Wang Zhen-Cheng, Wang Xue-Min, Jiao Bing-Hua, Jin You-Xin, Miao Ming-Yong, Zhu Ke-Jun, Ni Qing-Gui
Department of Basic Medicine, Department of Biochemistry and Molecular Biology, Second Military Medical University, Shanghai, 200433, People's Republic of China.
IUBMB Life. 2003 Mar;55(3):133-7. doi: 10.1080/1521654031000110181.
A new PCR based method was developed to detect deleted mitochondrial DNA (mtDNA). Peripheral blood cell DNA was obtained from a victim who was accidently exposed to a 60Co radiation source in 1990. Using the DNA as template, first PCR was performed to generate multiple products including true deletions and artifacts. The full length product was recovered and used as template of secondary PCR. The suspicious deletion product of mtDNA could be confirmed only if it was yielded by first PCR. Using either original primers or their nested primers, the suspicious deletion product was amplified and authenticated as a true deletion product. The template was recovered and determined to be a deletion by sequencing directly. The results show that a new mtDNA deletion, which spans 889 bp from nt 11688 to nt 12576, was detected in the peripheral blood cells of the victim. It indicates that this new PCR-based method was more efficient at detecting small populations of mtDNA deletion than other routine methods. MtDNA deletion was found in the victim, suggesting the relationship between the deletion and phenotypes of the disease.
开发了一种基于聚合酶链反应（PCR）的新方法来检测缺失的线粒体DNA（mtDNA）。从一名1990年意外暴露于60Co辐射源的受害者身上获取外周血细胞DNA。以该DNA为模板，进行第一轮PCR以产生多种产物，包括真正的缺失产物和假象产物。回收全长产物并用作第二轮PCR的模板。只有当线粒体DNA的可疑缺失产物由第一轮PCR产生时，才能得到确认。使用原始引物或其巢式引物对可疑缺失产物进行扩增，并鉴定为真正的缺失产物。回收模板并通过直接测序确定为缺失。结果表明，在受害者的外周血细胞中检测到一种新的线粒体DNA缺失，该缺失从第11688位核苷酸到第12576位核苷酸跨度为889 bp。这表明这种基于PCR的新方法在检测少量线粒体DNA缺失方面比其他常规方法更有效。在受害者中发现了线粒体DNA缺失，提示了该缺失与疾病表型之间的关系。
