APLP analysis for rapid detection of human mitochondrial DNA variations

线粒体是呼吸代谢中具有能量产生和重要功能的器官。线粒体DNA(MtDNA)的获得性(衰老)突变和遗传多态与该病密切相关。但目前还没有建立适用于编码区大规模人口调查的分析方法。本研究建立了一种简便、快速的多重APLP检测线粒体DNA SNPs的方法，选择了5个SNPs(nt3010、nt4386、nt5178、nt8794、nt10398)和1个9个碱基的缺失(nt8272-8289)。同时，用17种不同的引物组合进行扩增，用平板凝胶电泳法对6个碱基进行分型，然后进行SYBR-Gold染色。在多重APLP方法中，同时获得了75-151bp的扩增产物，可以准确地判断每个碱基的替换。在所有受试人群中均检出B1型和C1型。A2、B2、B3、C2和C4仅在亚洲人群中检出。尤其是B2型在所有日本人群中检测到相当高的频率。B5和C5是德国特有的单倍型。这种方法似乎是最适合检测mtDNA单倍型的方法，因为分型速度快，不需要昂贵的设备和试剂。

The mitochondrion is an organ with energy production and important function on respiratory metabolism. The acquired (aging) mutations and genetic polymorphisms of mitochondrion DNA (mtDNA) are deeply related to the disease. However, the analysis method suitable for large-scale population survey in coding region has not been established. This time, the convenient and quick method for detecting of SNPs of mtDNA by the multiplex APLP method was established.Five SNPs (nt3010, nt4386, nt5178, nt8794, nt10398) and a 9-bp deletion (nt8272-8289) were chosen. Simultaneously, the PCR was amplified using 17 kinds of primer mix, and 6 substitutions were typed by slab-gel electrophoresis, and then stained with SYBR-Gold. In the Multiplex APLP method, simultaneously, 75-151bp PCR product was got, and it was possible to accurately judge each base substitution. B1 and C1 types were detected from all populations tested. A2, B2, B3, C2 and C4 were detected only from the Asian groups. Especially, B2 type detects considerably high-frequent from all Japanese groups. B5 and C5 were the haplotype which were almost specific in Germany. This method seemed to be a most suitable for the detection of the mtDNA haplotype, because the typing is quickly performed without requiring expensive equipments and reagents.