218 Chapter 8 study by Lee et al., the magnetic bead-based MagMAX cfDNA isolation kit (Applied Biosystems, US) seemed most efficient in extracting fragments of 50-100 base pairs (73). However, according to the outcomes of Oreskovic et al., this kit is unable to recover short fragments of 40 base pairs (70). The outcomes of these comparison studies underline that the most optimal urinary cfDNA extraction method is yet to be defined. The choice of the optimal urine DNA extraction kit can also be influenced by specific test requirements and the particular urine fraction being utilized. For example, the Quick-DNA Urine kit used in this thesis extracts both short and long DNA fragments, which appeared suitable for endometrial cancer detection in full void urine. This could be explained by the fact that the endometrial cancer signal seems to derive from short transrenally excreted fragments and long fragments of shedded DNA, as shown in Chapter 2. By incorporating size selection during library preparation, sequencing of only short cfDNA fragments is still feasible using DNA extracted with the Quick-DNA Urine kit, as successfully performed for urine supernatant samples of ovarian cancer patients in Chapter 5. Similarly, this kit has shown utility across other cancer types in which the methylation signal most likely originated from short transrenally excreted DNA fragments, including colorectal (74) and lung cancer (Chapters 6 and 7). 8.3.4 Selective enrichment of methylated DNA Methylated DNA can also be selectively enriched before PCR or sequencing by leveraging the specific properties of 5-methylcytosine. This could be accomplished by using affinity approaches that specifically bind to methylated DNA, using either 5-methylcytosine antibodies (e.g. cell-free methylated DNA immunoprecipitation-sequencing [cfMeDIPseq] (75)), methyl-CpG binding domain proteins (e.g. methyl-binding domain sequencing [MBD-seq] (76) or methylated DNA capture by affinity purification [MethylCap-seq] (77)). Alternatively, methylation-dependent restriction enzymes could be used to enrich for methylated DNA (e.g. [MeD-seq] (61)). Methyl-binding proteins can also be immobilized on a capture surface to selectively enrich for methylated DNA from liquid biopsy samples (78). Such a microfluidic chip system would allow for the development of a lab-on-a-chip device for point-of-care diagnostics, which we are actively investigating in collaboration with the University of Twente. Point-of-care diagnostics enables healthcare providers to quickly and affordably run diagnostic tests in the presence of the patient, without sending a sample to the laboratory. In the future, such systems could be run by general practitioners which could in turn guide specialist referrals in case of a positive test outcome.
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