Multiplexed Target Detection Using DNA-Binding Dye Chemistry in Droplet Digital PCR
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- 作者：Geoffrey P. McDermott ; Duc Do ; Claudia M. Litterst ; Dianna Maar ; Christopher M. Hindson ; Erin R. Steenblock ; Tina C. Legler ; Yann Jouvenot ; Samuel H. Marrs ; Adam Bemis ; Pallavi Shah ; Josephine Wong ; Shenglong Wang ; David Sally ; Leanne Javier ; Theresa Dinio ; Chunxiao Han ; Timothy P. Brackbill ; Shawn P. Hodges ; Yunfeng Ling ; Niels Klitgord ; George J. Carman ; Jennifer R. Berman ; Ryan T. Koehler ; Amy L. Hiddessen ; Pramod Walse ; Luc Bousse ; Svilen Tzonev ; Eli Hefner ; Benjamin J. Hindson ; Thomas H. Cauly ; III ; Keith Hamby ; Viresh P. Patel ; John F. Regan ; Paul W. Wyatt ; George A. Karlin-Neumann ; David P. Stumbo ; Adam J. Lowe
- 刊名：Analytical Chemistry
- 出版年：2013
- 出版时间：December 3, 2013
- 年：2013
- 卷：85
- 期：23
- 页码：11619-11627
- 全文大小：600K
- 年卷期：v.85,no.23(December 3, 2013)
- ISSN：1520-6882
文摘
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 鈥渟econd generation鈥?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.