Double displacement reactions : novel strategies for nucleic acid detection

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This thesis describes the design, synthesis, and evaluation of two new classes of self-ligating oligonucleotide probes: Double Displacement and Sandwich probes. These probes are an extension of previous work in our laboratory with quenched autoligation (QUAL) probes, which produce a fluorescent signal by a template-dependent SN2 displacement of a fluorescence quencher, increasing the emission of a conjugated fluorophore. These probes are used to detect complementary RNA sequences in vitro and in cells, making them useful diagnostic agents for differentiating between different cell types based on their genetics. Both new classes of probes improve and expand on previous QUAL approaches by requiring two displacement reactions to fully unquench a nearby fluorophore. Three potential Double Displacement architectures, all possessing two fluorescence quencher/leaving groups (dabsylate groups) at the 5' end of a fluorescently-labeled oligonucleotide, were synthesized and evaluated for templated reaction with nucleophile probes both in vitro and in intact bacterial cells. All three DD probe designs provided substantially better initial quenching than a single quencher control. In isothermal templated reactions in vitro, Double Displacement probes yielded considerably lower background signal than previous single-displacement probes; investigation into the mechanism revealed that one dabsylate acts as a sacrificial leaving group, reacting nonspecifically with water, but yielding little signal because another quencher group remains. Templated reaction with the specific nucleophile probe is required to activate a signal. The best-performing probe architecture was demonstrated in a two-color, FRET-based two-allele discrimination system in vitro, and was shown to be capable of discriminating between two closely related species of bacteria differing by a single nucleotide at a ribosomal RNA target site. Sandwich probes also require two displacements to fully unquench an oligonucleotide-conjugated fluorophore, Unlike Double Displacement probes, these molecules have one fluorescence quencher at each end of the oligonucleotide, each of which is unquenched by a flanking nucleophile oligonucleotide. These probes also show improved initial quenching, excellent single nucleotide polymorphism discrimination and template-dependent activation in intact bacterial cells. Both of these new classes of template reactive oligonucleotide probes advance the field of reagent-free oligonucleotide ligation reactions and show promise for applications in detecting cellular RNA.


Type of resource text
Form electronic; electronic resource; remote
Extent 1 online resource.
Publication date 2010
Issuance monographic
Language English


Associated with Kleinbaum, Daniel Jerome
Associated with Stanford University, Department of Chemistry
Primary advisor Kool, Eric T
Thesis advisor Kool, Eric T
Thesis advisor Kanan, Matthew William, 1978-
Thesis advisor Khosla, Chaitan, 1964-
Advisor Kanan, Matthew William, 1978-
Advisor Khosla, Chaitan, 1964-


Genre Theses

Bibliographic information

Statement of responsibility Daniel Jerome Kleinbaum.
Note Submitted to the Department of Chemistry.
Thesis Ph.D. Stanford University 2010
Location electronic resource

Access conditions

© 2010 by Daniel Jerome Kleinbaum
This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).

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