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Specific single-cell isolation and genomic amplification of uncultured microorganisms

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Abstract

We in this study describe a new method for genomic studies of individual uncultured prokaryotic organisms, which was used for the isolation and partial genome sequencing of a soil archaeon. The diversity of Archaea in a soil sample was mapped by generating a clone library using group-specific primers in combination with a terminal restriction fragment length polymorphism profile. Intact cells were extracted from the environmental sample, and fluorescent in situ hybridization probing with Cy3-labeled probes designed from the clone library was subsequently used to detect the organisms of interest. Single cells with a bright fluorescent signal were isolated using a micromanipulator and the genome of the single isolated cells served as a template for multiple displacement amplification (MDA) using the Phi29 DNA polymerase. The generated MDA product was afterwards used for 16S rRNA gene sequence analysis and shotgun-cloned for additional genomic analysis. Sequence analysis showed >99% 16S rRNA gene homology to soil crenarchaeotal clone SCA1170 and shotgun fragments had the closest match to a crenarchaeotal BAC clone previously retrieved from a soil sample. The system was validated using Methanothermobacter thermoautotrophicus as single-cell test organism, and the validation setup produced 100% sequence homology to the ten tested regions of the genome of this organism.

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Acknowledgements

This study was partially financed by The Danish Natural Science Council and performed within the Danish Archaea Centre. Thomas Leser is acknowledged for running the t-RFLP analyses.

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Authors and Affiliations

  1. Environmental Microbiology and Biotechnology Group (EMB), BioCentrum—Technical University of Denmark, Building 227, 2800, Lyngby, Denmark

    Thomas Kvist, Birgitte K. Ahring & Peter Westermann

  2. The Institute for Genomic Research, 9712 Medical Center Dr., Rockville, MD, 20805, USA

    Roger S. Lasken

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  1. Thomas Kvist
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  2. Birgitte K. Ahring
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  3. Roger S. Lasken
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  4. Peter Westermann
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Correspondence to Peter Westermann.

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Kvist, T., Ahring, B.K., Lasken, R.S. et al. Specific single-cell isolation and genomic amplification of uncultured microorganisms. Appl Microbiol Biotechnol 74, 926–935 (2007). https://doi.org/10.1007/s00253-006-0725-7

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  • DOI: https://doi.org/10.1007/s00253-006-0725-7

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