Serious overestimation in quantitative PCR by circular (supercoiled) plasmid standard: microalgal pcna as the model gene.
Serious overestimation in quantitative PCR by circular (supercoiled) plasmid standard: microalgal pcna as the model gene.
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DOI：
10.1371/journal.pone.0009545
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发表时间：
2010-03-05
期刊：
影响因子：
3.7
通讯作者：
Lin S
中科院分区：
文献类型：
--
作者：
Hou Y;Zhang H;Miranda L;Lin S
实时定量PCR（qPCR）已成为核酸和微生物丰度定量的金标准，其中携带靶基因的质粒DNA最常用作标准。最近的一项研究表明，DNA的超螺旋环状确认似乎抑制PCR扩增。然而，尚未评估用作标准品的不同结构类型的DNA（环状与线性）可能影响定量准确度的程度。在这项研究中，我们定量比较了基于环状质粒（主要是超螺旋形式）和线性DNA标准品（线性化质粒DNA或PCR扩增子）的qPCR准确性，使用增殖细胞核基因（pcna），普遍存在的真核基因，在五种海洋微藻作为模型基因。我们观察到，使用环状质粒作为模板的PCR比等摩尔线性标准品多出2.65-4.38个阈值循环数。虽然记录的硅藻海链藻的基因组序列显示了单个拷贝的pcna，但使用环状质粒作为标准品的qPCR产生了每个基因组7.77个拷贝的pcna的估计值，而使用线性标准品的qPCR产生了每个基因组1.02个拷贝。我们的结论是，环状质粒DNA是不适合作为标准，和线性DNA应代替，在绝对qPCR。环状质粒标准品的严重高估可能是由于在PCR的早期阶段，当超螺旋质粒是主导模板时，其扩增效率较低而未被检测到。
Quantitative real-time PCR (qPCR) has become a gold standard for the quantification of nucleic acids and microorganism abundances, in which plasmid DNA carrying the target genes are most commonly used as the standard. A recent study showed that supercoiled circular confirmation of DNA appeared to suppress PCR amplification. However, to what extent to which different structural types of DNA (circular versus linear) used as the standard may affect the quantification accuracy has not been evaluated. In this study, we quantitatively compared qPCR accuracies based on circular plasmid (mostly in supercoiled form) and linear DNA standards (linearized plasmid DNA or PCR amplicons), using proliferating cell nuclear gene (pcna), the ubiquitous eukaryotic gene, in five marine microalgae as a model gene. We observed that PCR using circular plasmids as template gave 2.65-4.38 more of the threshold cycle number than did equimolar linear standards. While the documented genome sequence of the diatom Thalassiosira pseudonana shows a single copy of pcna, qPCR using the circular plasmid as standard yielded an estimate of 7.77 copies of pcna per genome whereas that using the linear standard gave 1.02 copies per genome. We conclude that circular plasmid DNA is unsuitable as a standard, and linear DNA should be used instead, in absolute qPCR. The serious overestimation by the circular plasmid standard is likely due to the undetected lower efficiency of its amplification in the early stage of PCR when the supercoiled plasmid is the dominant template.
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