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Chapter 84 : Methods for Soil Metagenomics: Extraction and Cloning of Soil DNA
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.
DNA size comparison for LMW and HMW extraction methods. Soil cores were collected at the National Science Foundation Long-Term Ecological Research site at Bonanza Creek Experimental Forest (Alaska), and organic (~40% organic matter) and mineral (~10% organic matter) layers were treated as separate samples ( 110 ). The LMW and HMW methods described in this chapter were used to extract DNA. Pulsed-field gel electrophoresis (4 h, 8 V/cm, 100° angle, linear ramping factor, 14°C, 0.5× Tris-borate-EDTA buffer) was performed with a CHEF Mapper, and DNA was visualized with ethidium bromide. Lanes (left to right): 1kb DNA Ladder (Promega), MidRange II PFG Marker (New England Biolabs), LMW mineral DNA, LMW organic DNA, HMW mineral DNA, and HMW organic DNA.
Comparison of restriction enzyme digestion of LMW DNA preceding or subsequent to CTAB purification. Soil samples were collected at the West Madison Agricultural Research Station (Wisconsin) (described in references 8 , 55 , 77 , and 80 ), and a FastDNA SPIN Kit was used to extract DNA from 0.5 g of soil. The eluted DNA was divided into two portions and one remained untreated (A), while the other was purified with CTAB (B). Prior to gel electrophoresis, DNA was digested with 10 U of various restriction enzymes (Promega) for 1 h. Lanes: 1, undigested DNA; 2 to 5, DNA digested with BamHI, EcoRI, HindIII, or PstI, respectively. The size marker is 1kb DNA Ladder (Promega).
Vectors used for soil metagenomics