Solution Hybrid Selection Capture for the Recovery of Functional Full-Length Eukaryotic cDNAs From Complex Environmental Samples [electronic resource]
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Full Text (via OSTI) |
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| Format: | Electronic eBook |
| Language: | English |
| Published: |
Washington, D.C. : Oak Ridge, Tenn. :
United States. Department of Energy. Office of Science ; Distributed by the Office of Scientific and Technical Information, U.S. Department of Energy,
2014.
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| Abstract: | Eukaryotic microbial communities play key functional roles in soil biology and potentially represent a rich source of natural products including biocatalysts. Culture-independent molecular methods are powerful tools to isolate functional genes from uncultured microorganisms. However, none of the methods used in environmental genomics allow for a rapid isolation of numerous functional genes from eukaryotic microbial communities. We developed an original adaptation of the solution hybrid selection (SHS) for an efficient recovery of functional complementary DNAs (cDNAs) synthesized from soil-extracted polyadenylated mRNAs. This protocol was tested on the Glycoside Hydrolase 11 gene family encoding endo-xylanases for which we designed 35 explorative 31-mers capture probes. SHS was implemented on four soil eukaryotic cDNA pools. After two successive rounds of capture, >90% of the resulting cDNAs were GH11 sequences, of which 70% (38 among 53 sequenced genes) were full length. Between 1.5 and 25% of the cloned captured sequences were expressed in <em>Saccharomyces cerevisiae</em>. Sequencing of polymerase chain reaction-amplified GH11 gene fragments from the captured sequences highlighted hundreds of phylogenetically diverse sequences that were not yet described, in public databases. This protocol offers the possibility of performing exhaustive exploration of eukaryotic gene families within microbial communities thriving in any type of environment. |
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| Item Description: | Published through Scitech Connect. 10/03/2014. "Journal ID: ISSN 1340-2838." Bragalini, Claudia ; Ribir̈e, Cľine ; Parisot, Nicolas ; Vallon, Laurent ; Prudent, Elsa ; Peyretaillade, Eric ; Girlanda, Mariangela ; Peyret, Pierre ; Marmeisse, Roland ; Luis, Patricia |
| Physical Description: | Size: p. 685-694 : digital, PDF file. |