Metagenomics Methods and Protocols
Metagenomics has proven to be a powerful tool for exploring the ecology, metabolic profiling, and comparison of complex microbial communities as well as its important applications in the mining of metagenomes for genes encoding novel biocatalysts and drug
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1. Introduction The construction and screening of metagenomic libraries that have been generated from DNA directly isolated from environmental samples have been proven to be a powerful tool for the recovery of novel biomolecules of biotechnological importance (1, 2). In principle, metagenomic libraries provide access to the entire gene content of a habitat (2). The construction of metagenomic libraries involves the same steps as the cloning of genomic DNA derived from individual microorganisms. The required steps include fragmentation of environmental DNA by restriction digestion or shearing, insertion into an appropriate vector system, and transformation of the recombinant vectors into a suitable host, which is in almost all published studies on construction of metagenomic libraries Escherichia coli (3). Although the generation of metagenomic libraries is conceptually simple, the community
Wolfgang R. Streit and Rolf Daniel (eds.), Metagenomics: Methods and Protocols, Methods in Molecular Biology, vol. 668, DOI 10.1007/978-1-60761-823-2_2, © Springer Science+Business Media, LLC 2010
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sizes of most metagenomes such as those derived from soil and sediment samples and, correspondingly, the large number of clones that is necessary for a significant coverage of the metagenome are great technological challenges (4, 5). Two types of libraries with respect to average insert size can be generated: small-insert libraries in plasmid vectors (less than 10 kb) and large-insert libraries in cosmid and fosmid vectors (up to 40 kb) or BAC vectors (more than 40 kb). The selection of a vector system for library construction depends on the quality of the isolated environmental DNA, the desired average insert size of the library, the copy number required, the host, and the screening strategy that will be used (3, 5). Environmental DNA that is contaminated with humic or matrix substances after purification or DNA sheared during purification is only suitable for the generation of small-insert libraries (3). Small-insert metagenomic libraries are useful for the isolation of single genes or small operons encoding novel biomolecules. To identify complex pathways encoded by large gene clusters or large DNA fragments for the partial genomic characterization of uncultured microorganisms, the generation of large-insert libraries is the appropriate method. Here, we describe one protocol for the construction of small-insert libraries and one for large-insert fosmid libraries. Both methods have been proven to be suitable for cloning of DNA purified from various environmental samples, including soil, ice, and compost (6–8).
2. Materials 2.1. Metagenomic DNA
2.2. Generation of Small-Insert Metagenomic Libraries
The construction of metagenomic libraries derived from environmental samples and cloning of functional genes is dependent on the high quality of the extracted DNA, since the enzymatic modifications required during the construction of the libraries are sensitive to contamination by various biotic and abiotic com
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