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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 order to determine the purity of mitochondrial DNA (mtDNA) extracted from wheat by ordinary differential centrifugation, the effects of β-Ac-tin, Ribulose-1, 5-bisphosphate carboxylase / Rabbit and Cytochrome C oxidase subunit (COXIII) gene fragments were used as internal reference markers, and the extracted mtDNA was detected by polymerase chain reaction (PCR) and agarose gel electrophoresis. The results showed that nuclear DNA and chloroplast DNA (ctDNA) were mixed in mtDNA extracted by ordinary differential centrifugation. When the mtDNA was diluted and then detected by internal reference method, it was found that when the mtDNA was diluted to 300 ~ 400 times of the DNA stock solution, the mixed DNA did not reach the sensitivity of the PCR reaction, but the mtDNA and ctDNA still satisfied As a PCR reaction system template requirements. At this point, the extracted mtDNA can be regarded as a nuclear-free DNA-contaminated cytoplasmic genomic DNA.