Extracting single genomes from heterogenous DNA samples: a test case with Carsonella ruddii, the bacterial symbiont of psyllids (Insecta).
Analysis of many bacterial genomes is impeded by the inability to separate individual species from complex mixtures of cells or to propagate cells in pure culture. This problem is an obstacle to the study of many bacterial symbionts that live intracellularly in insects and other animals. To recover bacterial DNA from complex samples, we devised a method that facilitates the cloning of DNA fragments of distinctive G+C contents in order to generate shotgun DNA libraries enriched in inserts having a specific base composition. DNA preparations are first treated with a restriction enzyme having a common cleavage site in a particular genome and then shotgun cloned following size-fractionation. This method was applied to whole bacteriomes of the psyllid, Pachypsylla venusta, which harbors the bacterial symbiont Candidatus Carsonella ruddii. The resulting libraries were highly enriched in bacterial sequences. Through the use of alternate enzymes and partial digests, this technique can be adapted to yield virtually pure DNA libraries for individual bacterial species.
Dale
Colin
C
Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, USA.
Dunbar
Helen
H
Moran
Nancy A
NA
Ochman
Howard
H
D000818:Animals; D001482:Base Composition / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification*; D030561:Databases, Nucleic Acid; D020563:Gammaproteobacteria / Q000235:genetics*; D016680:Genome, Bacterial / Q000235:genetics; D015698:Genomic Library; D006430:Hemiptera / Q000382:microbiology*; D012689:Sequence Homology, Nucleic Acid; D013559:Symbiosis
D004269:DNA, Bacterial; D004262:DNA Restriction Enzymes