A tiling DNA microarray approach for functional gene analyses in microbial ecosystems
Abstract/Contents
- Abstract
- I have developed a novel and broad-ranging approach to the characterization of complex microbial communities by analyzing the abundance and expression of key functional genes. The method is based on a tiling oligonucleotide DNA microarray, implementing an unprecedented number of probes per gene by tiling probe sequences across genes of interest at 1X--2X coverage. This design favors the avoidance of false positive gene identification in samples of DNA or RNA extracted from complex microbial communities. I have implemented this method using hydrogenase genes to investigate anaerobic microbial communities where H2 is an important intermediate (the Hydrogenase Chip), reductive dehalogenase genes to investigate ecosystems containing organohalide-respiring microorganisms (the Reductive Dehalogenase Chip), and the genome of the colorless sulfur bacterium Thiovulum to study its in-situ gene expression (the Thiovulum Chip). The Hydrogenase Chip revealed key organohalide-respiring and sulfate-reducing microorganisms in laboratory microcosms that simulate the bioremediation of tetrachloroethene and identified Microcoleus chthonoplastes as a key H2-producing microbe in phototrophic microbial mats. The Reductive Dehalogenase Chip revealed population dynamics of the organohalide-respiring strains present in a long-term laboratory microcosm to an unprecedented level of detail, and showed that different Dehalococcoides strains may differ in their sensitivity to hydrogen sulfide. Experiments with the Thiovulum Chip were ultimately unsuccessful, but revealed mRNA degradation that correlated with secondary structure stability in a way that was informative for future environmental transcriptomic experiments. Independent quantitative PCR analysis on selected hydrogenase genes showed that the tiling DNA microarray approach is semiquantitative. We also determined that as microbial community complexity increases, specificity must be traded for sensitivity in analyzing data from tiling DNA microarrays. This work on a range of questions in different ecosystems has determined the necessary conditions for the successful implementation of the tiling DNA microarray approach.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2012 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Marshall, Ian Philip George |
|---|---|
| Associated with | Stanford University, Civil & Environmental Engineering Department |
| Primary advisor | Spormann, Alfred M |
| Thesis advisor | Spormann, Alfred M |
| Thesis advisor | Francis, Christopher |
| Thesis advisor | Huang, Kerwyn Casey, 1979- |
| Advisor | Francis, Christopher |
| Advisor | Huang, Kerwyn Casey, 1979- |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Ian Philip George Marshall. |
|---|---|
| Note | Submitted to the Department of Civil and Environmental Engineering. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2012. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2012 by Ian Philip George Marshall
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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