Competitive-Binding Based Optical DNA Mapping - From Bacterial Plasmids to the Human Genome
Significant advances within the field of DNA sequencing have allowed us to study DNA at a level of detail that was previously impossible. However, dynamic genomic regions with a high degree of structural variations, while being linked to disease in humans and increased resistance to antibiotics, are still challenging to characterize. Furthermore, DNA sequencing for bacterial diagnostics and detection of resistance genes is presently hampered by the excessive lead times associated with the overall complexity of the applied methods.
This Thesis describes the development of novel assays based on optical DNA mapping, which, although studying DNA at a lower resolution, is capable of rapid processing of significantly larger DNA fragments compared to sequencing. The fluorescent labeling in the assays presented here relies on competitive DNA binding between the emissive YOYO-1 and the sequence-specific, non-emissive, netropsin. The labeled DNA is then stretched in nanofluidic channels and imaged using fluorescence microscopy, enabling extraction of coarse-grained sequence information from ultralong DNA molecules at the single-molecule level.
The results demonstrate how competitive binding-based optical DNA mapping can be used to characterize and trace bacterial DNA, responsible for the spread of antibiotic resistance. The mapped bacterial DNA can also be used to identify bacterial species in complex mixtures and directly from clinical samples. Additionally, so-called long-range sequence information of the human genome can be obtained, with possible future applications including detection of disease-related structural variations and epigenetic profiling.
optical DNA mapping