Journal article
Specific single-cell isolation and genomic amplification of uncultured microorganisms
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.
Language: | English |
---|---|
Publisher: | Springer Berlin Heidelberg |
Year: | 2007 |
Pages: | 926-935 |
ISSN: | 14320614 and 01757598 |
Types: | Journal article |
DOI: | 10.1007/s00253-006-0725-7 |
Base Sequence Biodiversity Biotechnology Chemistry DNA Primers DNA, Archaeal DNA, Ribosomal Genome, Archaeal In Situ Hybridization, Fluorescence Methanobacteriaceae Microbial Genetics and Genomics Microbiology Micromanipulation Molecular Sequence Data Multiple displacement amplification Nucleic Acid Amplification Techniques Phylogeny RNA, Ribosomal, 16S Sequence Analysis, DNA Sequence Homology Single cell genomics Soil Microbiology