胡萝卜肉质根线粒体DNA提取方法研究

The Method for Isolating Mitochondrial DNA from Fleshy Root of Carrot

本研究以胡萝卜根为材料,通过高盐差速离心和蔗糖沉淀差速离心两种方法提取线粒体DNA。通过改变缓冲液、使用DNaseⅠ处理得到了无核DNA污染的线粒体。用SDS和蛋白酶K裂解线粒体,经酚:氯仿:异戊醇抽提除去蛋白,并用RNaseA消化从而得到单纯线粒体DNA。本实验还特别设计了叶绿体特异性引物检测线粒体DNA纯度。结果表明:利用优化后的蔗糖沉淀差速离心法提取胡萝卜线粒体DNA,所得线粒体DNA纯度高、产量高,每克肉质根能够得到27.60μg线粒体DNA。

We used the fleshy root of carrot to be as experiment material, and isolated mitochondrial DNA from the tested material by using high-salt differential centrifugation and sucrose sedimentating differential centrifuga- tion in this study. No nuclear DNA-contamined mitochondrial DNA was obtained by adding DNase I and changing the buffer solutions. Pure mitochondrial DNA was successfully obtained by using SDS and proteinase K to crack mitochon, phenol：chloroform：isoamyl alcohol extraction to remove protein, and digestion with RNase A. Chloroplast-specific primers were designed to detect mitochondrial DNA purity. The results showed that mitochondrial DNA with higher purity and yield were obtained by using the optimized method of sucrose sedimentating differential centrifugation from the fleshy root of carrot, with 27.60 ug mitochondrial DNA was obtained from per gram of fleshy carrot root.