土壤微生物总DNA提取方法的优化

Optimization of soil microbial DNA isolation

摘要:

摘要:【目的】土壤中未培养微生物约占总量的99%，这就意味着绝大多数微生物资源还未得到开发和利用。本研究通过优化土壤微生物总DNA 的提取方法，获得较高质量的DNA，为后期研究土壤微生物的多样性及构建大插入片段的宏基因组文库奠定基础。【方法】通过综合比较已报道的微生物DNA 提取方法的优缺点，我们设计出一种新的提取方案。对提取过程中的几个关键步骤进行了优化，包括联合使用SDS-CTAB和溶菌酶一起来破细胞，利用氯仿除蛋白，使用PVPP柱纯化DNA等。比较分析了优化后的方法和3 种已报道方法所获得的土壤总DNA的产量、纯度及片段大小。【结果】优化后的方法所获得的土壤DNA质量明显有所提高:每克土壤最高能提取95μgDNA，A260/A280和A260/A230 比值更接近理想水平，PCR 扩增能够得到明显的目标条带，DNA 片段最大能达到100 kb左右。【结论】通过比较分析，最终确立了一种较理想的土壤微生物总DNA提取方法，为更好地开发利用土壤未培养微生物资源提供了有力工具。

Abstract:

Abstract:［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 μg 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.