DNA Extraction and Construction of a Metagenomic Fosmid Library of Alpine Meadow Soil from the Mila Mountains in Tibet, China*

Abstract

The Qinghai-Tibetan Plateau represents an extreme environment for special microbial resources. Extraction of total microbial DNA with cultured-in-dependent method and construction of metagenomic library is an alternative method to exploit uncultured microorganisms. The meadow soil from the Mila Mountains in Tibet was selected as alpine soil type and was collected. A pre-experiment indicated that the soil had high organic matter and humic acid content,which resulted in a great difficulty in DNA extraction. Our results showed that the DNA obtained by direct extraction from the soil was in small DNA fragments with high contamination,and it was not suitable for constructing big insert fragment library. Comparison with direct extraction,the DNA extracted from cells separated from combing low speed centrifugation and Nycodenz gradient centrifugation had larger molecular weigh and higher purity with great diversity. A microbial metagenomic library,which contained 30 624 clones and a total genomic storage capacity over 1 Gb,was constructed with the DNA fragments from indirect method. Bioassay demonstrated that the constructed Fosmid library had large capability of insert DNA with unknown species. The method for constructing library provides a powerful tool for soil samples from extreme environment and the constructed library provides resources for finding and utilizing new functional genes from the Tibetan Plateau. Fig 7,Tab 3,Ref 27