoa by using autosomal STRs as well as X- and Y-chromosomal STRs. The single spermatozoa DNA profiling method described in this thesis was successfully used to identify male contributors from mock intimate swabs with a mixture of semen from multiple male contributors. Different dyes were analysed to develop a staining method to discriminate skin flakes from other particles including particles such as those from hair cosmetic products. From all dyes tested, Orange G was the only dye which successfully discriminated skin flakes from hair product particles. Also, an alkaline based lysis protocol was developed that allowed PCR to be carried out directly on the lysates of single skin flakes. Furthermore, REPLI-g Single Cell WGA was tested on single skin flakes. In contrast to the single spermatozoa, REPLI-g Single Cell WGA was not successful in DNA profiling of single skin flakes. The single skin flake DNA profiling method described in this thesis was successfully used in correctly identifying contributors from mock mixed DNA evidence. Additionally, a small amplicon-based NGS method was tested on single skin flakes. Compared to the PCR and CE approach, the small amplicon-based NGS method improved DNA profiling of single skin flakes, giving a significant increase in allele recovery. In conclusion, this study shows circumventing mixtures is possible by DNA profiling of single spermatozoa, using micromanipulation and WGA. Furthermore, DNA profiling of single skin flakes has been improved by the staining of tape liftings methodology with Orange G, alkaline lysis, direct-PCR and a small amplicon-based NGS approach. Nonetheless, future work is required to assess the performance of the single spermatozoa method on mock swabs with more diluted semen. Also, commercially available NGS kits should be tested with single skin flakes and compared with the in-house NGS method.
|Institution Citation||THEUNISSEN, G.M.G. 2020. DNA profiling of single sperm cells and single skin flakes in forensics using micromanipulation and whole genome amplification. Robert Gordon University [online], PhD thesis. Available from: https://openair.rgu.ac.uk|
|Keywords||DNA profiling; Skin cells; Skin flakes; Sperm cells; Spermatozoa; Whole genome amplification (WGA); Next generation sequencing (NGS)|
THEUNISSEN 2020 DNA profiling of single sperm cells
Copyright: the author and Robert Gordon University