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钉螺线粒体基因组DNA提取方法的优化及比较

Optimization and comparison of extraction methods of mitochondrial DNA of Oncomelania hupensis
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摘要 目的 优化提取钉螺线粒体基因组DNA(mtDNA)的方法.方法 应用高温裂解、蛋白酶K变温消化和醋酸钾纯化对传统的高盐沉淀法进行优化,并将优化的高盐沉淀法与蔗糖密度梯度离心法、传统的高盐沉淀法进行比较.用紫外分光光度计、琼脂糖凝胶电泳和线粒体COX1基因PCR扩增产物鉴定提取的mtDNA,并用核ITS基因PCR扩增产物检测有无核DNA污染.结果 优化的高盐沉淀法获得的mtDNA浓度和产量较高,差异有统计学意义(F=3032.65、10185.00,P均〈0.01).用凝胶电泳检测后未发现核DNA与蛋白质污染,质量可满足于PCR、测序等分子生物学研究.结论 优化的高盐沉淀法简易高效,成本低,获得的mtDNA能满足相关分析要求. Objective To optimize the extraction methods of mitochondrial genome DNA(mtDNA)of Oncomelania hupen-sis. Methods The pyrolysis,protein K variable-temperature digestion and high-concentration potassium acetate purification were applied to optimize the high-concentration-salt precipitation method,and then the optimized method was compared with two common extraction methods,the sucrose density gradient centrifugation method and traditional high-concentration-salt pre-cipitation method. The mtDNA samples were identified by using spectrophotometry,agarose gel electrophoresis and the amplifi-cation products of COX1. The nuclear DNA contamination was tested by the amplification products of ITS. Results The concen-tration and yield of the improved method was significantly higher than those of the traditional method(F=3032.65,10185.00, both P〈0.01). The mtDNA samples extracted were essentially free of nuclear DNA and protein,meeting PCR,sequence analy-sis and other molecular biology research requirements. Conclusions The improved high-concentration-salt precipitation meth-od for isolating mtDNA is simple,and it has high yield and low cost. The extracted mtDNA can meet relevant analysis require-ments.
作者 王康 许树俊 王逸枭 陈文捷 王忆楠 于芬芳 王少圣 孙恩涛 周书林 WANG Kang, XU Shu-jun, WANG Yi-xiao, CHEN Wen-jie, WANG Yi-nan, YU Fen-fang, WANG Shao-sheng, SUN En-tao, ZHOU Shu-lin (Department of Medical Parasitology, Wannan Medical College, Anhui Province, Wuhu 241002, China)
出处 《中国血吸虫病防治杂志》 CSCD 北大核心 2017年第2期159-162,共4页 Chinese Journal of Schistosomiasis Control
基金 安徽省高校优秀青年人才支持计划重点项目(gxyqZD2016174) 安徽省高校自然科学研究重点项目(KJ2016A725) 皖南医学院重点科研项目培育基金(WK2015Z05) 安徽省大学生创新创业项目(201510368073,201510368054)
关键词 钉螺 线粒体基因组DNA 优化的高盐沉淀法 Oncomelania hupensis Improved high-concentration-salt precipitation method
作者简介 王康,男,本科生。研究方向:分子病原生物学 通信作者E-mail:asdentao@126.com
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