Gene cassette PCR: Sequence-independent recovery of entire genes from environmental DNA

The vast majority of bacteria in the environment have yet to be cultured. C onsequently, a major proportion of both genetic diversity within known gene families and an unknown number of novel gene families reside in these uncu ltured organisms. Isolation of these genes is limited by lack of sequence i nformation. Where such sequence data exist, PCR directed at conserved seque nce motifs recovers only partial genes. Here we outline a strategy for reco vering complete open reading frames from environmental DNA samples. PCR ass ays were designed to target the 59-base element family of recombination sit es that flank gene cassettes associated with integrons. Using such assays, diverse gene cassettes could be amplified from the vast majority of environ mental DNA samples tested. These gene cassettes contained complete open rea ding frames, the majority of which were associated with ribosome binding si tes. Novel genes with clear homologies to phosphotransferase, DNA glycosyla se, methyl transferase, and thiotransferase genes were identified. However, the majority of amplified gene cassettes contained open reading frames wit h no identifiable homologues in databases. Accumulation analysis of the gen e cassettes amplified from soil samples showed no signs of saturation, and soil samples taken at 1-m intervals along transects demonstrated different amplification profiles. Taken together, the genetic novelty, steep accumula tion curves, and spatial heterogeneity of genes recovered show that this me thod taps into a vast pool of unexploited genetic diversity. The success of this approach indicates that mobile gene cassettes and, by inference, inte grons are widespread in natural environments and are likely to contribute s ignificantly to bacterial diversity.