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The sequencing of individual DNA strands with nanopores is being developed as a rapid, low-cost platform in which bases are identified in order as a DNA strand is transported through a pore under an electrical potential. Several challenges remain and this thesis focuses on one major area, the base identification properties of the a-hemolysin (aHL) nanopore. Under the potentials required for threading, DNA translocates too fast for single bases to be identified. However, immobilization of the DNA within the pore increases the residence time and therefore improves the precision of the electrical current reading and allows for the small differences in current flow, associated with different sequences, to be observed. DNA molecules with a 3'-terminal biotin-tag were complexed with streptavidin. Streptavidin is too large to be transported through the aHL pore and therefore the DNA-btnestreptavidin complex is not fully translocated; thus, the DNA strand is immobilized within the pore. Using this approach the nucleobase recognition properties of the aHL pore were mapped. The data suggest that the transmembrane 13 barrel domain of the pore contains at least three nucleobase recognition sites, termed R1, R2 and R3. Additional sequence information can be gained when multiple recognition sites are employed within a single aHL pore, as compared to the simple case of a single recognition site. Recognition site R1, which is located near the central constriction, can be modified by site-directed mutagenesis of Met-113. It was observed that amino acids with related side chains produce similar patterns of nucleobase recognition. Amino acids that provide an energy barrier to ion flow (e.g. bulky or hydrophobic residues) strengthen base identification, while amino acids that lower the barrier, weaken identification. Deletion and site-directed mutagenesis were used to remove one recognition site and generate an αHL pore. With truncated β barrel domain that contains only two recognition sites.

Randomisation of DNA using conventional methodology requires an excess of genes to be cloned, since with randomised codons NNN or NNG/T 64 genes or 32 genes must be cloned to encode 20 amino acids respectively. Thus, as the number of randomised codons increases, the number of genes required to encode a full set of proteins increases exponentially. Various methods have been developed that address the problems associated with excess of genes that occurs due to the degeneracy of the genetic code. These range from chemical methodologies to biological methods. These all involve the replacement, insertion or deletion of codon(s) rather than individual nucleotides. The biological methods are however limited to random insertion/deletion or replacement. Recent work by Hughes et al., (2003) has randomised three binding residues of a zinc finger gene. The drawback with this is the fact that consecutive codons cannot undergo saturation mutagenesis. This thesis describes the development of a method of saturation mutagenesis that can be used to randomise any number of consecutive codons in a DNA strand. The method makes use of “MAX” oligonucleotides coding for each of the 20 amino acids that are ligated to a conserved sequence of DNA using T4 DNA ligase. The “MAX” oligonucleotides were synthesised in such a way, with an MlyI restriction site, that restriction of the oligonucleotides occurred after the three nucleotides coding for the amino acids. This use of the MlyI site and the restrict, purify, ligate and amplify method allows the insertion of “MAX” codons at any position in the DNA. This methodology reduces the number of clones that are required to produce a representative library and has been demonstrated to be effective to 7 amino acid positions.

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.

This thesis describes the design, fabrication and characterisation of diffraction-based sensors on silicon (100) substrates for the detection of complementary DNA sequences using colloidal gold labels. In-depth analysis of variations of DNA sequence within the human genome and association with diseases is expected to lead to personalised medical treatment. There is a great need for DNA analysis technologies and for techniques to determine whether sequence variations occur on the same chromosomal strand with applications in disease screening and diagnosis. Novel two-dimensional diffraction structures are designed and fabricated. It is shown that two-dimensional diffraction approaches may potentially offer multiplexing of DNA detection assays.