Limits...
Solution hybrid selection capture for the recovery of functional full-length eukaryotic cDNAs from complex environmental samples.

Bragalini C, Ribière C, Parisot N, Vallon L, Prudent E, Peyretaillade E, Girlanda M, Peyret P, Marmeisse R, Luis P - DNA Res. (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences and Systems Biology, University of Turin, viale Mattioli 25, Turin 10125, Italy Ecologie Microbienne, UMR CNRS 5557, USC INRA 1364, Université de Lyon, Université Lyon 1, Villeurbanne 69622, France.

Show MeSH
Semi-quantitative PCR amplification of a 281-bp GH11 fragment using different quantities (from 10 to 0.01 ng) of BRH cDNA obtained before (H0) and after one (H1) or two (H2) cycles of hybridization. Before capture, PCR products could only be obtained using 10 ng of input cDNA. Amplifications of the PUE, BRE and BEW samples are illustrated in Supplementary Fig. S3.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4263301&req=5

DSU030F2: Semi-quantitative PCR amplification of a 281-bp GH11 fragment using different quantities (from 10 to 0.01 ng) of BRH cDNA obtained before (H0) and after one (H1) or two (H2) cycles of hybridization. Before capture, PCR products could only be obtained using 10 ng of input cDNA. Amplifications of the PUE, BRE and BEW samples are illustrated in Supplementary Fig. S3.

Mentions: Successful enrichment in GH11 sequences along the capture protocol was demonstrated by semi-quantitative PCR using GH11-specific PCR primers and different quantities of cDNA in the PCRs (from 10 to 0.01 ng). As illustrated in Fig. 2 for the Breuil beech forest (BRH sample) and for the other soil samples discussed in Supplementary Fig. S3, clear positive amplification of a GH11 fragment after two rounds of capture was always obtained using the lowest quantity of cDNA (0.01 ng), whereas no amplification could be observed for the same amount of cDNA prior to SHS.Figure 2.


Solution hybrid selection capture for the recovery of functional full-length eukaryotic cDNAs from complex environmental samples.

Bragalini C, Ribière C, Parisot N, Vallon L, Prudent E, Peyretaillade E, Girlanda M, Peyret P, Marmeisse R, Luis P - DNA Res. (2014)

Semi-quantitative PCR amplification of a 281-bp GH11 fragment using different quantities (from 10 to 0.01 ng) of BRH cDNA obtained before (H0) and after one (H1) or two (H2) cycles of hybridization. Before capture, PCR products could only be obtained using 10 ng of input cDNA. Amplifications of the PUE, BRE and BEW samples are illustrated in Supplementary Fig. S3.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263301&req=5

DSU030F2: Semi-quantitative PCR amplification of a 281-bp GH11 fragment using different quantities (from 10 to 0.01 ng) of BRH cDNA obtained before (H0) and after one (H1) or two (H2) cycles of hybridization. Before capture, PCR products could only be obtained using 10 ng of input cDNA. Amplifications of the PUE, BRE and BEW samples are illustrated in Supplementary Fig. S3.
Mentions: Successful enrichment in GH11 sequences along the capture protocol was demonstrated by semi-quantitative PCR using GH11-specific PCR primers and different quantities of cDNA in the PCRs (from 10 to 0.01 ng). As illustrated in Fig. 2 for the Breuil beech forest (BRH sample) and for the other soil samples discussed in Supplementary Fig. S3, clear positive amplification of a GH11 fragment after two rounds of capture was always obtained using the lowest quantity of cDNA (0.01 ng), whereas no amplification could be observed for the same amount of cDNA prior to SHS.Figure 2.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences and Systems Biology, University of Turin, viale Mattioli 25, Turin 10125, Italy Ecologie Microbienne, UMR CNRS 5557, USC INRA 1364, Université de Lyon, Université Lyon 1, Villeurbanne 69622, France.

Show MeSH