Quantitative analysis of total mitochondrial DNA: Competitive polymerase chain reaction versus real-time polymerase chain reaction
Quantitative analysis of total mitochondrial DNA: Competitive polymerase chain reaction versus real-time polymerase chain reaction
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DOI：
10.1002/jbt.20024
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发表时间：
2004-01-01
影响因子：
3.6
通讯作者：
Epelboym, I
中科院分区：
文献类型：
--
作者：
Bhat, HK;Epelboym, I
在线粒体 DNA (mtDNA) 的消耗可能是造成病理表型的影响因素的情况下，用于定量样品中所含少量核酸的高效且有效的方法将是确定线粒体 DNA (mtDNA) 含量的重要诊断工具。本研究比较了两种定量测定方法，通过扩增 mtDNA 分子上的 613 bp 区域，计算从人类血液中分离的每纳克总基因组 DNA 的总 mtDNA 分子数。在一种情况下，mtDNA拷贝数是通过标准竞争性聚合酶链式反应（PCR）技术计算的，该技术涉及目标DNA与具有与目标序列相同的引物结合位点的非同源内部竞争剂的各种稀释度的共同扩增，以及随后确定目标和竞争剂浓度的等当点。在第二种方法中，拷贝数的计算涉及从使用不同稀释度的目标扩增子序列的实时PCR生成的荧光与拷贝数标准曲线进行外推。虽然使用两种方法得到的 mtDNA 拷贝数相当（使用竞争性 PCR 时为 4.92 +/- 1.01 X 10(4) 分子/ng 总基因组 DNA，而使用实时 PCR 时为 4.90 +/- 0.84 x 10(4) 分子/ng 总基因组 DNA），但使用实时 PCR 分析时，实验间和实验内方差均显着降低。基于重现性、测定复杂性和整体效率，包括分析单个样品所需的时间要求和 PCR 反应数量，我们推荐此处描述的实时 PCR 定量方法，因为其多功能性和有效性无疑将在与线粒体 DNA 损伤和耗竭相关疾病相关的各种研究中发挥巨大作用。 (c) 2004 年 Wiley 期刊公司。
An efficient and effective method for quantification of small amounts of nucleic acids contained within a sample specimen would be an important diagnostic tool for determining the content of mitochondrial DNA (mtDNA) in situations where the depletion thereof may be a contributing factor to the exhibited pathology phenotype. This study compares two quantification assays for calculating the total mtDNA molecule number per nanogram of total genomic DNA isolated from human blood, through the amplification of a 613-bp region on the mtDNA molecule. In one case, the mtDNA copy number was calculated by standard competitive polymerase chain reaction (PCR) technique that involves co-amplification of target DNA with various dilutions of a nonhomologous internal competitor that has the same primer binding sites as the target sequence, and subsequent determination of an equivalence point of target and competitor concentrations. In the second method, the calculation of copy number involved extrapolation from the fluorescence versus copy number standard curve generated by real-time PCR using various dilutions of the target amplicon sequence. While the mtDNA copy number was comparable using the two methods (4.92 +/- 1.01 X 10(4) molecules/ng total genomic DNA using competitive PCR vs 4.90 +/- 0.84 x 10(4) molecules/ng total genomic DNA using real-time PCR), both inter- and intraexperimental variance were significantly lower using the real-time PCR analysis. On the basis of reproducibility, assay complexity, and overall efficiency, including the time requirement and number of PCR reactions necessary for the analysis of a single sample, we recommend the real-time PCR quantification method described here, as its versatility and effectiveness will undoubtedly be of great use in various kinds of research related to mitochondrial DNA damage- and depletion-associated disorders. (c) 2004 Wiley Periodicals, Inc.