Barcoding von genomischer DNA und Anwendung im Bereich des DNA-Mappings

  • Barcoding of genomic DNA and application in the area of DNA-mapping

Schilcher, Felix Maximilian; Weinhold, Elmar (Thesis advisor); Wöll, Dominik (Thesis advisor)

Aachen : RWTH Aachen University (2021)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021

Abstract

In the course of this work, the sequence specific modification of DNA using methyltransferase-directed Transfer of Activated Groups (mTAG) was further developed. One focus of this work was the synthesis of analogs of the natural cofactor of DNA methyltransferases (DNA-MTases) AdoMet. This includes AdoMet analogs for the transfer of reactive as well as fluorescent and sterically demanding groups. The latter includes analogs with oligodeoxynucleotide modifications. In addition, the enzymatic activity of the new AdoMet analogs was studied using the DNA-MTases M.TaqI and M.BseCI. Compared to that of reactive and fluorescent AdoMet analogs, the activity of ODN-modified AdoMet analogs was generally lower and differed greatly from one another. EMSA experiments were used to show the influence of the sterically demanding modification on electrophoretic mobility of modified DNA-fragments. On this basis, one-step modification reactions were performed using the newly developed AdoMet analogs. The modified DNA was then analyzed using different methods, either optically or electronically. This includes analysis using the so called C-Trap as well as nanopores, which were carried out externally by cooperation partners, as well as the analysis of stretched and immobilized DNA using fluorescence microscopy. Another focus of this work was the experimental development of DNA modification using the mTAG method. On one hand, new specificities were introduced implementing a newly developed labelling strategy called DNA-blocking. On the other hand, the mTAG method was extended to genomic DNA embedded in agarose plugs. Finally, standardized computational analysis routines were developed, which were then used to automate the analysis of the fluorescence microscopy of stretched and immobilized DNA to determine and compare the quality of different modification experiments on the basis of key figures.

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