高通量半自动基因组DNA提取仪的开发与应用  

Development and Application of High-Throughput Semi-automatic Genome DNA Extraction Instrument

作　　者：周悦 程溪柳 高英[2] 孙莹璐 王欢 刘君 白江平 ZHOU Yue;CHENG Xi-liu;GAO Ying;SUN Ying-lu;WANG Huan;LIU Jun;BAI Jiang-ping(College of Agronomy,Gansu Agricultural University/Gansu Provincial Key Laboratory of Aridland Crop Science/Gansu Provincial Key Laboratory of Crop Improvement&Germplasm Enhancement,Lanzhou 730070;Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081;Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081)

机构地区：[1]甘肃农业大学农学院/甘肃省干旱生境作物学重点实验室/甘肃省作物遗传改良与种质创新重点实验室,兰州730070 [2]中国农业科学院作物科学研究所,北京100081 [3]中国农业科学院生物技术研究所,北京100081

出　　处：《植物遗传资源学报》2022年第4期1224-1232,共9页Journal of Plant Genetic Resources

基　　金：中国科技部重点研发计划(2019YFD1000700,2019YFD1000703,2016YFD0101000,2016YFD0101001);国家自然科学基金(31901595)。

摘　　要：大规模高质量基因组DNA的提取是植物分子生物学研究的基础,是作物种质资源鉴定、重要农艺性状调控基因的正向定位克隆、作物基因组标记分子辅助育种的重要技术环节。然而,目前常规的单管离心法效率不能满足大规模提取需要,整板离心法及全自动高通量核酸提取仪对设备和耗材要求较高,实验室常用的传统离心方法步骤较为繁琐,涉及较多有机溶剂,耗时较长,成本较高,不太适用于大批量植物材料。为了满足一般实验室大规模、低成本、高质量DNA提取的需求,我们开发了一套半自动DNA提取仪、配套耗材和钢珠分样器,并开发相应DNA提取流程和试剂。应用高通量、高效率的磁珠法吸附DNA,目的是建立简化、快速、高效且安全的植物组织基因组DNA提取方法。通过对小麦、水稻、玉米等主粮作物,以及部分种类的杂粮、油料、蔬菜等植物组织进行DNA提取,并用琼脂糖凝胶电泳、Qubit等方法对DNA完整性和纯度检测,结果表明:(1)磁珠法提取的植物基因组DNA质量与纯度高、完整性好、无降解且无盐离子、有机物等污染;(2)提取过程无需反复对每个样品进行标注,试剂少、步骤少、操作简单快速且成本低,平均每人每天可提取2000个样品;(3)所得DNA样本可应用于基因克隆,转基因检测、CRISPR编辑检测等多种分子实验,也可用于多种NGS(Next Generation Sequencing)建库的高通量深度测序检测。因此,基于磁珠法的DNA纯化方案为植物研究提取DNA提供了一种简单、高效且经济的方法。The high-throughput extraction of high-quality genomic DNA is one of the basic analysis of plant molecular biology research,and an important technology for identifying genomic variation of crop germplasm resources,genetic mapping of important functional genes,crop molecular breeding based on genomic markerassisted selection.However,the efficiency of conventional method is inadequate for large-scale DNA extraction.The automatic high-throughput instruments are expensive and consumables,and the centrifugal-sedimentation method commonly used in laboratories are tedious and involve more organic solvents.To supply the demand of high-throughput,large-scale and less expensive DNA extraction,we have developed a 96-pool magnetic separation device DNA.Using this device,the genomic DNA extraction has been succeeded for wheat,rice,maize and other cereal crops,as well as vegetable and oil crops.The integrity and purity of DNA were detected by agarose gel electrophoresis,Qubit and PCR.The results showed that(1)the plant genomic DNA had high quality and purity,good integrity;(2)one person can handle 2000 samples per day;(3)the obtained DNA samples can be used in gene cloning,transgenic plant detection,CRISPR editing site detection and NGS library construction.

关 键 词：高通量 磁珠法 DNA提取 NGS 

分 类 号：S188[农业科学—农业基础科学]