University of Twente Student Theses
Increasing the selectivity of hypermethylated DNA enrichment by a two-step washing process
Reuver, I.K.E. (2023) Increasing the selectivity of hypermethylated DNA enrichment by a two-step washing process.
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| Abstract: | In the liquid biopsy urine the cancer biomarker hypermethylated (hmDNA) can be detected. Hypermethylated DNA (hmDNA) involves DNA methylation on certain parts of the genome. The methylation of specific genes can be associated with for example lung and cervical cancer. To determine methylation on a specific sequence, the hmDNA needs to be separated from the nonmethylated DNA. Separation is done using the hmDNA enrichment method. This method involves flushing a DNA solution over a surface modified with methyl binding domain 2 (MBD2) proteins. These MBD2 proteins have affinity for the hmDNA, resulting into surface binding of hmDNA. After the enrichment method, the surface bound DNA is eluted by means of a solution of NH4OH. The achieved DNA concentration is provided by AccuBlue. Using methyl sensitive restriction enzyme digest and quantitative polymerase chain reaction, the ratio of hmDNA/non-methylated DNA was determined after hmDNA enrichment. Currently, a ratio of 10:3 unmethylated DNA/hmDNA is achieved starting with a DNA solution containing 1% hmDNA. The aim of the research is to increase the selectivity of the hmDNA enrichment method further by applying a second washing step with a higher salt concentration. Compared to Ruben Kolkman's research, the achieved amount DNA was 10 times lower than planned, so it was necessary to investigate why. It was eventually found that the hmDNA was not properly methylated and therefore could not bind to the MBD2 proteins. It's important for the next time to use fresh reagents for the hmDNA enrichment experiment and check if the hmDNA is well methylated. Learned from these experiments is what chemistry toke place to enable the hmDNA enrichment experiment and how to determine the concentration from the captured DNA with AccuBlue. Future work should focus on whether the hmDNA enrichment selectivity could be increased by applying a second washing step with a higher salt concentration. |
| Item Type: | Essay (Bachelor) |
| Faculty: | EEMCS: Electrical Engineering, Mathematics and Computer Science |
| Subject: | 50 technical science in general |
| Programme: | Biomedical Technology BSc (56226) |
| Link to this item: | https://purl.utwente.nl/essays/94034 |
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