Improvement of speed and sensitivity of DNA hybridization using isotachophoresis
Abstract/Contents
- Abstract
- DNA hybridization is essential to a wide range of diagnostics and molecular biology techniques including genetic profiling, pathogen identification, and single-nucleotide polymorphism. In particular, microarray hybridization has expedited many scientific discoveries by enabling high throughput analysis of millions of sequences in parallel. However, slow second order hybridization kinetics at low concentration DNA targets, often the most interesting, results in long analysis time (order 15 h). This limits the applicability of state-of-the art techniques in point-of-care settings. The common methods to speed up DNA hybridization typically involve trade-offs between speed and specificity (e.g. by varying concentration and type of cation or incubation temperature). Microarray hybridization (DNA hybridized to an array of surface probes) holds an additional challenge due to diffusion limitations, and this has been well addressed by chaotic mixing. However, even hybridizations with perfect mixing still poses a slow reaction limitation at trace target concentrations. This dissertation discusses a method based on isotachophoresis (ITP) to significantly improve kinetically limited DNA hybridization through focusing of reactants. ITP is an electrokinetic technique, which enables greater than 10,000-fold preconcentration at its moving interface between heterogeneous electrolytes. We leverage a locally increased concentration to enhance hybridization rate either between suspended species or between suspended and immobilized species. ITP-based DNA hybridization was demonstrated with molecular beacon probes in free solution. But until recently, analyses on reaction kinetics coupled with ITP focusing dynamics and qualitative studies on improvement of reaction rates and sensitivity were not well-established for both homogeneous and heterogeneous DNA hybridization by ITP. This thesis focuses on development and validation of theoretical models for ITP-enhanced hybridization, and experimental demonstration of its acceleration and sensitivity increases.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2015 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Han, Crystal |
|---|---|
| Associated with | Stanford University, Department of Mechanical Engineering. |
| Primary advisor | Santiago, Juan G |
| Thesis advisor | Santiago, Juan G |
| Thesis advisor | Mani, Ali, (Professor of mechanical engineering) |
| Thesis advisor | Quake, Stephen Ronald |
| Advisor | Mani, Ali, (Professor of mechanical engineering) |
| Advisor | Quake, Stephen Ronald |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Crystal Han. |
|---|---|
| Note | Submitted to the Department of Mechanical Engineering. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2015. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2015 by Crystal Mijung Han
- License
- This work is licensed under a Creative Commons Attribution 3.0 Unported license (CC BY).
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