Institute of Legal Medicine, Innsbruck Medical University, Innsbruck, Austria.
PLoS One. 2009 Dec 18;4(12):e8374. doi: 10.1371/journal.pone.0008374.
For large scale studies aiming at a better understanding of mitochondrial DNA (mtDNA), sequence variation in particular mt haplogroups (hgs) and population structure, reliable low-cost high-throughput genotyping assays are needed. Furthermore, methods facilitating sensitive mixture detection and relative quantification of allele proportions are indispensable for the study of heteroplasmy, mitochondrial sequence evolution, and mitochondrial disorders. Here the properties of a homogeneous competitive duplex allele specific PCR (ARMS) assay were scrutinized in the light of these requirements.
METHODOLOGY/PRINCIPAL FINDINGS: A duplex ARMS assay amplifying either the ancestral mtDNA 2706G allele (non-hg H samples) or the derived 7028C allele (hg H samples) in the presence of SYBR Green fluorescent reporter dye was developed and characterized. Product detection, allele calling, and hg inference were based on the amplicon-characteristic melting-point temperatures obtained with on-line post-PCR fluorescent dissociation curve analysis (DCA). The analytical window of the assay covered at least 5 orders of magnitude of template DNA input with a detection limit in the low picogram range of genomic DNA. A set of forensically relevant test specimens was analyzed successfully. The presence of mtDNA mixtures was detected over a broad range of input DNA amounts and mixture ratios, and the estimation of allele proportions in samples with known total mtDNA content was feasible with limitations. A qualified DNA analyst successfully analyzed approximately 2,200 DNA extracts within three regular working days, without using robotic lab-equipment. By performing the amplification on-line, the assay also facilitated absolute mtDNA quantification.
Although this assay was developed just for a particular purpose, the approach is general in that it is potentially suitable in a broad variety of assay-layouts for many other applications, including the analysis of mixtures. Homogeneous ARMS-DCA is a valuable tool for large-volume studies targeting small numbers of single nucleotide polymorphisms (SNPs).
对于旨在更好地了解线粒体 DNA（mtDNA）的大规模研究，特别是 mt 单倍群（hgs）和群体结构的序列变异，需要可靠的低成本高通量基因分型检测方法。此外，对于异质性、线粒体序列进化和线粒体疾病的研究，还需要能够方便地检测混合样本和相对定量等位基因比例的方法。本文根据这些要求，详细研究了一种均相竞争双链等位基因特异性 PCR（ARMS）检测方法的性能。
方法/主要发现：开发并鉴定了一种在 SYBR Green 荧光报告染料存在的情况下扩增原始 mtDNA 2706G 等位基因（非 hg H 样本）或衍生的 7028C 等位基因（hg H 样本）的双链 ARMS 检测方法。通过在线荧光解链曲线分析（DCA）获得的扩增子特征熔点温度，进行产物检测、等位基因调用和 hg 推断。该检测方法的分析窗口至少覆盖 5 个模板 DNA 输入数量级，检测限低至皮克数量级的基因组 DNA。成功分析了一组法医学相关的测试样本。该检测方法可以在广泛的输入 DNA 量和混合比例下检测到 mtDNA 混合物的存在，并且在已知总 mtDNA 含量的样本中估计等位基因比例是可行的，但存在一定限制。一位合格的 DNA 分析人员在三个正常工作日内，无需使用机器人实验室设备，成功分析了大约 2200 个 DNA 提取物。通过在线进行扩增，该检测方法还便于进行绝对 mtDNA 定量。
虽然该检测方法是专为特定目的开发的，但该方法具有通用性，因为它可能适用于多种不同检测方法的布局，用于许多其他应用，包括混合样本的分析。均相 ARMS-DCA 是针对少数单核苷酸多态性（SNP）进行大规模研究的有价值工具。
