Solution hybrid selection capture for the recovery of functional full-length eukaryotic cDNAs from complex environmental samples.
Identifieur interne : 001825 ( PubMed/Corpus ); précédent : 001824; suivant : 001826Solution hybrid selection capture for the recovery of functional full-length eukaryotic cDNAs from complex environmental samples.
Auteurs : Claudia Bragalini ; Céline Ribière ; Nicolas Parisot ; Laurent Vallon ; Elsa Prudent ; Eric Peyretaillade ; Mariangela Girlanda ; Pierre Peyret ; Roland Marmeisse ; Patricia LuisSource :
- DNA research : an international journal for rapid publication of reports on genes and genomes [ 1756-1663 ] ; 2014.
English descriptors
- KwdEn :
- DNA, Complementary (chemistry), DNA, Complementary (genetics), DNA, Complementary (isolation & purification), Databases, Nucleic Acid, Eukaryotic Cells, Metagenome, RNA, Messenger (chemistry), RNA, Messenger (genetics), RNA, Messenger (isolation & purification), Saccharomyces cerevisiae (genetics), Soil (chemistry), Soil Microbiology.
- MESH :
- chemical , chemistry : DNA, Complementary, RNA, Messenger, Soil.
- chemical , genetics : DNA, Complementary, RNA, Messenger.
- chemical , isolation & purification : DNA, Complementary, RNA, Messenger.
- genetics : Saccharomyces cerevisiae.
- Databases, Nucleic Acid, Eukaryotic Cells, Metagenome, Soil Microbiology.
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 Saccharomyces cerevisiae. 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.
DOI: 10.1093/dnares/dsu030
PubMed: 25281543
Links to Exploration step
pubmed:25281543Le document en format XML
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<front><div type="abstract" xml:lang="en">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 Saccharomyces cerevisiae. 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. </div>
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<Abstract><AbstractText>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 Saccharomyces cerevisiae. 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. </AbstractText>
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<ForeName>Claudia</ForeName>
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