Applied Microbiology and Biotechnology

, Volume 74, Issue 4, pp 926–935

Specific single-cell isolation and genomic amplification of uncultured microorganisms

  • Thomas Kvist
  • Birgitte K. Ahring
  • Roger S. Lasken
  • Peter Westermann
Methods

DOI: 10.1007/s00253-006-0725-7

Cite this article as:
Kvist, T., Ahring, B.K., Lasken, R.S. et al. Appl Microbiol Biotechnol (2007) 74: 926. doi:10.1007/s00253-006-0725-7

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.

Keywords

Single cell genomicsArchaeaMultiple displacement amplification

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Thomas Kvist
    • 1
  • Birgitte K. Ahring
    • 1
  • Roger S. Lasken
    • 2
  • Peter Westermann
    • 1
  1. 1.Environmental Microbiology and Biotechnology Group (EMB)BioCentrum—Technical University of DenmarkLyngbyDenmark
  2. 2.The Institute for Genomic ResearchRockvilleUSA