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 precipitation method. The mtDNA samples were identified by using spectrophotometry, agarose gel electrophoresis and the amplification products of COX1. The nuclear DNA contamination was tested by the amplification products of ITS.
Results: The concentration and yield of the improved method was significantly higher than those of the traditional method (F = 3 032.65, 10 185.00, both P < 0.01). The mtDNA samples extracted were essentially free of nuclear DNA and protein, meeting PCR, sequence analysis and other molecular biology research requirements.
Conclusions: The improved high-concentration-salt precipitation method for isolating mtDNA is simple, and it has high yield and low cost. The extracted mtDNA can meet relevant analysis requirements.
［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 = 3 032.65、10 185.00, 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。.
Keywords: Improved high-concentration-salt precipitation method; Mitochondrial genome DNA (mtDNA); Oncomelania hupensis.