Cloning

DNA sequence information is obtained from environmental samples in two main ways, (i) cloning DNA extracted from soil directly or (ii) cloning of PCR-amplified DNA, followed in both cases by sequencing of the cloned DNA. Direct cloning involves isolating DNA from the soil, ligating the DNA into a vector (most frequently a self-replicating plasmid), and transforming (moving) the vector into a competent host bacterium, such as commercially available E. coli competent cells, where it can be maintained and multiplied. In this way, a recombinant DNA clone library is produced. Once a clone library is obtained, DNA inserts contained in the clones can be reisolated from the host cells, purified, and sequenced. The clone library can also be screened for biological activity expressed directly in E. coli or probed for sequences of interest using various genomics applications. This approach circumvents the need to culture microorganisms from environmental samples, and it also provides a relatively unbiased sampling of the genetic diversity of sampled environments.

It has become possible to clone large fragments (100-300 kb) of DNA into bacterial artificial chromosome (BAC) vectors (Rondon et al., 2000). BAC vectors are low-copy-number plasmids that can readily maintain large DNA inserts. When some of the BAC libraries were analyzed, sequences homologous to the low-G + C, Grampositive Acidobacterium, Cytophagales, and Proteobacteria were found. Rondon et al. (2000) also identified clones that expressed lipase, amylase, nuclease, and hemolytic activities. Hence, the library could be used both for phylogenetic studies and as a tool for natural product discovery. Probing metagenomic libraries from a range of environments led to the discovery that uncultivated members of the archaeal lineage Crenarchaeota contain gene sequences with homology to the ammonia monooxygenase genes in nitrifying bacteria (Schleper et al., 2005). This discovery suggests that the Crenarchaeota may be playing a more significant role in the global cycling of N than thought previously (Schleper et al., 2005; Nicol and Schleper, 2006). Such metagenomic libraries are powerful tools for exploring soil microbial diversity and will form the basis of future genomic studies that link phylogenetic information with soil microbial function.

An alternative approach for creating large clone libraries from soil sequences that allows subsequent profiling of microbial communities is called serial analysis of ribosomal sequence tags (SARST). In this approach, a region of the 16S rRNA gene is amplified by PCR, such as the V1 region (variable in sequence between taxo-nomic groups). Through a series of enzymatic and ligation (linking) steps, the various V1 region amplicons are joined together. The resulting concatemers are then purified, cloned, screened, and sequenced. The sequences of the individual V1 amplicons are deduced by ignoring the linking sequences and analyzing each sequence tag individually (Neufeld et al., 2004). Several other PCR-based community analysis methods (see Partial Community Analyses—PCR-Based Assays) allow DNA fragments to be retrieved in a selective manner and these can then be cloned using the methods described above.

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