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%0 Thesis
%A Eiden, Michael
%T Entwicklung enzymatischer Methoden zur DNA-Methylierungssequenzierung
%I RWTH Aachen University
%V Dissertation
%C Aachen
%M RWTH-2024-07022
%P 1 Online-Ressource : Illustrationen
%D 2024
%Z Veröffentlicht auf dem Publikationsserver der RWTH Aachen University
%Z Dissertation, RWTH Aachen University, 2024
%X The overall aim of this work was to develop different methods to enable reliable CpG methylation detection. The first aim was to find out whether a higher modification yield of the target sequences on the DNA under investigation could be achieved using two-step labeling with sterically demanding ODN and the DNA adenine N6 MTase M.TaqI (WT), which in turn should be reflected in an increase in the blocking observed during Sanger sequencing. This increase in blocking or the increase in intensity decreases in the electropherograms could be registered in most cases of the sequences examined or sequenced. A further topic investigated in this work was the question of the extent to which other sterically demanding groups transferred to DNA, such as those carrying fluorophore groups, can influence the course or result of Sanger sequencing. In particular, the properties of the structure of fluorophores played a role here, as these often consist of extended aromatic systems with charges. Initially, four different fluorescent cofactors were tested for their activity using the DNA adenine N6 MTase M.TaqI (WT) and the DNA cytosine C5 MTase M.HhaI (tm) by means of modification/restriction tests. If this could be detected by protecting the DNA, corresponding modification samples were prepared and subjected to Sanger sequencing. In addition to CpG methylation detection by transferring large groups to corresponding recognition sequences with subsequent Sanger sequencing, a further approach was taken to detect CpG methylation. For this purpose, a cofactor that could be suitable for nanopore sequencing was to be identified, synthesized and tested for its activity. This cofactor or the group to be transferred should have two main properties. On the one hand, it should not be too large in order to prevent possible blocking of the nanopore used, and on the other hand, it should carry a charge in order to exert the greatest possible influence on the ion current measured during nanopore sequencing. Based on these considerations, the cofactor Carboxy-AdoMet was synthesized, which carries a carboxyl group. First, activity tests were performed with λ-DNA and with different CpG methylation-dependent DNA MTases, namely the wild types of M.TaqI, M.MpeI and M.SssI as well as different variants. A methylation-dependent DNA MTase with a 5`-CG-3` recognition sequence should be used for nanopore sequencing. Therefore, the DNA-MTase M.MpeI (dm) was used for the carboxymethylation of T7 DNA to be analyzed, because it showed the highest activity with the cofactor carboxy-AdoMet. Since λ-DNA was used in the previous modification/restriction tests, a modification/restriction test was also carried out with T7-DNA and M.MpeI (dm) for verification purposes, and the activity was also similar here. Based on this result, T7 DNA was modified and subjected to nanopore sequencing. The results of this sequencing showed that carboxymethylation leads to an altered course of the voltage signals around and within the CpG sequences compared to unmodified DNA.
%F PUB:(DE-HGF)11
%9 Dissertation / PhD Thesis
%R 10.18154/RWTH-2024-07022
%U https://publications.rwth-aachen.de/record/989927