Kovalenko S A, Harms P J, Tanaka M, Baumer A, Kelso J, Ozawa T, Linnane A W
Centre for Molecular Biology and Medicine, Austin and Repatriation Medical Centre, Victoria, Australia.
Hum Mutat. 1997;10(6):489-95. doi: 10.1002/(SICI)1098-1004(1997)10:6<489::AID-HUMU12>3.0.CO;2-W.
A number of mitochondrial DNA (mtDNA) deletions have been recently identified in the tissues of patients with mitochondrial diseases and in elderly individuals. To investigate the distribution of mutant mitochondrial genomes within any particular tissue, we have developed a sensitive method based on indirect in situ PCR. Our experiments have shown that the new method had the advantage of selectively amplifying only mtDNA bearing the 4,977 bp deletion. We show that this method is more sensitive than in situ hybridization for detecting the 4977 bp mtDNA deletion while using only a low number of PCR cycles that minimize damage to tissue architecture. By using this method, we have demonstrated that the mutation does not occur uniformly among the cells of a given tissue/organ. This technique will be useful studying the distribution/localization of mtDNA mutations in individual cells of tissues and when combined with enzyme histochemical procedures in adjacent sections will enable the correlation between mtDNA mutations and bioenergy defects in single cells.
最近在患有线粒体疾病的患者组织和老年人组织中发现了许多线粒体DNA（mtDNA）缺失。为了研究突变线粒体基因组在任何特定组织中的分布，我们开发了一种基于间接原位PCR的灵敏方法。我们的实验表明，新方法具有仅选择性扩增携带4977 bp缺失的mtDNA的优势。我们表明，在仅使用少量PCR循环以最小化对组织结构的损伤的情况下，该方法在检测4977 bp mtDNA缺失方面比原位杂交更灵敏。通过使用这种方法，我们证明了给定组织/器官的细胞中突变并非均匀发生。这项技术将有助于研究组织单个细胞中mtDNA突变的分布/定位，并且当与相邻切片中的酶组织化学程序结合使用时，将能够揭示单细胞中mtDNA突变与生物能量缺陷之间的相关性。
