BackgroundThe DNA modification 5-hydroxymethylcytosine (5hmC) is now referred to as the sixth base of DNA with evidence of tissue-specific patterns and correlation with gene regulation and expression. This epigenetic mark was recently reported as a potential biomarker for multiple types of cancer, but its application in the clinic is limited by the utility of recent 5hmC quantification assays. We use a recently developed, ultra-sensitive, fluorescence-based single-molecule method for global quantification of 5hmC in genomic DNA. The high sensitivity of the method gives access to precise quantification of extremely low 5hmC levels common in many cancers.MethodsWe assessed 5hmC levels in DNA extracted from a set of colon and blood cancer samples and compared 5hmC levels with healthy controls, in a single-molecule approach.ResultsUsing our method, we observed a significantly reduced level of 5hmC in blood and colon cancers and could distinguish between colon tumor and colon tissue adjacent to the tumor based on the global levels of this molecular biomarker.ConclusionsSingle-molecule detection of 5hmC allows distinguishing between malignant and healthy tissue in clinically relevant and accessible tissue such as blood and colon. The presented method outperforms current commercially available quantification kits and may potentially be developed into a widely used, 5hmC quantification assay for research and clinical diagnostics. Furthermore, using this method, we confirm that 5hmC is a good molecular biomarker for diagnosing colon and various types of blood cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-017-0368-9) contains supplementary material, which is available to authorized users.

Epigenetic transformations may provide early indicators for cancer and other disease. Specifically, the amount of genomic 5‐hydroxymethylcytosine (5‐hmC) was shown to be globally reduced in a wide range of cancers. The integration of this global biomarker into diagnostic workflows is hampered by the limitations of current 5‐hmC quantification methods. Here we present and validate a fluorescence‐based platform for high‐throughput and cost‐effective quantification of global genomic 5‐hmC levels. We utilized the assay to characterize cancerous tissues based on their 5‐hmC content, and observed a pronounced reduction in 5‐hmC level in various cancer types. We present data for glioblastoma, colorectal cancer, multiple myeloma, chronic lymphocytic leukemia and pancreatic cancer, compared to corresponding controls. Potentially, the technique could also be used to follow response to treatment for personalized treatment selection. We present initial proof‐of‐concept data for treatment of familial adenomatous polyposis.

5-Hydroxymethylcytosine (5hmC), a modified form of the DNA base cytosine, is an important epigenetic mark linked to regulation of gene expression in development, and tumorigenesis. We have developed a spectroscopic method for a global quantification of 5hmC in genomic DNA. The assay is performed within a multiwell plate, which allows simultaneous recording of up to 350 samples. Our quantification procedure of 5hmC is direct, simple, and rapid. It relies on a two-step protocol that consists of enzymatic glucosylation of 5hmC with an azide-modified glucose, followed by a "click reaction" with an alkyne-fluorescent tag. The fluorescence intensity recorded from the DNA sample is proportional to its 5hmC content and can be quantified by a simple plate reader measurement. This labeling technique is specific and highly sensitive, allowing detection of 5hmC down to 0.002% of the total nucleotides. Our results reveal significant variations in the 5hmC content obtained from different mouse tissues, in agreement with previously reported data.

Knowing the amount and type of DNA damage is of great significance for a broad range of clinical and research applications. However, existing methods are either lacking in their ability to distinguish between types of DNA damage or limited in their sensitivity and reproducibility. The method described herein enables rapid and robust quantification of type-specific single-strand DNA damage. The method is based on repair-assisted damage detection (RADD) by which fluorescent nucleotides are incorporated into DNA damage sites using type-specific repair enzymes. Up to 90 DNA samples are then deposited on a multiwell glass slide, and analyzed by a conventional slide scanner for quantification of DNA damage levels. Accurate and sensitive measurements of oxidative or UV-induced DNA damage levels and repair kinetics are presented for both in vitro and in vivo models.

