Nucleic acid purification and analysis by isotachophoresis
Abstract/Contents
- Abstract
- Microfluidics has recently enabled new capabilities in life sciences research. By leveraging physics at the microscale, novel miniaturized methods and devices provide fundamental improvements over traditional assays including higher sensitivity, massive parallelization, speed and automation. For example, nucleic acid analyses such as PCR or capillary electrophoresis are now commonly executed on microfluidic platforms. However, extracting and isolating a set of molecules of interest from a biological sample remains a widespread challenge, in particular when the target is a nucleic acid; such sample preparation has been identified as the "weak link" of microfluidics. Moreover, the discovery of classes of small RNA such as microRNA (miRNA) has revealed the limitations of benchtop preparation methods. This work tackles these issues by leveraging an electrophoretic focusing method, isotachophoresis (ITP), to perform selective focusing of specific nucleic acid molecules contained in complex mixtures. This includes extraction of genomic DNA from blood and isolation of miRNA from total RNA. Also, we leverage the unique physics of ITP to perform simultaneous purification and analysis of these molecules, thus enabling automated analysis at unprecedented speed. ITP is a robust electrophoretic preconcentration technique which generates strong electric field gradients, and enables selective focusing and separation of charged species based on their electrophoretic mobilities. In this work, we show that we can extract and purify genomic DNA from chemically lysed whole blood samples by carefully controlling ITP electrolyte chemistry to achieve selective focusing of genetic material. We show that ITP outputs PCR-compatible DNA with high efficiency in about 1 min. This novel sample preparation technique allows for efficient, fast, and automated purification of DNA from 10 nL to 1 [Mu]L of biological fluids. We also demonstrate that ITP focusing of microRNA -- short (~22 nt), non-coding RNA regulating gene expression -- enables quantification and sequence specific detection. We leverage both the selective and preconcentration capability of ITP for the quantification of global miRNA abundance. This allows for the measurement of RNA silencing activity in specific cells or tissues. We have optimized ITP chemistry and used a multi-stage injection strategy to selectively preconcentrate and quantify RNA shorter than 40 nt. We discuss results of miRNA quantification for a wide variety of samples. Additionally, we show that the combination of selective ITP focusing with simultaneous hybridization with molecular beacons is an efficient method for detection and quantitation of specific miRNA sequences. We demonstrate the efficacy of this assay for the detection of a liver specific miRNA. Finally, we show that we can use ITP to perform polymerase chain reaction in isothermal conditions by creating a cycles of chemicals mimicking thermal cycling.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Copyright date | 2011 |
| Publication date | 2010, c2011; 2010 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Persat, Alexandre Louis Andre |
|---|---|
| Associated with | Stanford University, Department of Mechanical Engineering |
| Primary advisor | Santiago, Juan G |
| Thesis advisor | Santiago, Juan G |
| Thesis advisor | Quake, Stephen Ronald |
| Thesis advisor | Shaqfeh, Eric S. G. (Eric Stefan Garrido) |
| Advisor | Quake, Stephen Ronald |
| Advisor | Shaqfeh, Eric S. G. (Eric Stefan Garrido) |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Alexandre Louis Andre Persat. |
|---|---|
| Note | Submitted to the Department of Mechanical Engineering. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2011. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2011 by Alexandre Louis Andre Persat
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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