Multiplexed Target Detection Using DNA-Binding Dye Chemistry in Droplet Digital PCR
Multiplexed Target Detection Using DNA-Binding Dye Chemistry in Droplet Digital PCR
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DOI：
10.1021/ac403061n
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发表时间：
2013-12-03
影响因子：
7.4
通讯作者：
Lowe, Adam J.
中科院分区：
文献类型：
--
作者：
McDermott, Geoffrey P.;Do, Duc;Lowe, Adam J.
两年前，我们描述了第一个液滴数字PCR（ddPCR）系统，旨在为所有研究人员提供一种工具，消除与使用模拟标准定量实时PCR（qPCR）相关的大量不确定性。该系统能够进行基于TaqMan水解探针的测定，用于核酸的绝对定量。由于液滴化学的显著进步以及与基于染料的靶标检测相关的多种益处，我们已经创建了与TaqMan探针和DNA结合染料检测化学相容的“第二代”ddPCR系统。在这里，我们描述了DNA结合染料的ddPCR的操作特性，并提供了并排比较TaqMan探针检测。通过在热循环之前对每个样品进行分区，我们证明了现在可以使用DNA结合染料从单个反应中定量多个靶物质。与分区相关的分辨率提高也使得可以可视化和解释非特异性扩增产物产生的信号。我们期望，将ddPCR的精确性与DNA结合染料和TaqMan探针检测化学物质联合收割机相结合的能力将进一步使研究界能够回答复杂和多样化的遗传问题。
Two years ago, we described the first droplet digital PCR (ddPCR) system aimed at empowering all researchers with a tool that removes the substantial uncertainties associated with using the analogue standard, quantitative real-time PCR (qPCR). This system enabled TaqMan hydrolysis probe-based assays for the absolute quantification of nucleic acids. Due to significant advancements in droplet chemistry and buoyed by the multiple benefits associated with dye-based target detection, we have created a "second generation" ddPCR system compatible with both TaqMan-probe and DNA-binding dye detection chemistries. Herein, we describe the operating characteristics of DNA-binding dye based ddPCR and offer a side-by-side comparison to TaqMan probe detection. By partitioning each sample prior to thermal cycling, we demonstrate that it is now possible to use a DNA-binding dye for the quantification of multiple target species from a single reaction. The increased resolution associated with partitioning also made it possible to visualize and account for signals arising from nonspecific amplification products. We expect that the ability to combine the precision of ddPCR with both DNA-binding dye and TaqMan probe detection chemistries will further enable the research community to answer complex and diverse genetic questions.