Novel strategies for DNA detection and assay.

Bourin, Stephanie

Abstract

The work carried out during this project was aimed at developing new strategies for DNA bioassays, using alternative detection techniques and new labelling methods. Firstly, a new labelling technique for DNA sequencing was developed using lanthanide chelates. These compounds were identified as likely candidates to replace traditional dyes in four colour sequencing strategies because of their advantageous spectroscopic properties. Several chelates were synthesised and their fluorescent properties tested in presence of various lanthanide ions. None of the chelates tested was fluorescent in presence of the four ions (Tb3+, Eu3+, Sm3+, D3+). A triphosphate amino- thymidine derivative was synthesised. The triphosphate was convalently bound to a linear chelate, and was tested for incorporation in the PCR reaction as this reaction uses a polymerase similar to that of Sanger’s sequencing reaction. There was no detectable incorporation of the compound. Studies of the toxicity of those derivatives, of the chelates and of the free lanthanide ions on the same polymerase enzyme were carried out. They showed a high sensitivity of the enzyme to unbound metal ions and to the chelates themselves in a smaller proportion. These effects were studied in more detail, and the hypothesis that lanthanide ions might prevent polymerisation by acting on the DNA itself was raised. Thermal stability of synthetic DNA duplexes was studied in the presence of lanthanide ions. NMR spectrometry was used to further improve the knowledge on the interaction, and this work was done in collaboration with King’s college London. The results obtained showed that about 1 lanthanide ion binds to 4 nucleotide bases. They bind first to the phosphate back bone, increasing the stability of the double chain. Once these sites are saturated, the lanthanide ions bind on the bases, and their bulkiness causes distortions that prevent proper Watson and Crick hydrogen bonding, thus decreasing the Tm. It was also found, using MALDl mass spectrometry, that lanthanide ions did not all bind to DNA with the same stoichiometry, which may be due to a difference in affinity between the metal ion and the nucleic acid. As well as confirming that this interaction with DNA is certainly the cause of the inhibition of polymerisation (PCR), this study was used as a preliminary study for the development of a DNA hybridisation assay, using lanthanides as fluorescent labels. Finally, work was carried out on the characterisation of DNA coated surfaces by second harmonic generation. This work was done to assess the feasibility of a non labelling DNA hybridisation assay using surface second harmonic generation (SSHG) detection techniques. It was found that the density of DNA monolayers deposited on the surface affected strongly the second harmonic signal and that the structure and size of the nucleic acid also affected the intensity of the signal. A detection limit of 10-13 mol nucleic acids/mm2 was achieved. It was therefore concluded that SSHG was a suitable method for solid surface hybridisation assays, although it would probably be necessary to improve further its sensitivity to make it a commercially viable diagnostic tool.

Citation

BOURIN, S. 1998. Novel strategies for DNA detection and assay. Robert Gordon University, PhD thesis. Hosted on OpenAIR [online]. Available from: https://doi.org/10.48526/rgu-wt-2807307

• Thesis Type: Thesis
• Deposit Date: May 29, 2025
• Publicly Available Date: May 29, 2025
• DOI: https://doi.org/10.48526/rgu-wt-2807307
• Public URL: https://rgu-repository.worktribe.com/output/2807307
• Award Date: Dec 31, 1998