576 Genetik und Evolution
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Polymerase Chain Reaction
(2021)
DNA Sequencing
(2021)
Isolation of DNA and RNA
(2021)
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.
Survival of patients with pediatric acute lymphoblastic leukemia (ALL) after allogeneic hematopoietic stem cell transplantation (allo-SCT) is mainly compromised by leukemia relapse, carrying dismal prognosis. As novel individualized therapeutic approaches are urgently needed, we performed whole-exome sequencing of leukemic blasts of 10 children with post–allo-SCT relapses with the aim of thoroughly characterizing the mutational landscape and identifying druggable mutations. We found that post–allo-SCT ALL relapses display highly diverse and mostly patient-individual genetic lesions. Moreover, mutational cluster analysis showed substantial clonal dynamics during leukemia progression from initial diagnosis to relapse after allo-SCT. Only very few alterations stayed constant over time. This dynamic clonality was exemplified by the detection of thiopurine resistance-mediating mutations in the nucleotidase NT5C2 in 3 patients’ first relapses, which disappeared in the post–allo-SCT relapses on relief of selective pressure of maintenance chemotherapy. Moreover, we identified TP53 mutations in 4 of 10 patients after allo-SCT, reflecting acquired chemoresistance associated with selective pressure of prior antineoplastic treatment. Finally, in 9 of 10 children’s post–allo-SCT relapse, we found alterations in genes for which targeted therapies with novel agents are readily available. We could show efficient targeting of leukemic blasts by APR-246 in 2 patients carrying TP53 mutations. Our findings shed light on the genetic basis of post–allo-SCT relapse and may pave the way for unraveling novel therapeutic strategies in this challenging situation.
Die Diskussion über Nutzen und Risiken der Biound Gentechnik beherrschte die Jahre von 1998 bis 2001. Die „grüne Gentechnik“ kämpfte auch 1997 gegen wachsende Widerstände. Erst als sich im Jahr 2001 die Medien mit der „roten“ Gentechnik befassten, sank der Anteil kritischer Vorbehalte und stieg die Akzeptanz gegenüber gentechnisch herstellten Medikamenten in der Bevölkerung. Die Entschlüsselung der menschlichen Erbanlage und die Diskussion um das Klonen von Menschen sowie die Forschung mit embryonalen Stammzellen führte neue Aspekte in die öffentliche Diskussion ein. Die Debatte findet seither auf allen politischen Ebenen statt. Im Zusammenhang mit der Genforschung werden in den Medien immer häufiger Moral und Ökonomie gegenübergestellt und gegeneinander abgewogen. In diesem Wirrwarr von unterschiedlichen und häufig auch widerstreitenden Interessen ist es nicht einfach, sich zurechtzufinden und schließlich zu einer eigenen Position zu gelangen. Umso wichtiger ist daher eine nüchterne Darstellung der Sachverhalte.