DNA molecules seen at the femtogram level
The DNALIGHTMAP (Mapping structural variation on native chromosomal DNA – a single molecule approach) project has developed a toolbox to view and analyse DNA at the femtogram level (1 mg is 100 million femtograms). One focus was the stretch of genetic material responsible for Facioscapulohumeral muscular dystrophy (FSHD). DNALIGHTMAP researchers have developed a technique to clone long bacterial genomic sequences – up to 110 kb in one step. Cloning long gene segments is essential in the quest to engineer pharmaceuticals – a tough and expensive proposition by traditional methods. To unravel and stretch DNA, the new method uses DNA methyltransferases in combination with synthetic cofactors to produce a fluorescent reference map. The team worked on extensively mapping the human genome and analysed the gene causing FSHD. As a tool for comparison between healthy and mutated genes, researchers cut out specific regions from DNA, enabling genetic and epigenetic analysis. The new technique has been patented and licensed and has been published in Nature Communications(opens in new window). A new technique was devised for fluorescent labelling of 5-hydroxymethycytosine (5-hmC), a key gene expression modulator in the epigenetics arena. Further development resulted in a high-throughput, multi-well plate based assay for the simultaneous analysis of over 300 samples. DNA damage is inextricably linked with disease, notably cancer. Adapting the techniques already developed, researchers generated a single molecule technique to directly visualise and quantify the damaged sites on stretched DNA molecules. For UV irradiation damage, the researchers also followed the repair dynamics in cells from Xeroderma pigmentosum patients who lack just one enzyme. The protocol is being used in several labs around the world to study DNA damage. Identification of bacteriophages is now possible using a new fluorescent profiling method for a molecular fingerprint that can be extended to any DNA sample. Details of these ground-breaking new approaches to DNA analysis can be found in the journals American Chemical Society(opens in new window) and Nucleic Acids Research(opens in new window). As DNA structure and its epigenetics lies at the very heart of disease, DNALIGHTMAP research has wide-ranging applications in diagnosis and therapeutics.
