Parr Ryan L, Maki Jennifer, Reguly Brian, Dakubo Gabriel D, Aguirre Andrea, Wittock Roy, Robinson Kerry, Jakupciak John P, Thayer Robert E
Genesis Genomics Inc, 1294 Balmoral Street, Thunder Bay, Ontario, P7B 5Z5, Canada.
BMC Genomics. 2006 Jul 21;7:185. doi: 10.1186/1471-2164-7-185.
Nuclear mitochondrial pseudogenes (numts) are a potential source of contamination during mitochondrial DNA PCR amplification. This possibility warrants careful experimental design and cautious interpretation of heteroplasmic results.
Here we report the cloning and sequencing of numts loci, amplified from human tissue and rho-zero (rho0) cells (control) with primers known to amplify the mitochondrial genome. This paper is the first to fully sequence 46 paralogous nuclear DNA fragments that represent the entire mitochondrial genome. This is a surprisingly small number due primarily to the primer sets used in this study, because prior to this, BLAST searches have suggested that nuclear DNA harbors between 400 to 1,500 paralogous mitochondrial DNA fragments. Our results indicate that multiple numts were amplified simultaneously with the mitochondrial genome and increased the load of pseudogene signal in PCR reactions. Further, the entire mitochondrial genome was represented by multiple copies of paralogous nuclear sequences.
These findings suggest that mitochondrial genome disease-associated biomarkers must be rigorously authenticated to preclude any affiliation with paralogous nuclear pseudogenes. Importantly, the common perception that mitochondrial template "swamps" numts loci precluding detectable amplification, depends on the region of the mitochondrial genome targeted by the PCR reaction and the number of pseudogene loci that may co-amplify. Cloning and relevant sequencing data will facilitate the correct interpretation. This is the first complete, wet-lab characterization of numts that represent the entire mitochondrial genome.
核线粒体假基因（numts）是线粒体DNA PCR扩增过程中潜在的污染来源。这种可能性需要谨慎的实验设计以及对异质性结果进行审慎的解读。
在此我们报告了numts位点的克隆与测序情况，这些位点是用已知可扩增线粒体基因组的引物从人类组织和ρ零（rho0）细胞（对照）中扩增得到的。本文首次对代表整个线粒体基因组的46个同源核DNA片段进行了全序列测定。这一数量出奇地少，主要归因于本研究中使用的引物组，因为在此之前，通过BLAST搜索表明核DNA中含有400至1500个同源线粒体DNA片段。我们的结果表明，多个numts与线粒体基因组同时被扩增，并且增加了PCR反应中假基因信号的负荷。此外，整个线粒体基因组由多个同源核序列的拷贝所代表。
这些发现表明，必须对与线粒体基因组疾病相关的生物标志物进行严格鉴定，以排除与同源核假基因的任何关联。重要的是，认为线粒体模板“淹没”numts位点从而排除可检测到的扩增这一普遍观念，取决于PCR反应所靶向的线粒体基因组区域以及可能共同扩增的假基因位点数量。克隆和相关测序数据将有助于正确解读。这是对代表整个线粒体基因组的numts进行的首次完整的湿实验室表征。
