Accurate measurement of the relative abundance of different DNA species in complex DNA mixtures.
Accurate measurement of the relative abundance of different DNA species in complex DNA mixtures.
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准确测量复杂 DNA 混合物中不同 DNA 物种的相对丰度。
DOI：
10.1093/dnares/dss002
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Pfeifer,Karl
中科院分区：
文献类型：
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作者：
Jeong,Sangkyun;Yu,Hyunjoo;Pfeifer,Karl
一种可以比较不同DNA序列丰度的分子工具对于比较基因间或种间基因表达是必要的。我们设计并验证了这样一个工具，使用定量竞争性聚合酶链反应方法。对于这种方法，我们采用了竞争对手阵列，一个人工制作的质粒DNA，其中所有的竞争对手模板的目标DNA的安排与一个定义的比例，和解链分析等位基因定量的分数比的竞争性扩增的DNA的准确定量。对具有明确已知的测试序列组成结构的两组DNA混合物进行测定。所得的平均相对误差为0.059和0.021，强调了该方法的高度准确性。此外，该方法的能力，获得生物学数据的事实证明，它可以说明的组织特异性定量表达特征的三个管家基因G6pdx，Ubc和Rps27通过使用其转录本的相对丰度的形式，和Igf2增强子的不同偏好的多个Igf2启动子的转录。
A molecular tool that can compare the abundances of different DNA sequences is necessary for comparing intergenic or interspecific gene expression. We devised and verified such a tool using a quantitative competitive polymerase chain reaction approach. For this approach, we adapted a competitor array, an artificially made plasmid DNA in which all the competitor templates for the target DNAs are arranged with a defined ratio, and melting analysis for allele quantitation for accurate quantitation of the fractional ratios of competitively amplified DNAs. Assays on two sets of DNA mixtures with explicitly known compositional structures of the test sequences were performed. The resultant average relative errors of 0.059 and 0.021 emphasize the highly accurate nature of this method. Furthermore, the method's capability of obtaining biological data is demonstrated by the fact that it can illustrate the tissue-specific quantitative expression signatures of the three housekeeping genesG6pdx,Ubc, andRps27by using the forms of the relative abundances of their transcripts, and the differential preferences ofIgf2enhancers for each of the multipleIgf2promoters for the transcription.