一种可直接用于PCR扩增的聚合氯化铝提取土壤总DNA的方法  

One Polyaluminium Chloride-Based Method of Soil Total DNA Extraction for Direct PCR amplification

作　　者：涂祖新[1,2] 王小红[2,3] 张莉莉[1] 金平 郑国华[1,2] 

机构地区：[1]江西省科学院微生物研究所,南昌330096 [2]江西省科学院鄱阳湖重点实验室,南昌330096 [3]江西省科学院科技战略研究所,南昌330096 [4]南昌水业集团南昌工贸有限公司,南昌330025

出　　处：《江西科学》2016年第3期305-310,共6页Jiangxi Science

基　　金：国家863项目<微生物资源及技术数据集成整合的关键技术研发>子课题<微生物资源库及数据标准>;江西省科学院"省部共建鄱阳湖国家重点实验室培育基地"计划(赣科院字[2013]19号-9)

摘　　要：为了研究湿地土壤微生物群落结构及其因时间和环境改变而产生的变化,找出与环境因子密切相关的微生物,有必要建立一种土壤微生物群落多样性代表全、提取率高的土壤总DNA提取方法。这有利于完成实验后,同一个土壤样品还留有足够的总DNA供多次使用,并可构建特定时空下的总DNA样品库,为科技水平更发达的未来留下一批不可再得的全息DNA样品。通过综合比较已报道的微生物总DNA提取方法的优缺点,设计出一种新的提取方案,关键步骤包括用焦磷酸钠溶解并洗脱样品的腐植酸;联合使用蛋白酶K-SDSCTAB和热激裂解细胞;加入聚合氯化铝再次沉淀去除残存的腐植酸并同步去除蛋白。结果用聚合氯化铝提取土壤总DNA的方法所获得的DNA量明显高于常规试剂盒法,每个1.5 m L提取管可得到25.831μg DNA;OD260nm/OD280nm比值达到或接近理想水平;OD260nm/OD230nm比值在0.550.73之间,说明还有盐污染,但可直接用于PCR扩增。通过实验和Illumina Mi Seq高通量测序应用验证,确立了一种土壤微生物群落多样性代表全、完整性好、提取率高、经济、安全、快捷的土壤总DNA提取方法,为今后的相关工作提供了一种技术手段。To investigate wetland soil microbes community structure and its variation on time and environmental change and to find out the microbes highly associated with environmental factors,it is essential to establish one highly efficient soil total DNA extraction method. The extracted total DNA from this method allows for several uses and the remaining sample can be stored for future use as val-uable DNA sample library of specific time or environment. Through comprehensive comparison of previously reported microbes total DNA extraction method,we designed one novel extraction protocol. This protocol incudes several main steps： Dissolve and elute humic acids from the sample using sodium pyrophosphate; Lyse the cells through protease K-SDS-CTAB and heat shock. Precipitate again to remove the remaining humic acids and proteins by adding polyaluminium chloride. The yield of soil DNA extracted from this polyaluminium chloride-based method is significantly higher than cannonical kit method： 25. 8 to 31 μg DNA will be obtained from one 1. 5 m L eppendorf tube; The OD260nm/ OD280 nmratio reaches or is near the ideal level; The OD260nm/ OD230 nmratio is between 0. 55 and 0. 73,indicating that there is salt contamination,but the sample can still be directly used for PCR amplification. Through experiment and illumina Mi Seq high throughout DNA sequencing,we established one highly efficient,ecnomic,safe,fast soil total DNA extraction method which might serve as one technical alternative for future work.

关 键 词：聚合氯化铝 腐植酸 DNA提取 PCR扩增 DNA样品库 

分 类 号：S154.34[农业科学—土壤学]