Fixation-free fluorescence in situ hybridization for targeted enrichment of microbial populations

Yilmaz, S, Haroon, MF, Rabkin, BA, Tyson, GW and Hugenholtz, P (2010) Fixation-free fluorescence in situ hybridization for targeted enrichment of microbial populations. The ISME Journal, 4 10: 1352-1356. doi:10.1038/ismej.2010.73


Author Yilmaz, S
Haroon, MF
Rabkin, BA
Tyson, GW
Hugenholtz, P
Title Fixation-free fluorescence in situ hybridization for targeted enrichment of microbial populations
Formatted title
Fixation-free fluorescence in situ hybridization for targeted enrichment of microbial populations
Journal name The ISME Journal   Check publisher's open access policy
ISSN 1751-7362
1751-7370
Publication date 2010-10-27
Year available 2010
Sub-type Article (original research)
DOI 10.1038/ismej.2010.73
Open Access Status Not yet assessed
Volume 4
Issue 10
Start page 1352
End page 1356
Total pages 4
Place of publication New York, United States
Publisher Nature Publishing Group
Language eng
Subject 2404 Microbiology
1105 Ecology, Evolution, Behavior and Systematics
Abstract We modified the standard ribosomal RNA-targeted fluorescence in situ hybridization (FISH) protocol by removing the fixation steps to allow recovery of unmodified nucleic acids. Using this method, hybridized cells could be visualized in two bioreactor sludges and termite hindgut samples by epifluorescence microscopy. We then targeted one bacterial and one archaeal population in the sludge samples with group-specific oligonucleotide probes using in-solution fixation-free FISH and sorted hybridized populations using fluorescence-activated cell sorting (FACS). We could show that sorted populations were highly enriched for the target organisms based on 16S rRNA gene sequencing, thus confirming probe specificity using the modified FISH protocol. This approach should facilitate subsequent genomic sequencing and analysis of targeted populations as DNA is not compromised by crosslinking during fixation.
Formatted abstract
We modified the standard ribosomal RNA-targeted fluorescence in situ hybridization (FISH) protocol by removing the fixation steps to allow recovery of unmodified nucleic acids. Using this method, hybridized cells could be visualized in two bioreactor sludges and termite hindgut samples by epifluorescence microscopy. We then targeted one bacterial and one archaeal population in the sludge samples with group-specific oligonucleotide probes using in-solution fixation-free FISH and sorted hybridized populations using fluorescence-activated cell sorting (FACS). We could show that sorted populations were highly enriched for the target organisms based on 16S rRNA gene sequencing, thus confirming probe specificity using the modified FISH protocol. This approach should facilitate subsequent genomic sequencing and analysis of targeted populations as DNA is not compromised by crosslinking during fixation.
Keyword Fluorescence in situ hybridization (FISH)
Fluorescence-activated cell sorting (FACS)
Cell fixation
Genomics
Q-Index Code C1
Q-Index Status Confirmed Code
Grant ID DE-AC02-05CH11231
Institutional Status UQ
Additional Notes Published under Short Communications.

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2011 Collection
Advanced Water Management Centre Publications
 
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Created: Tue, 29 Jun 2010, 21:44:40 EST by Hong Lee on behalf of Advanced Water Management Centre