Trembath-Reichert, Elizabeth and Green-Saxena, Abigail and Orphan, Victoria J. (2013) Whole Cell Immunomagnetic Enrichment of Environmental Microbial Consortia Using rRNA-Targeted Magneto-FISH. In: Microbial Metagenomics, Metatranscriptomics, and Metaproteomics. Methods in Enzymology. No.531. Academic Press , pp. 21-44. ISBN 9780124078635 http://resolver.caltech.edu/CaltechAUTHORS:20131002-134144694
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Magneto-FISH, in combination with metagenomic techniques, explores the middle ground between single-cell analysis and complex community characterization in bulk samples to better understand microbial partnerships and their roles in ecosystems. The Magneto-FISH method combines the selectivity of catalyzed reporter deposition– fluorescence in situ hybridization (CARD–FISH) with immunomagnetic capture to provide targeted molecular and metagenomic analysis of co-associated microorganisms in the environment. This method was originally developed by Pernthaler et al. (Pernthaler et al., 2008; Pernthaler & Orphan, 2010). It led to the discovery of new bacterial groups associated with anaerobic methane-oxidizing (ANME-2) archaea in methane seeps, as well as provided insight into their physiological potential using metagenomics. Here, we demonstrate the utility of this method for capturing aggregated consortia using a series of nested oligonucleotide probes of differing specificity designed to target either the ANME archaea or their Deltaproteobacteria partner, combined with 16S rRNA and mcrA analysis. This chapter outlines a modified Magneto-FISH protocol for largeand small-volume samples and evaluates the strengths and limitations of this method predominantly focusing on (1) the relationship between FISH probe specificity and sample selectivity, (2) means of improving DNA yield from paraformaldehyde-fixed samples, and (3) suggestions for adapting the Magneto-FISH method for other microbial systems, including potential for single-cell recovery.
|Item Type:||Book Section|
|Additional Information:||© 2013 Elsevier Inc. We acknowledge Annelie (Pernthaler) Wendeberg, Rachel Poretsky, Joshua Steele for their contributions towards the development and optimization of Magneto-FISH. Stephanie Cannon, Jen Glass, Kat Dawson, Hiroyuki Imachi, and Caltech Genomics Center are also acknowledged for their assistance with various aspects of this project. Funding for this work was provided by the Gordon and Betty Moore foundation and a DOE early career grant (to V. J. O.) and NIH/NRSA training grant 5 T32 GM07616 (to E. T. R.).|
|Subject Keywords:||Cell separation; Metagenomics; Syntrophy; CARD–FISH; ANME; Microbial association; Symbiosis|
|Official Citation:||Elizabeth Trembath-Reichert, Abigail Green-Saxena, Victoria J. Orphan, Chapter Two - Whole Cell Immunomagnetic Enrichment of Environmental Microbial Consortia Using rRNA-Targeted Magneto-FISH, In: Edward F. DeLong, Editor(s), Methods in Enzymology, Academic Press, 2013, Volume 531, Pages 21-44, ISSN 0076-6879, ISBN 9780124078635, http://dx.doi.org/10.1016/B978-0-12-407863-5.00002-2. (http://www.sciencedirect.com/science/article/pii/B9780124078635000022)|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Tony Diaz|
|Deposited On:||02 Oct 2013 20:59|
|Last Modified:||09 Jan 2016 03:27|
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