Studies On Detection Of Deletion Mutation Of Human Mtiochondrial DNA

Human mitochondria contain multiple copies of a 16,569bp closed circular DNA molecule, encoding 13 polypeptides, 2 rRNAs and 22 tRNAs that are involved in the generation of bioenergy for cell's functioning. So, any alteration of mitochondrial genome may affect the function of mitochondrial oxidative phosphorylation and cause the further pathological changes of cells. It has been discovered that the oxidative damage to mtDNAs can result in DNA deletion, base modification and insertion mutation etc, among which deletion mutation is the most common. In the past ten years, it has been generally accepted that mtDNA deletions were associated with some degenerative diseases and aging. Meanwhile,some mtDNA deletions have also been detected in some other patients who suffer from mitochondrial diseases , cancer , familial deaf and so on.To develop such reseaches, it is quite necessary to detect mtDNA deletions accurately and roundly. At present, there still exist a great deal of questions in mtDNA deletion detection. Presented methods have pitfalls considering accuracy and systematicness in a sense. Therefore, though it has been noticed that mtDNA deletions play an important role in aging and many kinds of human diseases, there is still no consentaneous opinion about what a role the mtDNA deletions play. Even some contradictory experimental datas or results have been gained. To define the meaning of mtDNA deletions in aging process and some other pathologic states, we must guarantee the reliability and scientificalness of experimental results above all. And so far as the detection of mtDNA deletions, we should ensure the accuracy and systematicness of our results.Our study focused on searching a solution to ensure the accuracy and systematicness of mtDNA deletions screening. And on the basis of this, we make a tentative investigation on the occurrence of mtDNA deletions in cancer and aging. 1 .Preparation of mtDNA template by a modified methodTo develop a simple and rapid method to isolate high quality mitchondrial DNA from human peripheral blood and tissues, Mitochondron are isolated by differential velocityDNAcentrifugation,the membrane of mitochondron is broken up by alkaline lysis.MtDNA is extracted by phonl and chlorodorm. Then we identify the mtDNA and compare it with other mtDNAs obtained by routine methods when handling long PCR.The results show that the nuclear DNA can't be detected in mtDNA prepared by using the improved method and the 8kb product can be obtained efficiently using it as long PCR template. This work suggests the improved method is simple and rapid, the prepared mtDNAs charater as high purity and good for long PCR. 2.The further perfection to "the modified PCR method"we have developedWe have detected a new 889-bp mtDNA deletion in the peripheral blood cells of the victim exposed to a ^Co radiation source by our modified PCR-based method. In this two-step PCR process, we can screen out some suspicious products by comparing the sizes of the first PCR products and then comfirm the real deletions by comparing the sizes of the next PCR products. Each sussuspicious product was recovered as template and PCRs were performed using either original primer pair or nested primer pair. We ever took it for granted that as long as the band, whose size was almost equal to the template, could be found in both amplfication mixture, the template should be derived from real deletion of mtDNA. However,the real deletion of mtDNA may be missed using this kind of modified PCR method in one exceptive instance.That is if the breakpoint of deletion is close(less than 10-bp) or even proximate to 3 ' end of primer, though the product of the first PCR was derived form real deletion of mtDNA, the band,whose size was almost equal to the template,could not be found in PCR mixtures using nested primer pair for reason of no-matching of one primer. Thereby, we may regard such product of the first PCR as "artifact product" due to the mismatching of one of primer pair.Considering such a situation, the original sample mtDNA...