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Final Report Summary - DNALIGHTMAP (Mapping structural variation on native chromosomal DNA – a single molecule approach)

The main goal of our research project is to unmask genomic variation. We aim to achieve this goal by developing and utilizing a toolbox of physical barcoding techniques that highlight and read-out information from genomic DNA. Our lab specializes in many areas of optical imaging and spectroscopy with emphasis on single molecule detection and development of imaging based techniques. In addition, we have great interest in developing unique biochemistries for genomic analysis. The combination of these techniques is applied to genomic studies and biomarker detection.
Within the DNALightMap project we have addressed the project goals in three fields of interest:

1 Single molecule genomics by optical mapping:

The principle of this approach is to unravel long genomic DNA molecules and to stretch them such that information can be extracted along the molecule. We create optical barcodes containing genetic and epigenetic information by labeling these long chromosomal DNA molecules with fluorescent tags. Nano-fluidic channels are used to stretch the DNA by flow or electric field and the barcode is directly visualized by single molecule imaging. We are also applying super-resolution imaging techniques in order to increase the resolution and allow detection of genomic aberrations.
In this context we have shown that we can use DNA methyltransferases in combination with synthetic cofactors to introduce a fluorescent tag at specific locations in the genome. This approach creates a fluorescence reference map on the genome relative to which we could later map the positions of genomic aberrations and epigenetic marks.
In order to access more complex genomes we have started utilizing silicon nano-channel arrays that allow stretching and imaging massive amounts of DNA molecules. We are currently working extensively on mapping epigenetic marks and structural variations in the human genome and already have single molecule maps of several regions in the sequenced genome (unpublished). In addition, and in line with the goals of this project we were able to analyze the exact repeat copy number causing the common muscular dystrophy FSHD. In order to further facilitate these studies we have recently been able to demonstrate that we can cut-out a specific region of interest from the genome. This will allow us to study and compare multiple DNA molecules from a specific genomic region, thus enabling us to characterize the genetic and epigenetic variation in this region (Cas9-Assisted Targeting of CHromosome segments (CATCH) enables one-step targeted cloning of large gene clusters (2015) / Nature Communications - accepted). This technique has also been patented and licensed in order to allow future applications in targeted genomics.

2 Epigenetic analysis technologies:

Epigenetics is one of the most exciting and fast growing fields in biology. It constitutes several layers of genomic information that do not involve changes in the underlying DNA sequence and links biological signatures with mental or environmental conditions. We develop new methods for sequencing, as well as targeted and global analysis of various epigenetic markers. We use these novel methods in order to study epigenetic alterations related to disease.
The epigenetic modification 5-hydroxymethycytosine (5-hmC) was discovered in mammalian genomic DNA in 2009. Research related to this mysterious modification has been growing exponentially in an effort to reveal its function and relation to development and disease. We have recently developed a very efficient chemo-enzymatic method for fluorescent labeling of 5-hmC. We use the specific attachment of glucose to 5-hmC by β-glucosyltransferase which transfers a glucose from UDP-glucose onto the hydroxyl group of 5-hmC. By using a chemically modified, azide containing UDP-glucose we could attach a fluorescent molecule to the glucose by copper-free click chemistry. The result is that 5-hmC residues “light-up” and create a fluorescent pattern along the DNA molecules. These patterns could be used for mapping as well as for ultra-sensitive global quantification of 5-hmC (Optical detection of epigenetic marks: Sensitive quantification and direct imaging of individual hydroxymethylcytosine bases (2013) / Chem. Commun., 49 (77), 8599 – 8601). We further developed this technique to a high-throughput, multi-well plate based assay which allows analysis of over 300 samples simultaneously. We demonstrated the quantification of tissue specific 5-hmC levels in 190 mouse tissue samples and demonstrated performance that is superior to most reported methods (Spectroscopic quantification of global 5’ hydroxymethyl cytosine content in genomic DNA (2014) / Analytical Chemistry 86 (16), pp 8231–8237). Our lab is now funded through several agencies for promoting the study of 5-hmC levels in cancer.
In addition to 5-hmC, we have used a similar approach for labeling sites of damage along DNA molecules. DNA damage is a fundamental process linked to almost every field of biological and biomedical research. Most of this damage exists in the form of single-strand damage lesions. Quantifying the global levels of DNA damage lesions in the genomes of malignant cells has the potential to emerge as an informative biomarker for determining predisposition to disease, early diagnostics and assessment of response to therapy. Modulations in the global levels of DNA damage lesions have been shown to correlate with various cancers but are difficult to quantify in a cost effective and timely manner. To address this issue we developed a single molecule technique that enables direct visualization and quantification of damage sites on stretched DNA molecules. We use a cocktail of bacterial repair enzymes to perform in-vitro repair of genomic DNA extracted from cells. Fluorescent nucleotides are incorporated into damage sites in this repair process. When the DNA is stretched on glass slides and imaged, damage sites are seen as fluorescent spots along the DNA contour and can be counted directly. We used this technique to follow DNA damage caused by UV irradiation and exposure to hydrogen peroxide as well as to follow repair dynamics in several cell types including XP deficient cells derived from Xeroderma Pigmentosum patiants (People that lack one of the UV damage repair enzymes and cannot be exposed to sunlight). In order to demonstrate the multiplexing potential of our method we used two different colors to label both DNA damage and 5-hydroxymethyl cytosine on DNA extracted from mouse brain and showing possible correlation between the two (Lighting Up Individual DNA Damage Sites by In Vitro Repair Synthesis (2014). JACS 136, (21), 7771–7776.)
Our protocol is now used by several labs in the world to study various aspects of DNA damage. Our lab has recently been selected to coordinate a Horizon 2020 consortium project where we are applying our ability to detect DNA damage and 5-hmC to novel medical diagnostics platforms.

3 High-throughput single molecule detection:

Another area of interest in the lab is to develop optical methods for detection of rare analytes and weakly interacting biomolecules. Our emphasis is on ultrasensitive detection and quantification of clinical biomarkers. For many of our experiments we are utilizing single-molecule imaging in order to size and classify DNA molecules. By imaging in multiple spectral bands, various genomic tags may be co-localized in order to obtain quantitative sample properties such as the labeling percentage or the distribution of DNA fragment sizes from femtogram amounts of DNA (Sizing femtogram amounts of dsDNA by single-molecule counting (2016) / Nucl. Acids Res., doi:10.1093/nar/gkv904)
In addition, we have recently developed a method for bacteriophage identification based on optical analysis of individual bacteriophage genomes. Specifically, we utilized for the first time a synthetic cofactor for DNA methyltransferase enzymes, that enables a single-step, covalent attachment of a fluorophore to the DNA methylation site. The labelling was used in order to generate a fluorescent profile along DNA molecules stretched in nanochannel arrays. The result is a sequence-specific, continues intensity profile that can be used as a molecular fingerprint for DNA/organism identification. As proof of concept we labelled the genomes of lambda and T7 phages and obtained consistent and unique intensity profiles for both. Using cross correlation analysis we were also able to identify these phages from a background phage library. The outcome of the described work is a new approach for DNA identification and strain typing of samples containing unknown and mixed populations (Bacteriophage strain typing by rapid single molecule analysis (2015) / Nucl. Acids Res., doi: 10.1093/nar/gkv563).

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