Methyltransferase-directed labelling for visualisation and identification of DNA

[摘要] New techniques for rapid identification of complex mixtures of viral and bacterial DNA and for visualising multi-copy plasmids in single bacterial cells have been developed using a combination of methyltransferase-directed labelling, molecular combing, and widefield microscopy. In Chapter 2 the protocol for methyltransferase-directed labelling was optimised. A maximum labelling efficiency of 50% (as measured by single molecule counting results) was obtained for Atto 647N-labelled pUC19. In Chapters 3 and 4 the optimised labelling protocol was used to label genomic DNA for optical mapping and identification. The in silico results in Chapter 3 show that the combination of techniques used in this thesis represents the ‘sweet-spot’ for optical mapping and identification of microorganisms. In Chapter 4 this combination of techniques was used along with a new algorithm, for rapid identification of bacteriophages, Adenovirus A, resistance plasmids and bacterial strains in complex mixtures of genomic DNA. Finally, Chapter 5 uses methyltransferase-directed labelling to investigate the mechanisms bacteria use to maintain and transfer resistance plasmids. Atto647N-labelled pUC19 and pRSET B plasmids were visualised in E. coli bacteria and diffusion over several seconds or plasmid segregation over many hours was studied.

[发布机构] University:University of Birmingham;Department:School of Chemistry

[关键词] Q Science;QD Chemistry