Methods for Soil Metagenomics: Extraction and Cloning of Soil DNA

Abstract

Metagenomics (synonymous with environmental or community genomics) is the construction and analysis of libraries containing random DNA fragments cloned from naturally occurring microbial communities. The goals are to (i) describe the genomic structure of microbial communities, (ii) decipher the physiology and ecology of uncultured prokaryotes, and (iii) identify novel genes, enzymes, and molecules for biotechnology. Most methods for extracting DNA from soil are intended for PCR-based applications such as the amplification of 16S rRNA or other genes rather than direct cloning, which is especially challenging because of humic acids in soil that coextract with DNA and inhibit restriction enzymes. This chapter provides detailed methods to obtain DNA of sufficient purity for cloning into plasmid, cosmid, fosmid, or bacterial artificial chromosome (BAC) vectors. The most common techniques for soil metagenomic library construction entail partial digestion of soil DNA with various restriction enzymes. Chemical and enzymatic lysis methods are normally employed, which may bias the extraction towards easily lysed cells. There are two basic approaches to high-molecular- weight (HMW) DNA extraction from soil: (i) direct lysis of cells in the soil matrix and (ii) separation of cells from soil followed by lysis. Cell separation is more time-consuming than direct lysis, but larger DNA can be extracted when the cells are embedded and lysed within an agarose plug.