内参法在线粒体DNA纯度鉴定中的应用

Application of Internal Reference Method in Identification of Mitochondrial DNA Purity

为了确定普通差速离心法提取的小麦线粒体DNA（mtDNA）的纯度，以小麦肌动蛋白β亚基（β-Ac～tin）、1，5-二磷酸核酮糖羧化酶／加氧酶大亚基（RabcL）和细胞色素C氧化酶第三亚基（COXⅢ）基因的片段作为内参标记，依靠聚合酶链式反应（PCR）及琼脂糖凝胶电泳技术，对提取的mtDNA进行了检测。结果表明，普通差速离心法提取的mtDNA中混杂有核DNA和叶绿体DNA（ctDNA）。对提取的mtDNA稀释后再进行内参法检测，结果发现，当mtDNA稀释到DNA原液的300～400倍时，混杂其中的核DNA已达不到PCR反应的敏感度，但mtDNA、ctDNA仍能满足作为PCR反应体系模板的要求。此时，提取到的mtDNA可视为无核DNA污染的细胞质基因组DNA。

In this study, the genes β-Actin, RabcL and COXIII were used as the internal reference markers to detect the purity of mitochondrial DNA extracted using differential centrifugation through polymerase chain reaction （PCR） and agarose gel electrophoresis. The result showed that mitochondrial DNA extrac- ted using differential centrifugation contained impurities, which included nuclear and chloroplast DNA （ctDNA）. Then the mtDNA was diluted and the purity of the diluted mtDNA was detected using the same method. It was found that the nuclear DNA became undetectable by PCR when the mtDNA was diluted 300-400 times. However, the mtDNA and ctDNA could still be amplified by PCR. The mtDNA obtained in this way could be used as mtDNA without contamination of nuclear DNA.