DNA methylation patterns create distinct gene-expression profiles. These patterns are maintained after cell division, thus enabling the differentiation and maintenance of multiple cell types from the same genome sequence. The advantage of this mechanism for transcriptional control is that chemical-encoding allows to rapidly establish new epigenetic patterns 'on-demand' through enzymatic methylation and demethylation of DNA. Here we show that this feature is associated with the fast response of macrophages during their pro-inflammatory activation. By using a combination of mass spectroscopy and single-molecule imaging to quantify global epigenetic changes in the genomes of primary macrophages, we followed three distinct DNA marks (methylated, hydroxymethylated and unmethylated), involved in establishing new DNA methylation patterns during pro-inflammatory activation. The observed epigenetic modulation together with gene-expression data generated for the involved enzymatic machinery may suggest that demethylation upon LPS-activation starts with oxidation of methylated CpGs, followed by excisionrepair of these oxidized bases and their replacement with unmodified cytosine.

DNA methylation patterns create distinct gene expression profiles. These patterns are maintained after cell division, thus enabling the differentiation and maintenance of multiple cell types from the same genome sequence. The advantage of this mechanism for transcriptional control is that chemical-encoding allows to rapidly establish new epigenetic patterns "on-demand" through enzymatic methylation and de-methylation of DNA. Here we show that this feature is associated with the fast response of macrophages during their proinflammatory activation. By using a combination of mass spectroscopy and single-molecule imaging to quantify global epigenetic changes in the genomes of primary macrophages, we followed three distinct DNA marks (methylated, hydroxymethylated and unmethylated), involved in establishing new DNA methylation patterns during pro-inflammatory activation.The observed epigenetic modulation together with gene expression data generated for the involved enzymatic machinery, may suggest that de-methylation upon LPS-activation starts with oxidation of methylated CpGs, followed by excision-repair of these oxidized bases and their replacement with unmodified cytosine.

DNA combing is a widely used method for stretching and immobilising DNA molecules on a surface. Fluorescent labelling of genomic information enables high-resolution optical analysis of DNA at the single-molecule level. Despite its simplicity, the application of DNA combing in diagnostic workflows is still limited, mainly due to difficulties in analysing multiple smallvolume DNA samples in parallel. Here, we report a simple and versatile microfluidic DNA combing technology (μDC), which allows manipulating, stretching and imaging of multiple, microliter scale DNA samples by employing a manifold of parallel microfluidic channels. Using DNA molecules with repetitive units as molecular rulers, we demonstrate that the μDC technology allows uniform stretching of DNA molecules. The stretching ratio remains consistent along individual molecules as well as between different molecules in the various channels, allowing simultaneous quantitative analysis of different samples loaded into parallel channels. Furthermore, we demonstrate the application of μDC to characterise UVB-induced DNA damage levels in human embryonic kidney cells and the spatial correlation between DNA damage sites. Our results point out the potential application of μDC for quantitative and comparative single-molecule studies of genomic features. The extremely simple design of μDC makes it suitable for integration into other microfluidic platforms to facilitate high-throughput DNA analysis in biological research and medical point-of-care applications.

Mapping DNA damage and its repair has immense potential in understanding environmental exposures, their genotoxicity, and their impact on human health. Monitoring changes in genomic stability also aids in the diagnosis of numerous DNA-related diseases, such as cancer, and assists in monitoring their progression and prognosis. Developments in recent years have enabled unprecedented sensitivity in quantifying the global DNA damage dose in cells via fluorescence-based analysis down to the single-molecule level. However, genome-wide maps of DNA damage distribution are challenging to produce. Here, we describe the localization of DNA damage and repair loci by repair-assisted damage detection sequencing (RADD-seq). Based on the enrichment of damage lesions coupled with a pull-down assay and followed by next-generation sequencing, this method is easy to perform and can produce compelling results with minimal coverage. RADD-seq enables the localization of both DNA damage and repair sites for a wide range of single-strand damage types. Using this technique, we created a genome-wide map of the oxidation DNA damage lesion 8-oxo-7,8-dihydroguanine before and after repair. Oxidation lesions were heterogeneously distributed along the human genome, with less damage occurring in tight chromatin regions. Furthermore, we showed repair is prioritized for highly expressed, essential genes and in open chromatin regions. RADD-seq sheds light on cellular repair mechanisms and is capable of identifying genomic hotspots prone to mutation.