Methyltransferase-directed labelling for visualisation and identification of DNA

Wand, Nathaniel Ovelin (2019). Methyltransferase-directed labelling for visualisation and identification of DNA. University of Birmingham. Ph.D.

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Abstract

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.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):