Real-time cdPCR opens a window into events occurring in the first few PCR amplification cycles
Real-time cdPCR opens a window into events occurring in the first few PCR amplification cycles
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DOI：
10.1007/s00216-015-9073-8
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发表时间：
2015-12-01
影响因子：
4.3
通讯作者：
Romsos, Erica L.
中科院分区：
文献类型：
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作者：
Duewer, David L.;Kline, Margaret C.;Romsos, Erica L.
聚合酶链式反应 (PCR) 终点有限稀释技术统称为“数字 PCR (dPCR)”，已被提议为 DNA 定量提供潜在的主要方法。我们正在评估几种市售 dPCR 系统，用于验证人类基因组 DNA 参考材料中的质量浓度。为了更好地了解腔室和液滴 dPCR（cdPCR 和 ddPCR）系统结果中观察到的异常情况，我们开发了一种图形工具，用于评估和记录实时 cdPCR 系统中 PCR 检测的性能：尖形图，交叉阈值的累积分布。尖顶结构似乎嵌入了有关早期扩增事件的信息。我们使用四阶段扩增模型成功地模拟了在不同测定和反应条件下观察到的结果，该模型通过创建完整的 1) 来自原始 DNA 靶标的不确定长度的第一代“长”扩增子，2) 来自长扩增子的第二代定义长度扩增子，以及 3) 来自另一个定义长度扩增子的定义长度扩增子的概率进行参数化。我们正在利用该模型的见解来优化 dPCR 测定设计和反应条件，并帮助验证提议用于分配 DNA 参考材料的测定。
Polymerase chain reaction (PCR) end-point limiting dilution techniques, collectively termed "digital PCR (dPCR)", have been proposed as providing a potentially primary method for DNA quantification. We are evaluating several commercially available dPCR systems for use in certifying mass concentration in human genomic DNA reference materials. To better understand observed anomalies among results from chamber- and droplet-dPCR (cdPCR and ddPCR) systems, we have developed a graphical tool for evaluating and documenting the performance of PCR assays in real-time cdPCR systems: the ogive plot, the cumulative distribution of crossing threshold values. The ogive structure appears to embed information about early amplification events. We have successfully simulated ogives observed with different assays and reaction conditions using a four-stage amplification model parameterized by the probability of creating an intact 1) first generation "long" amplicon of indeterminate length from an original DNA target, 2) second generation defined-length amplicon from a long amplicon, and 3) defined-length amplicon from another defined-length amplicon. We are using insights from this model to optimize dPCR assay design and reaction conditions and to help validate assays proposed for use in value-assigning DNA reference materials.