The development and initial testing of the vertical comet assay, a novel technique for the study of DNA damage and repair

Thesis (M.S.) University of Alaska Fairbanks, 2021Gene-specific repair is the idea that certain segments of the genome repair at a faster rate than others. This idea, if demonstrated with adequate evidence, would have large implications for the field of biology as a whole, with special significance for the fields of oncology, gerontology, and molecular and cell biology. The concept of gene-specific repair is not new, with the earliest references in the literature dating back to 1985, and there is a small volume of evidence derived over the years. However, the evidence generated so far is not enough to conclusively prove the existence of gene-specific DNA repair. Generally, the reason for the lack of evidence is that currently available assays and techniques are not adequate for the study of gene-specific repair on a large scale as the techniques that are available require a great deal of time, funding, and skill to generate a reliable and conclusive data set for a single gene, let alone the entire genome. The vertical comet technique described here-in is a response to the perceived need for a robust and relatively high-throughput technique for the study of gene-specific DNA repair. In the traditional comet assay, cells are fixed in agarose gel. Electrophoresis is performed, following several treatment steps, to create a ball of nuclear material embedded in the agarose gel with a 'tail' of smaller pieces of nominally damaged DNA extending to one side. The vertical comet captures this tail DNA in a buffer, allowing for its further analysis with processes such as quantification, PCR/qPCR, and sequencing. The capture of the tail DNA not only makes genespecific repair studies possible, it also allows the vertical comet to fulfill the role of the traditional comet assay with a number of advantages - a reduction in human bias, a reduction in labor-hours required for work, and a reduction in inter-lab variability of results.National Institute Of General Medical Sciences of the National Institutes of Health under Award Numbers UL1GM118991, TL4GM118992, or RL5GM118990General introduction -- Chapter 1: Gene specific repair. Possible mechanisms and implications -- Chapter 2: - Vertical comet: a new technique to measure DNA damage and repair -- General conclusion