Objective: The vast majority of microbial resources has not been exploited and utilized because more than 99% of microorganisms in soil cannot be cultured by conventional means. The quantity and quality of environmental DNA (eDNA) are the most important issues in metagenomic study. Here we set up an optimized method for high quality soil microbial DNA isolation, which can be applied for microbial diversity study and metagenomic library construction containing large eDNA inserts.
Methods: After comprehensive comparison of strengths and weaknesses of the commonly used methods for microbial DNA isolation, we proposed a new method by optimizing several key procedures of isolation, including combination of sodium dodecyl sulfate (SDS)-hexadecyltrimethylammonium bromide (CTAB) and lysozyme to lysis cells, usage of chloroform in stead of phenol to remove protein contamination, filtration with the polyvinylpolipyrrolidone (PVPP) column to purify DNA. Using three different soil samples, we compared the optimized method with three reported methods in the literature by analyzing soil eDNA yield, rate of purity, size and ability of PCR amplification.
Results: The quality of soil eDNA isolated by the optimized method was significantly improved. We obtained 95 microg DNA per gram of soil. OD A260/A280 and A260/A230 ratios of DNA were much closer to the ideal level. More desired PCR product was amplified and maximum size of eDNA reached 100 kb.
Conclusion: High quality and quantity of soil microbial DNA can be isolated by the optimized method we established, which provides a powerful tool for metagenomic research to exploit uncultured microbial resources in soil.