硅羟基磁珠的制备及全基因组DNA提取优化  

Optimization of Genomic DNA Extraction with Silica Hydroxyl Magnetic Beads

作　　者：李海洋[1] 王飞[1] 雷红涛[2] 张璇[3,2] 陈珂[1] 

机构地区：[1]西南科技大学生命科学与工程学院,绵阳621000 [2]广东省食品质量安全重点实验室农业部农产品贮藏保鲜质量安全风险评价实验室华南农业大学食品学院,广州510642 [3]广东顺德创新设计研究院,佛山528000

出　　处：《生物技术通报》2017年第6期223-229,共7页Biotechnology Bulletin

基　　金：国家自然科学基金项目(U1301214);广东省自然科学基金项目(S2013030013338);四川省教育厅重点项目(15zd1117);四川省科技厅应用基础项目(2015JY0015);四川省生物质改性重点实验室项目(14tdgc02)

摘　　要：旨在制备硅羟基功能化纳米磁珠与核酸提取试剂,并对提取过程进行优化。采用溶剂热法制备硅羟基功能化纳米磁珠,并用表面化学修饰法在磁珠表面包裹SiO_2;在自制磁珠提取过程中分别对比缓冲液浓度、裂解液浓度、磁珠量以及洗脱温度等因素对DNA提取效率的影响,使用紫外分光光度计定量测定和琼脂糖凝胶电泳定性验证,并与商品化磁珠试剂盒提取效果进行对比。结果显示,通过水热法成功合成了粒径在200 nm左右的Fe_3O_4@SiO_2磁珠,当磁珠量为1.5 mg,裂解液为含5 mol/L的盐酸胍溶液(p H4.9),缓冲液体系为含60%无水乙醇的60 mmol/L盐酸胍缓冲液(pH7.5),洗脱温度为65℃时,所提取的核酸效果达到最优,且4个因素对浓度的影响均存在统计学差异(P<0.05)。利用自制磁珠和试剂体系可高效的完成全血中全基因组DNA的提取,且优于商业化试剂盒。This study aims to prepare silicon hydroxyl functionalized magnetic nano magnetic beads and nucleic acid extraction reagents,and to optimize the extraction process. Solvent thermal method was employed to prepare the silicon hydroxyl functionalized nano magneticbeads,then coating SiO_2 on the surface of magnetic beads by surface chemical modification method. The effects of buffer concentration,lysateconcentration,dose of magnetic beads and elution temperature on the efficiency of DNA extraction were analyzed by self-made magnetic beadsextraction kit,respectively. All the experimental results,quantitatively by ultraviolet spectrophotometer,and qualitatively by agarose gelelectrophoresis,were validated;meanwhile,the extraction results were compared with commercial kit. Results showed that we successfullysynthesized the Fe_3O_4@SiO_2 magnetic beads of approximate 200 nm. The extraction efficiency of nucleic acid achieved the best on the conditionsof magnetic beads was 1.5 mg,lysate contained 5 mol/L guanidine hydrochloride buffer（p H4.9）,the buffer system was 60 mmol/L guanidinehydrochloride（pH7.5）with 60% ethanol,and elution temperature was 65℃. Moreover,there were statistical differences on the effects bythe four factors（P 〈0.05）. In conclusion,using self-made magnetic beads and reagent system may efficiently carry out the complete genomicDNA extraction of whole blood,even it is better than the commercial kit.

关 键 词：纳米磁珠 表面修饰 全基因组DNA提取 

分 类 号：Q78[生物学—分子生物学]