α-Hemolysin (αHL) is a biological nanopore, which is currently under investigation for implementation into a new method for DNA sequencing. It has been established that αHL is capable of discriminating the canonical bases (adenine, cytosine, guanine and thymine) when they are immobilised within a pore by means of a biotin•streptavidin complex. Work in this thesis develops this procedure for the discrimination of these standard nucleobases from the the epigenetic modifications of cytosine – 5-methylcytosine (5mC) and 5 hydroxymethylcytosine (hmC). Strategies to selectively modify and detect the modified bases are also explored. Introduction. DNA sequencing strategies from the initial methods employed by Sanger up to current techniques utilised for the sequencing of entire human genomes, are reviewed. The initial discoveries of the epigenetic modifications 5-methylcytosine and 5 hydroxymethylcytosine are discussed. The proposed effects these bases have in vivo and a number of methods for their detection are also covered. Finally, methods for the selective chemical modification of the DNA bases are reviewed. Results and Discussion. Initially, the biotin•strepatvidin immobilisation strategy is implemented for the discrimination of the epigenetically modified analogues of cytosine (5mC and hmC), without prior chemical modification. It is subsequently observed that an α-hemolysin mutant is capable of discriminating all six bases – adenine, cytosine, guanine, thymine, 5 methylcytosine and 5-hydroxymethylcytosine. A number of different chemical reactions are then investigated on the bis-TBS protected deoxynucleoside model system, for their ability to selectively modify one of the DNA bases. Two selective oxidation reactions are then further optimised for use on ssDNA. Finally, electrical recording experiments were used to investigate two selective chemical reactions, as well as the modification of the bases, by the cancer drug temozolomide.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:558431 |
| Date | January 2011 |
| Creators | Wallace, Emma Victoria Bristowe |
| Contributors | Donohoe, T. J. : Bayley, H. |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:e30564de-0a84-4665-b7af-66a8ea589801 |
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