A novel approach for screening metagenomic libraries by merging both metagenomic and microarray platforms was developed and optimized. This high-throughput screening strategy termed "metagenomic microarrays" involved the construction of two Arctic soil large-insert libraries and the high density arraying of the clone plasmid DNA (~50 kb) onto glass slides. A standard alkaline lysis technique used for the purification of plasmid DNA was adapted and optimized to function efficiently in a 96-well format, providing an economically viable means of producing sufficient high-quality plasmid DNA for direct printing onto microarrays. The amounts of printed material and probe, required for maximal clone detection, were optimized. To examine catabolic clone detection libraries were first screened by PCR for catabolic genes of interest. Two PCR-positive clones were printed onto microarrays, and detection of these specific clones in the printed libraries was achieved using labeled probes produced from PCR fragments of known sequence. Also, hybridizations were performed using labeled PCR fragments derived from the amplification of a catabolic gene from the total community DNA. The ability of selected probes to specifically target clones of interest was demonstrated. This merger of metagenomics and microarray technologies has shown great promise as a tool for screening the natural microbial community for catabolic potential and could also be used to profile microbial diversity in different environments.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.98518 |
| Date | January 2006 |
| Creators | Whissell, Gavin. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Natural Resource Sciences.) |
| Rights | © Gavin Whissell, 2006 |
| Relation | alephsysno: 002479862, proquestno: AAIMR24825, Theses scanned by UMI/ProQuest. |
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