title: Multiplexed target detection using DNA-binding dye chemistry in droplet digital PCR.
abstract: 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.
authors.affiliation: The Digital Biology Center, Bio-Rad Laboratories, Inc. , 5731 West Las Positas Boulevard, Pleasanton, California 94566, United States.
authors.forename: Geoffrey P
authors.initials: GP
authors.lastname: McDermott
authors.forename: Duc
authors.initials: D
authors.lastname: Do
authors.forename: Claudia M
authors.initials: CM
authors.lastname: Litterst
authors.forename: Dianna
authors.initials: D
authors.lastname: Maar
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Hindson
authors.forename: Erin R
authors.initials: ER
authors.lastname: Steenblock
authors.forename: Tina C
authors.initials: TC
authors.lastname: Legler
authors.forename: Yann
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authors.lastname: Jouvenot
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Marrs
authors.forename: Adam
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authors.lastname: Bemis
authors.forename: Pallavi
authors.initials: P
authors.lastname: Shah
authors.forename: Josephine
authors.initials: J
authors.lastname: Wong
authors.forename: Shenglong
authors.initials: S
authors.lastname: Wang
authors.forename: David
authors.initials: D
authors.lastname: Sally
authors.forename: Leanne
authors.initials: L
authors.lastname: Javier
authors.forename: Theresa
authors.initials: T
authors.lastname: Dinio
authors.forename: Chunxiao
authors.initials: C
authors.lastname: Han
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Brackbill
authors.forename: Shawn P
authors.initials: SP
authors.lastname: Hodges
authors.forename: Yunfeng
authors.initials: Y
authors.lastname: Ling
authors.forename: Niels
authors.initials: N
authors.lastname: Klitgord
authors.forename: George J
authors.initials: GJ
authors.lastname: Carman
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Berman
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Koehler
authors.forename: Amy L
authors.initials: AL
authors.lastname: Hiddessen
authors.forename: Pramod
authors.initials: P
authors.lastname: Walse
authors.forename: Luc
authors.initials: L
authors.lastname: Bousse
authors.forename: Svilen
authors.initials: S
authors.lastname: Tzonev
authors.forename: Eli
authors.initials: E
authors.lastname: Hefner
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Hindson
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Cauly
authors.forename: Keith
authors.initials: K
authors.lastname: Hamby
authors.forename: Viresh P
authors.initials: VP
authors.lastname: Patel
authors.forename: John F
authors.initials: JF
authors.lastname: Regan
authors.forename: Paul W
authors.initials: PW
authors.lastname: Wyatt
authors.forename: George A
authors.initials: GA
authors.lastname: Karlin-Neumann
authors.forename: David P
authors.initials: DP
authors.lastname: Stumbo
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Lowe
mesh_terms: D004247:DNA / Q000032:analysis* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D011485:Protein Binding / Q000502:physiology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D004247:DNA