Kakuda Tsuneo, Shojo Hideki, Tanaka Mayumi, Nambiar Phrabhakaran, Minaguchi Kiyoshi, Umetsu Kazuo, Adachi Noboru
Department of Legal Medicine, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Yamanashi 409-3898, Japan.
Department of General Dental Practice and Oral & Maxillofacial Imaging, Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, Malaysia.
PLoS One. 2016 Jun 29;11(6):e0158463. doi: 10.1371/journal.pone.0158463. eCollection 2016.
Mitochondrial DNA (mtDNA) serves as a powerful tool for exploring matrilineal phylogeographic ancestry, as well as for analyzing highly degraded samples, because of its polymorphic nature and high copy numbers per cell. The recent advent of complete mitochondrial genome sequencing has led to improved techniques for phylogenetic analyses based on mtDNA, and many multiplex genotyping methods have been developed for the hierarchical analysis of phylogenetically important mutations. However, few high-resolution multiplex genotyping systems for analyzing East-Asian mtDNA can be applied to extremely degraded samples. Here, we present a multiplex system for analyzing mitochondrial single nucleotide polymorphisms (mtSNPs), which relies on a novel amplified product-length polymorphisms (APLP) method that uses inosine-flapped primers and is specifically designed for the detailed haplogrouping of extremely degraded East-Asian mtDNAs. We used fourteen 6-plex polymerase chain reactions (PCRs) and subsequent electrophoresis to examine 81 haplogroup-defining SNPs and 3 insertion/deletion sites, and we were able to securely assign the studied mtDNAs to relevant haplogroups. Our system requires only 1×10-13 g (100 fg) of crude DNA to obtain a full profile. Owing to its small amplicon size (<110 bp), this new APLP system was successfully applied to extremely degraded samples for which direct sequencing of hypervariable segments using mini-primer sets was unsuccessful, and proved to be more robust than conventional APLP analysis. Thus, our new APLP system is effective for retrieving reliable data from extremely degraded East-Asian mtDNAs.
线粒体DNA（mtDNA）因其多态性本质和每个细胞中的高拷贝数，成为探索母系系统发育地理祖先以及分析高度降解样本的有力工具。完整线粒体基因组测序技术的最新出现，带来了基于mtDNA的系统发育分析技术的改进，并且已经开发出许多多重基因分型方法用于系统发育重要突变的分层分析。然而，很少有用于分析东亚mtDNA的高分辨率多重基因分型系统能够应用于极度降解的样本。在此，我们展示了一种用于分析线粒体单核苷酸多态性（mtSNP）的多重系统，该系统依赖于一种新型的扩增产物长度多态性（APLP）方法，该方法使用含次黄苷的引物，并且专门为极度降解的东亚mtDNA的详细单倍群分类而设计。我们使用14个6重聚合酶链反应（PCR）及随后的电泳来检测81个定义单倍群的SNP和3个插入/缺失位点，并且我们能够将所研究的mtDNA可靠地分配到相关的单倍群。我们的系统仅需要1×10-13克（100飞克）的粗DNA就能获得完整图谱。由于其扩增子尺寸小（<110 bp），这种新的APLP系统成功应用于使用微型引物对高变区进行直接测序未成功的极度降解样本，并且证明比传统的APLP分析更稳健。因此，我们的新APLP系统对于从极度降解的东亚mtDNA中检索可靠数据是有效的。
