Abstract Isolation of High Molecular Weight DNA from Forest Topsoil for Metagenomic Analysis

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Abstract.
In this study, we employed a modified method to extract DNA from forest topsoil that was suitable for construction of large insert soil metagenomic library. The DNA extraction method used produced considerable DNA yield with DNA fragments ranging from 48 kb up to 290 kb. The recovery of soil DNA suitable for PCR and metagenomic library construction is difficult because soil DNA is often co-purified with polyphenolics and contaminants that interfere with the downstream applications.
PCR amplification of 16S and fungal 18S SSU rRNA genes from the extracted soil DNA suggesting that the DNA isolated using this modified method contained low concentration of PCR inhibitory substances and had sufficient purity for PCR without the need of further purification. Sequence analysis of PCR amplicons revealed this extraction method can efficiently capture a wide range of microorganisms including the hard-to-lyse Gram-positive bacteria and fungi. We have also successfully constructed a metagenomic fosmid library with insert size of between 23.1 kb – 40 kb. This metagenomic library will serve as basis for screening of novel biocatalysts from the soil metagenome.