Enzymatic DNA modifications for genetic diagnostics

Rushton, Ashleigh E (2020). Enzymatic DNA modifications for genetic diagnostics. University of Birmingham. Ph.D.

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Abstract

Techniques for genetic diagnostics are advancing at a rapid pace, with new technologies constantly emerging as we understand an increasing amount about the human genome. Methods of visualising DNA – such as fluorescence in situ hybridisation (FISH) – have been used for decades, but novel approaches are now opening up new applications for the technique, such as rapid hybridisation times for faster results, or investigation of areas of the genome that have previously been inaccessible due limitations in the current technology. This thesis shows how methyltransferase (MTase) enzymes can be used as a means to explore different regions of the human genome for various clinical applications.

Chapter three sees the optimisation of the expression of the MTase, M.TaqI. This protein is used throughout this thesis, alongside natural cofactor AdoMet and cofactor analogue AdoHcy-6-N3, to label DNA site specifically. This technology is used for various experiments in the following chapters. Chapter three also attempts to produce mutated versions of other MTases for similar labelling experiments.

Chapter four uses the M.TaqI labelling technology to label oligoprobes, short sequences of DNA, for potential use in FISH diagnostics; specifically looking at aneuploidy, which can be indicative of certain cancers. Different conditions are tested to obtain the highest signal to noise ratio, to ensure confident detection of centromeres of the chromosome 17 in patients. These results are used to design a probe set that can simultaneously detect the loss of chromosomes 1, 7 or 17 – which is associated with poor prognosis in acute lymphocytic leukaemia – by labelling each probe with a different colour dye. As oligoprobes can detect
highly homologous sequences, this chapter also explores the use of this technique in potentially detecting single nucleotide polymorphisms in the human genome, which are associated with many diseases such as spinal muscular atrophy.

In Chapter five, this MTase labelling technology is used to produce probes for single genes as opposed to centromeric regions. Focus is on the BCR gene, as it is associated with the BCR/ABL translocation, prevalent in most cases of chronic myeloid leukaemia.

Finally, Chapter six explores DNA mapping as an approach to detect small DNA mutations, by investigating the pattern in fluorescence intensity of two highly similar sequences labelled with the MTase technology. This could enable certain carriers of spinal muscular atrophy to be identified.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):