Development and Application of a Modular Enzymatic Labeling Process for the Analysis of Diverse DNA Base Modifications
出版項
2021
說明
1 online resource (140 pages)
文字
text
無媒介
computer
成冊
online resource
附註
Source: Dissertations Abstracts International, Volume: 82-08, Section: B
Advisor: Hall, Adam R
Thesis (Ph.D.)--Wake Forest University, 2021
Includes bibliographical references
There exists a large diversity of DNA base modifications, but the ability to study these modifications and their effects on disease development and progression has thus far been limited. Most techniques are only able to examine a few specific modifications, and some methods are destructive to the sample, precluding the possibility of additional analysis.However, cells have evolved enzymes called glycosylases that are used during base excision repair in order to remove these modifications and replace them with appropriate unmodified bases. Thus, we sought to develop a modular enzymatic labeling process that takes advantage of these naturally occurring enzymes and pathways to expand the range of modifications that can be studied.In this work, we have accomplished several aims. First, we have developed a methodology to successfully label a variety of modifications, including uracil, 8-oxoguanine, T:G mismatches, 1,N6-ethenoadenine, and the products of the cytosine demethylation pathway, with several glycosylases that display different types of activity (Chapters 3 and 4).We then validated the success of this labeling method and applied it in two analytical techniques: solid-state nanopores and qPCR. With solid-state nanopores, we were able to measure DNA containing different base modifications by incorporating a biotinylated nucleotide during the labeling process, which produced positive results in our system with concentrations as low as 250 nM (Chapter 5).Following additional optimizations in our protocols (Chapters 6 and 7), we then applied our labeling process to qPCR and examined a more complex system: oxidative damage generated following irradiation in head and neck cancer cell line models. We were able to determine differences in the trends in the accumulation and repair of oxidative damage in these cell lines (Chapter 8).Through this work, we have developed a modular labeling method that can facilitate the study of a wide range of DNA base modifications without the limitations present in traditional methods. We have also demonstrated the applicability of this method to different analytical techniques. This may be of great use in determining the roles of less-studied modifications in organism growth and development, the development and progression of disease, and other genetic studies
Electronic reproduction. Ann Arbor, Mich. : ProQuest, 2021
