适于基因组测序的高纯度的大豆线粒体基因组DNA提取方法

Extraction of High Purity Mitochondrial DNA from Glycine max for Genome Sequencing

为满足高通量二代测序要求,本研究采用大豆黄花苗为试材,结合差速离心、蔗糖密度梯度离心及超速离心方法提取高纯度大豆线粒体基因组DNA(mt DNA)。结果表明,差速离心能够有效去除核基因组掺杂;超速离心与蔗糖密度梯度离心结合能够有效去除叶绿体污染。提取的mt DNA经琼脂糖凝胶电泳、紫外光度计检测及叶绿体和细胞核特异性引物检测表明,该方法提取的大豆mt DNA无叶绿体DNA及核DNA污染,且纯度高,可满足测序等对线粒体高纯度的要求,为研究大豆线粒体相关性状的机理奠定了坚实基础。

An efficient method, combination of differential centrifugation, sucrose gradient centrifugation and ultracentrifugation, was used to extract mitochondrial from soybean etiolated tissue of soybean for sequencing. The results showed that differential centrifugation was a good method to remove the nuclear DNA. Combining sucrose gradient centrifugation and ultracentrifugation is better way to remove chloroplasts. The mt DNA was detected by gelose electrophoresis and ultra violet spectrophotometer, furthermore, the chloroplast and nuclear-specific primers were designed to detect mt DNA purity. The results showed that the mt DNA extracted in this way is high purity and had no contamination. It laid a foundation for the study of mt DNA relatived mechanism on Glycine max.