Thesis
A novel method of oligonucleotide labelling and detection by surface enhanced resonance Raman scattering
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2004
- Thesis identifier
- T10916
- Person Identifier (Local)
- 682270
- Qualification Level
- Qualification Name
- Abstract
- A number of methods for detecting specific DNA sequences have been used to provide data for diagnosis of diseases and examination of gene expression. The most favoured is fluorescence detection although the addition of fluorescent labels can be disadvantageous due to the complex and expensive labelling chemistry. A novel method of oligonucleotide labelling was devised and surface enhanced resonance Raman scattering (SERRS) used as a detection technique. Single and dual labelled oligonucleotides were produced by first tagging the oligonucleotide with a furan or butadiene moiety at the 5’ terminus to act as a diene in the subsequent Diels Alder cycloaddition with specifically designed SERRS active dienophiles. Four benzotriazole azo maleimide dyes were synthesised. Benzotriazole group is known to complex strongly to the silver metal surface, which is one of the requirements for SERRS. The maleimide group in turn serves as a dienophile to undergo the cycloaddition with the furan or butadiene modified oligonucleotide to generate a SERRS active probe. These probes gave excellent SERRS signals from both silver- polyvinyl alcohol (PVA) discs and silver colloid enabling the detection of ultra low concentrations of DNA. Benzotriazole maleimide dye and fluorescein were used in the design of a novel class of biosensor, SERRS Beacons. In this type of molecular probe both SERRS and fluorescence were used as the detection techniques. The significant changes in the SERRS spectra of the Beacon indicated the presence of complementary sequence, while there were just slight changes when single base mismatch sequence was added. Both silver-PVA discs and silver colloid were used as SERRS surfaces, which demonstrated the flexibility of the SERRS Beacon to be used in different formats. Low detection limits, simplicity of both the labelling chemistry and SERRS surface preparation, makes SERRS an excellent technique for oligonucleotide detection with the possibility of use in the rapidly growing DNA microarray and proteomic area.
- Advisor / supervisor
- Smith, W. E.
- Graham, D.
- Resource Type
- Note
- Contents of the Appendix CD 1. Fluorescence Quenching and FRET 2. Common Fluorophores and Their Properties 3. Crystallographic Data 4. DFT Calculation and Raman Spectra of Dye Maleimide 10 5. Publication (1) 6. Presentation 1: Euroanalysis Conference, Dortmund 2002 7. Presentation 2: Final Year Presentation, Glasgow 2003 8. Poster 1: IR and Raman Discussion Meeting, Glasgow 2002 9. Poster 2: Symposium on Nucleic Acid Chemistry and Biology, Cambridge 2003
- DOI
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PDF of thesis T10916 | 2023-06-06 | Public | Download |
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ZIP file of T10916 contents of thesis appendix directory | 2023-10-06 | Public | Download |