Optical Mapping of Bacterial Plasmids

Abstract: Bacteria that have acquired resistance to antibiotics represent one of the largest threats to human health and modern health care. The genes encoding resistance are frequently spread via the transfer of plasmids, which are circular double stranded DNA molecules separate from the chromosomal DNA of the bacteria. The increasing prevalence of resistant bacteria, in combination with the absence of new major discoveries of antimicrobial drugs, means that health care could soon be entering a “post-antibiotic er". Besides developing new drugs, methods capable of rapidly detecting bacteria that have acquired resistance to antibiotics are of paramount importance for clinical point of care applications.In this Thesis, the development of an assay for rapid plasmid characterization is described. The method is based on optical DNA mapping using competitive binding of the fluorophore YOYO-1 and the sequence-specific, non-fluorescent, molecule netropsin, to DNA. The fluorescently labeled DNA is stretched in nanofluidic channels and imaged using fluorescence microscopy, enabling coarse-grained sequence information to be read from the intact plasmid at the single plasmid level. Additionally, an approach for gene detection on individual plasmids is described, combining the CRISPR/Cas9 system with optical DNA mapping.The results demonstrate how the assay can be used to obtain the number of different plasmids in a sample, the size of each plasmid, an optical barcode for tracing and identification, as well as information about which plasmid that carries a specific (resistance) gene. Overall, the assay shows great potential as a first step of plasmid characterization in point of care diagnostics.