| Abstract [eng] |
DNA is furnished with a variety of epigenetic modifications conferring an alternation in gene expression during development and beyond. To enhance and supplement current knowledge on how genomic methylation is established and maintained during development, senescence, and disease, it is essential to track methyltransferase activity. Therefore, one method to employ is the modification of Cytosine bases using engineered MTases capable of attaining synthetic groups from their corresponding synthetic cofactors. Further, detection of said modifications by nanopore sequencing would be advantageous for more extensive, higher-throughput research. Therefore, this project aimed at synthetically modifying murine gene fragments to train and test nanopore detection of synthetic cytosine modifications. The genes selected are of murine origin with some implications in mouse neurogenesis during embryonic development. Three objectives were set in place to be met. Firstly, the optimization of PCR conditions for the gene fragments. Secondly, modifying the selected PCR products, having the highest quality and yield, with synthetic and natural modifications. And finally, nanopore sequencing of selected modified fragments with direct modification detection. Results revealed high and mostly specific PCR product yields when amplifying the selected gene promoter regions. With regards to optimizing the synthetic modifications, it was achieved to produce selected fragments with either natural (mC and hmC) or synthetic modifications, and all in combination. Finally, detection of the synthetic modifications was validated upon nanopore sequencing with varying degrees of base-calling confidence and reads coverage. We drew from these results that further optimizations would be required for the PCR and modification reactions, in addition to further training of the nanopore sequencing algorithm for more consolidated identification of synthetic modifications. |
