摘要
[Objective] The objective of this study was to report an improved method for rapid DNA extraction from black-order sediments, without any purification step. [Methods] Sediments in eutrophic lake are complex ecosystems and soil microbes involved in anthropogenic nutrient cycling are very important. DNA-based molecular methods offer new tools for characterization of these mixed communities of mi- croorganisms. However, it is very difficult to remove humic substances, heavy met- als that co-existed with genome DNA representing the microbial community directly from these complex systems and can interfere with subsequent genetic analysis. The potassium dichromate solution was firstly used to remove humic substances. [Results] The steps of removing humic substances and DNA extraction were per- formed simultaneously that improved the speed of extraction to approximately 1 hour and the nucleic acids that were obtained with this method did not need to be washed with 70% ethanol and dissolved directly in sterile water for total bacterial 16S rDNA, nosZ gene of denitrifying bacteria, pmoA of methanotrophs, nifH of nitro- gen-fixing bacteria, amoA of ammonia-oxidizing bacteria and ammonia-oxidizing ar- chaea molecular ecology analyses. [Conclusion] This method could provide a plat- form for preparing a fast sediments DNA extraction.
[目的]建立一种从黑臭沉积物中快速、不需要任何纯化步骤的基因组DNA提取方法。[方法]富营养化湖泊沉积物是一种复杂的生态系统,土壤微生物在其氮循环过程中起着重要的作用。基于DNA的分子生物学方法为描述这些复杂微生物群落提供了新途径。但是,很难去除基因组DNA(代表了这些复杂系统中的微生物群落)的共提取物腐殖酸、重金属等物质,其能够干扰后续的基因分析。[结果]腐殖酸去除和DNA提取同步进行,提取时间大约为1 h,所得到的核酸不需要用70%的乙醇洗,直接溶解于灭菌水即可用于总细菌16S r DNA、反硝化细菌nos Z基因、甲烷氧化菌pmo A基因、固氮细菌nif H基因、氨氧化细菌和古菌的amo A基因的分子生态学分析。[结论 ]该方法为快速制备沉积物DNA提供了一个框架。
基金
Supported by the Recruitment Program of Beifang Univesity of Nationality(Grant No.44/4400302502)~~
