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Extraction of inhibitor-free metagenomic DNA from soil
Sponsored,vendor-submitted protocol    Sponsored by Zymo Research Corporation    Published in November 2009 (p.23) DOI: 10.2144/000113292

Abstract

Metagenomics (otherwise known as environmental genomics, ecogenomics, or community genomics) is the study of genetic material recovered directly from environmental samples (e.g., soil). This relatively new field of genetic research focuses on the study of organisms that are not easily cultured in the laboratory and studies of organisms in their natural environment. Quintessential to the success of such studies is the isolation of PCR-quality, inhibitor-free DNA from sampled materials. The ZR Soil Microbe DNA Kit™ from Zymo Research has been designed for the rapid (15 min), efficient isolation of microbial DNA from soil. It features a unique Fast Spin column and includes technologies for the thorough removal of polyphenolic substances (e.g., humic acids) that often contaminate DNA preparations and confound PCR. Its application is demonstrated below.

Materials

Figure 1. The ZR Soil Microbe DNA Kit™ can be used to isolate high-quality DNA from a variety of soil types, which yields robust products following PCR. (A) Physical characteristics of sampled soils (lanes 1–5). (B) Microbial DNA was isolated from soil samples (lanes 1–5) using the ZR Soil Microbe DNA Kit™. Approximately 10% of the eluted DNA was then separated in a 0.8% (w/v) agarose/ethidium bromide gel. (C and D) The results of PCR of microbial DNA isolated from the samples with primers specific for prokaryotic 16S rRNA (C) or eukaryotic rRNA (D). In the panels, the 1-kb size marker (NEB) is as indicated and the arrows show the prokaryotic 16S rRNA.

ZR Soil Microbe DNA Kit™ (Cat. no. D6001; Zymo Research Corp.)

FastPrep-24 instrument (MP Biomedicals)

ZymoTaq™ PreMix (Cat. no. E2005; Zymo Research Corp.)

Primers specific for prokaryotic and eukaryotic rRNA

Method

1. 0.25 g of five different soil samples (ranging 5−8% organic, 10−50% silt/clay composition) were added to five individual ZR BashingBead™ lysis tubes with lysis solution and processed for 45 s using a FastPrep-24 instrument.

2. Following centrifugation of the tubes, supernatants were filtered using the provided Zymo-Spin™ IV columns (red caps), and the filtrates were mixed with soil DNA binding buffer at a 1:3 ratio.

3. DNA was bound via centrifugation using Zymo-Spin™ II Fast Spin columns and then washed twice with soil DNA wash buffer.

4. DNA was eluted with DNA elution buffer and then filtered with Zymo-Spin™ IV-HRC columns (green caps) to remove any remaining polyphenolic substances.

5. PCR was performed using ZymoTaq™ with primers specific for prokaryotic and eukaryotic rRNA.

Results

Figure 1 shows the results of processing five different samples with the ZR Soil Microbe DNA Kit™. Various amounts of DNA were recovered from all samples. The DNA was subsequently used for PCR (results shown in panels C and D). In all cases both prokaryotic- and eukaryotic-specific primers resulted in amplicon formation. PCR was significantly inhibited with the omission of Zymo-Spin™ IV-HRC filtration, or in the case of using DNAs isolated with the kits of some other suppliers (data not shown).

Conclusions

The following prerequisites are important for the efficient isolation of PCR-quality DNA for metagenomic study: (i) efficient lysis of microbes (including those tough-to-lyse organisms) in the sampled material; (ii) integrity and yield of DNA in the lysate; and (iii) purity of the eluted DNA. Many techniques have been developed over the years for the isolation of DNA from environmental sources, but many have been plagued by inefficient lysis and/or inadequate removal of polyphenolic inhibitors of PCR. Zymo Research has developed a family of kits that can be used for routine spin column−based (micro, mini, midi) or high-throughput (96-well) processing of environmental samples. Lysis is thorough, DNA yield is high, and inhibitors to PCR are removed at various steps during the purification procedure. Zymo-Spin™ IV-HRC columns are part of the ZR Soil Microbe DNA Kit™ but are available separately for impure DNA clean-up as the OneStep™ PCR Inhibitor Removal Kit (Cat. no. D6030).

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