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@PHDTHESIS{Kver:843084,
author = {Käver, Larissa},
othercontributors = {Weinhold, Elmar and Albrecht, Markus},
title = {{E}ntwicklung einer orthogonalen {M}arkierungsmethode für
die {F}luoreszenzdetektion epigenetischer
{DNA}-{M}odifikationen},
school = {RWTH Aachen University},
type = {Dissertation},
address = {Aachen},
publisher = {RWTH Aachen University},
reportid = {RWTH-2022-02863},
pages = {1 Online-Ressource : Illustrationen, Diagramme},
year = {2022},
note = {Veröffentlicht auf dem Publikationsserver der RWTH Aachen
University; Dissertation, RWTH Aachen University, 2022},
abstract = {5-Methylcytosin (5mC) and its oxidation product
5-Hydroxymethylcytosin (5hmC) are epigenetic DNA
modifications responsible for gene regulation processes and
also known for different types of cancer because of
alterations in DNA methylation and DNA hydroxymethylation.
[1–4] Parallel analysis of the levels of 5mC and 5hmC can
potentially serve as biomarkers for early cancer detection,
monitoring of disease progression and response to treatment.
Current techniques for global quantification of 5mC and 5hmC
are not yet suitable for clinical applications because this
would require very rapid quantification of both
modifications in the specific tissue using two different
fluorophores. Therefore, the aim of this work was to develop
an orthogonal method for parallel global fluorescent
labeling of the epigenetic modifications 5mC and 5hmC. First
focus of this work was the synthesis of analogs of the
natural cofactor AdoMet (1) that exhibited different vinyl
modifications and could be applied in two-step labeling
reactions. Furthermore, the enzymatic activity of the new
cofactor analogues was investigated in activity assays with
different adenine- and cytosine-specific DNA-MTases. Based
on this, the two-step DNA labeling reaction for methylation
detection was studied using the different vinylic cofactor
analogues and several tetrazine conjugates by analysis of
fluorescent labeling with high and low label density. The
analysis of the fluorescently labeled DNA was performed on
immobilized and stretched single DNA-molecules (DNA combing)
using the institute's own fluorescence microscope.
Furthermore, methylation detection was additionally examined
with one-step labeling reaction by sequence-specific
modification of different DNA substrates using fluorescent
cofactor analogues. Due to the important role of the
epigenetic modifications 5mC and 5hmC in the early detection
of cancer, a genomic human DNA sample (breast cancer cell
line, UK Aachen) was also labeled and studied by our
cooperation partners at Tel Aviv University in Israel with
regard to epigenetic information. Besides, in addition to
methylation detection, hydroxymethylation was analyzed in a
two-step labeling reaction, as well as the orthogonal
determination of 5mC and 5hmC by fluorescence microscopy.
Both single detections as well as the orthogonal
determination were additionally quantified with respect to
reaction yields on short duplex ODN by rp-HPLC.[1]P. A.
Jones, S. B. Baylin, Cell 2007, 128, 683.[2]M. C. Haffner,
A. Chaux, A. K. Meeker, D. M. Esopi, J. Gerber, L. G.
Pellakuru, A. Toubaji, P. Argani, C. Iacobuzio-Donahue, W.
G. Nelson, G. J. Netto, A. M. de Marzo, S. Yegnasubramanian,
Oncotarget 2011, 2, 627.[3]M. Ko, Y. Huang, A. M. Jankowska,
U. J. Pape, M. Tahiliani, H. S. Bandukwala, J. An, E. D.
Lamperti, K. P. Koh, R. Ganetzky, X. S. Liu, L. Aravind, S.
Agarwal, J. P. Maciejewski, A. Rao, Nature 2010, 468,
839.[4]A. P. Feinberg, R. Ohlsson, S. Henikoff, Nat. Rev.
Genet. 2006, 7, 21.},
cin = {152620 / 150000},
ddc = {540},
cid = {$I:(DE-82)152620_20140620$ / $I:(DE-82)150000_20140620$},
typ = {PUB:(DE-HGF)11},
doi = {10.18154/RWTH-2022-02863},
url = {https://publications.rwth-aachen.de/record/843084},
}
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