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.

Comparison of 3 Methodologies for Genotyping of Small Deletion and Insertion Polymorphisms

“…In ddPCR, each PCR reaction is compartmentalized to allow the direct measurement of target sequences based on limiting dilution (23 ). The use of TaqMan hydrolysis probes in ddPCR assays enables genotyping by allelic discrimination and, if necessary, absolute quantification (24 -26 ).…”

“…The probe-free ddPCR workflow adopted for the genotyping of DIPs in this study was similar to that used in a previously published protocol (23 ). Conditions were standardized across all 6 assays.…”

“…The fluorescence emission from EvaGreen is proportional to the amount of DNA present in solution and, in the absence of PCR bias, will reflect the amplicon sizes (23 ).…”

Quantification of Circulating miRNAs by Droplet Digital PCR: Comparison of EvaGreen- and TaqMan-Based Chemistries

“…The plate was heat-sealed with foil and placed in a conventional thermal cycler. Thermal cycling conditions for EvaGreen assays were as follows: 95 C for 5 minutes, then 40 cycles of 95 C for 30 seconds and 58 C for 1 minute (ramping rate reduced to 2%), and three final steps at 4 C for 5 minutes, 90 C for 5 minutes, and a 4 C indefinite hold to enhance dye stabilization (11). Thermal cycling conditions for TaqMan assays were as follows: 95 C for 10 minutes, then 40 cycles of 95 C for 15 seconds and 60 C for 1 minute (ramping rate reduced to 2%), and a final inactivation step at 98 C for 10 minutes.…”

“…In particular, Hindson and colleagues (10) found that, for quantifying circulating miRNAs, a prototype of the QX100 Droplet Digital PCR system (Bio-Rad Laboratories) was superior to qPCR carried out with TaqMan miRNA assays (Applied Biosystems and Life Technologies). Now, a second-generation instrument (named QX200) has been developed that is able to detect both TaqMan probe and DNA-binding dye chemistries with comparable precision and accuracy (11). In that work, ddPCR using the recently commercialized EvaGreen DNA-binding dye was shown to be comparable with TaqMan assays for quantification of mRNA; however, miRNA was not investigated.…”