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Title: Strand exchange for duplex DNA detection
Author: Ho, F. M.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2004
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The phenomenon of strand exchange between an unlabelled double-stranded target oligonucleotide and a single-stranded, fluorophore labelled probe oliognucleotide was investigated. This behaviour was characterised using fluorescence resonance energy transfer (FRET). The individual fluorescence characteristics of the fluorophores the minor-groove binder Hoechst 33258 and the dye Oregon Green 488 were studied, as well as their properties in combination as a FRET pair. These dyes allowed the use of FRET for the study of duplex DNA without the need for covalently attaching two labels on the component strands. Two strategies were studied for the detection of a duplex target. Firstly, detection could be by monitoring the FRET process as a function of time, monitoring the fluorescence intensities at both the donor and acceptor emission peak wavelengths. Single base pair discrimination was achieved, with very high reproducibility, especially if ratiometric analysis of the emission signals was employed. The mechanism of this process was examined using mathematical modelling, and comparisons made with the experimental results. Secondly, an in-gel detection technique was investigated for the detection of the target duplex within a complex mixture. The target sequence was successfully detected from within the enzyme digestion products of plasmids extracted from cloned E. coli cells. This was performed directly from a polyacrylamide electrophoresis gel without the need for blotting, and was possible with or without polymerase chain reaction amplification. Multiplexing was also demonstrated using this in-gel strategy, giving simultaneous detection of two targets of different base sequences and lengths. Finally, the synthesis of an acceptor fluorophore labelled dendrimer was proposed. This opened up the prospect of exploiting the properties of the dendrimer to enhance the FRET signal upon strand exchange.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available