Development of oligonucleotide probes for genetic analysis
Development of oligonucleotide probes for genetic analysis
Fluorescent oligonucleotide probes are of great value in genetic analysis. These include TaqMan®, molecular beacons and scorpion primers. The efficacy of an oligonucleotide probe is dependent on the specific detection of a target and the emission of an intense signal. In this thesis the optimisation and development of novel oligonucleotide probes are discussed.
Most fluorescent oligonucleotide probes consist of two key elements; a fluorescent reporter and a quenching moiety. Conventionally, non-fluorescent diazo-dyes, known as ‘universal’ quenchers, perform the role of the quencher. However, these do not nullify the fluorescence of longer wavelength dyes sufficiently. Hence, the use of alternative chromophores as quenchers, more efficient at long wavelengths, is discussed. We have designed and synthesised diaminoanthraquinone derivatives, which absorb in the 500-700nm region. The relative efficiency of quenching of a variety of fluorophores has been determined in a number of different probe systems.
Stabilisation of the interaction between an oligonucleotide and its specific complementary sequence, without increasing non-specific binding, is also investigated. The stabilities of oligonucleotide duplexes containing a variety of nucleoside modifications have been studied by UV-melting. Synthesis of a novel 2’-aminoethyl-2’-deoxyuridine nucleoside is discussed.
University of Southampton
May, Jonathan Paul
bf1def44-0f31-4beb-b9fc-49c6823b3fba
2003
May, Jonathan Paul
bf1def44-0f31-4beb-b9fc-49c6823b3fba
May, Jonathan Paul
(2003)
Development of oligonucleotide probes for genetic analysis.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Fluorescent oligonucleotide probes are of great value in genetic analysis. These include TaqMan®, molecular beacons and scorpion primers. The efficacy of an oligonucleotide probe is dependent on the specific detection of a target and the emission of an intense signal. In this thesis the optimisation and development of novel oligonucleotide probes are discussed.
Most fluorescent oligonucleotide probes consist of two key elements; a fluorescent reporter and a quenching moiety. Conventionally, non-fluorescent diazo-dyes, known as ‘universal’ quenchers, perform the role of the quencher. However, these do not nullify the fluorescence of longer wavelength dyes sufficiently. Hence, the use of alternative chromophores as quenchers, more efficient at long wavelengths, is discussed. We have designed and synthesised diaminoanthraquinone derivatives, which absorb in the 500-700nm region. The relative efficiency of quenching of a variety of fluorophores has been determined in a number of different probe systems.
Stabilisation of the interaction between an oligonucleotide and its specific complementary sequence, without increasing non-specific binding, is also investigated. The stabilities of oligonucleotide duplexes containing a variety of nucleoside modifications have been studied by UV-melting. Synthesis of a novel 2’-aminoethyl-2’-deoxyuridine nucleoside is discussed.
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Published date: 2003
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Local EPrints ID: 464924
URI: http://eprints.soton.ac.uk/id/eprint/464924
PURE UUID: a5c8e393-f10e-4b59-bf0b-8f0473ad45f8
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Date deposited: 05 Jul 2022 00:11
Last modified: 16 Mar 2024 19:49
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Author:
Jonathan Paul May
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