Urinary Cell-free DNA Preparation For Detection Of Epigenetic DNA Modifications

5-methylcytosine?5mC?is widely present in mammalian genomic DNA.5mC can be oxidized by TET protein to form 5-hydroxymethylcytosine?5hmC?,5-formylcytosine?5fC?and 5-carboxycytosine?5caC?.5mC and 5hmC play important role in diverse biologicalprogress in mammals,such as embryonic development and regulation of gene expression.It's also found that the aberrant levels of the four DNA epigenetic modifications are closely related to the occurrence and development of cancer.Essentially,it is a potential biomarker for cancer diagnosis,treatment,and prognosis.With the advantages of obtaining easily and ultra-noninvasive,urine cell free DNA?ucfDNA?has been widely used for disease diagnosis and prognosis.However,it's little known about ucfDNA epigenetic modification.Due to the presence of a limited amount of cfDNA in urine,a large volume of urine is needed to obtain sufficient DNA sample.However,the widely used commercial kits have a limited urineextraction capacity,and are expensive,so it is difficult to meet the needs of extracting a large number of urine samples.In this work,we attempted to establish a new method forpreparation of ucfDNA and further develop a ultrahigh-performance liquid chromatography tandem mass spectrometry?HPLC-MS/MS?method for detection of epigeneticmodifications in ucfDNA.First,we prepared a Graphitic Carbon-based solid phase extraction?SPE?cartridge and developed a SPE method for ucfDNA extraction.This method is easy to operate and has a high adsorption capacity.A single SPE column can meet the requirement for extraction of ucfDNA from 200 mL urine.We can obtain 21 ng cfDNA from 80 mL urine.However,under current conditions,some unknown impurities cannot be removed.Next,SiO₂,as an adsorbent,was used to prepare SPE cartridge and to develop a method for extracting ucfDNA.This method operates easily,with high purity of extracted DNA.At the same urine volume,the amount of cfDNA extracted is higher than that obtained using the commercial kit.In addition,5mC,5hmC and 5fC were detected from the extracted humanucfDNA.The relative standard deviation?RSD?is about 5.0%-15.8%.Finally,the SiO₂ extraction method combined with UHPLC-MS/MS detection was used to detect 5mC,5hmC and 5fC in the ucfDNA extracted from volunteers.The results showed that the average levels of these modifications are present in the ucfDNA as 5mC?4.5±0.3%?,5hmC?0.121±0.003%?,and 5fC?0.0079±0.0006%?,respectively.Of note,there is no significant difference in 5mC,5hmC and 5fC levels between male and female samples.In summary,we develop a simple and economic SiO₂ method for the preparation of ucfDNA with high purity.The extracted ucfDNA can be used for the analysis of DNA epigenetic modification.