Multiplexed Target Detection Using DNA-Binding Dye Chemistry in Droplet Digital PCR

被引：147

作者：

McDermott, Geoffrey P. ^{[1
]}

Do, Duc ^{[1
]}

Litterst, Claudia M. ^{[1
]}

Maar, Dianna ^{[1
]}

Hindson, Christopher M. ^{[2
]}

Steenblock, Erin R. ^{[1
]}

Legler, Tina C. ^{[1
]}

Jouvenot, Yann ^{[1
]}

Marrs, Samuel H. ^{[1
]}

Bemis, Adam ^{[1
]}

Shah, Pallavi ^{[1
]}

Wong, Josephine ^{[1
]}

Wang, Shenglong ^{[1
]}

Sally, David ^{[1
]}

Javier, Leanne ^{[1
]}

Dinio, Theresa ^{[1
]}

Han, Chunxiao ^{[1
]}

Brackbill, Timothy P. ^{[1
]}

Hodges, Shawn P. ^{[1
]}

Ling, Yunfeng ^{[1
]}

Klitgord, Niels ^{[1
]}

Carman, George J. ^{[1
]}

Berman, Jennifer R. ^{[1
]}

Koehler, Ryan T. ^{[3
]}

Hiddessen, Amy L. ^{[4
]}

Walse, Pramod ^{[1
]}

Bousse, Luc ^{[1
]}

Tzonev, Svilen ^{[1
]}

Hefner, Eli ^{[1
]}

Hindson, Benjamin J. ^{[2
]}

Cauly, Thomas H., III ^{[1
]}

Hamby, Keith ^{[1
]}

Patel, Viresh P. ^{[1
]}

Regan, John F. ^{[1
]}

Wyatt, Paul W. ^{[1
]}

Karlin-Neumann, George A. ^{[1
]}

Stumbo, David P.

Lowe, Adam J. ^{[1
]}

机构：

[1] Biorad Labs Inc, Digital Biol Ctr, Pleasanton, CA 94566 USA

[2] 10X Technol Inc, Pleasanton, CA 94566 USA

[3] VerdAscend Sci, West Linn, OR 97068 USA

[4] Purigen Biosyst Inc, Alviso, CA 95002 USA

来源：

ANALYTICAL CHEMISTRY | 2013年 / 85卷 / 23期

关键词：

REAL-TIME PCR; QUANTITATION;

D O I：

10.1021/ac403061n

中图分类号：

O65 [分析化学];

学科分类号：

070302 ; 081704 ;

摘要：

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.

引用

页码：11619 / 11627

页数：9

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