APLP analysis for rapid detection of human mitochondrial DNA variations

线粒体是产生能量的器官，在呼吸代谢中起着重要作用。线粒体DNA （mtDNA）的获得性（老化）突变和遗传多态性与该病密切相关。然而，目前还没有适合于编码区大规模人口调查的分析方法。本文建立了一种简便、快速的多重APLP法检测mtDNA snp的方法。五个snp （nt3010, nt4386, nt5178, nt8794, nt10398）和一个9 bp的缺失（nt8272-8289）被选中。同时，用17种不同的引物组合扩增PCR，用平板-凝胶电泳分型6个代位，并用SYBR-Gold染色。Multiplex APLP方法可同时获得75-151bp的PCR产物，并能准确判断每个碱基置换。所有检测人群均检测到B1和C1型。A2、B2、B3、C2和C4仅在亚洲人群中检测到。特别是B2型在所有日本人群中检测频率相当高。B5和C5是德国特有的单倍型。由于分型速度快，不需要昂贵的设备和试剂，这种方法似乎是最适合检测mtDNA单倍型的方法。

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.