Abstract
Environmental DNAs from 21 samples of saltern soil in Taiwan were isolated by using the SDS-lysis method, resulting in yields ranging from 0.03 ng to 8.06 µg per gram of soil. However, sample 143 collected from saltern soil near a crystallizer had a low yield of 1.2 ng per gram of soil. Comparative analyses of the sequence data of representative clones with other 16S rRNA samples indicated that not all clones for sample 143 were closely related to the soil bacteria. A minute amount of DNA (0.15 ng) was amplified 100,000-times to 15 µg by multiple displacement amplification (MDA). The MDA method was validated by analysis of amplified bacteriorhodopsin (bR) genes. Two clone libraries were constructed from DNA samples before and after amplification and were compared. The result suggests that bR diversity was relatively conserved during whole-genome amplification (WGA). The constructed metagenome fosmid library consists of 1.7 × 106clones with an average insert size of 26.1 kb. Taken together, WGA of metagenomic DNA from very minute microbial sources allows for construction of metagenomic libraries that are previously inaccessible. Key words: Saltern soil, DNA extraction, metagenomic DNA, multiple displacement amplification, fosmid library construction.
Highlights
Metagenomics, the study of genetic material recovered directly from environmental samples, is a new and rapidly developing field
Environmental DNAs from 21 samples of saltern soil in Taiwan were isolated by using the SDS-lysis method, resulting in yields ranging from 0.03 ng to 8.06 μg per gram of soil
whole-genome amplification (WGA) of metagenomic DNA from very minute microbial sources allows for construction of metagenomic libraries that are previously inaccessible
Summary
Metagenomics, the study of genetic material recovered directly from environmental samples, is a new and rapidly developing field. Metagenomic techniques have been used to analyze the complex genomes contained within microbial communities (Kowalchuk et al, 2007; Schmeisser et al, 2007) and are based on the direct isolation of DNA from environmental samples from which metagenomic libraries are generated. Isolation of metagenomic DNA is difficult because coextracted polyphenolic substances found in soil interferes with downstream applications (Tsai and Olson, 1992) and a few studies have attempted to quantify the efficiency of various DNA extraction protocols using environmental samples (Frostegard et al, 1999; Bertrand et al, 2005). Isolation of high molecular weight (HMW DNA) is important to reduce the risk of chimera formation during PCR
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