title: Specific single-cell isolation and genomic amplification of uncultured microorganisms.
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.
authors.affiliation: Environmental Microbiology and Biotechnology Group, BioCentrum-Technical University of Denmark, Building 227, Lyngby, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Kvist
authors.forename: Birgitte K
authors.initials: BK
authors.lastname: Ahring
authors.forename: Roger S
authors.initials: RS
authors.lastname: Lasken
authors.forename: Peter
authors.initials: P
authors.lastname: Westermann
mesh_terms: D001105:Archaea / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D044822:Biodiversity; D017931:DNA Primers / Q000235:genetics; D019641:DNA, Archaeal / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D020745:Genome, Archaeal / Q000235:genetics*; D017404:In Situ Hybridization, Fluorescence; D017013:Methanobacteriaceae / Q000235:genetics; D008846:Micromanipulation; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D012988:Soil Microbiology
chemical_list: D017931:DNA Primers; D019641:DNA, Archaeal; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S