2 research outputs found
International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant
This study tested the claim that
digital PCR (dPCR) can offer highly reproducible quantitative measurements
in disparate laboratories. Twenty-one laboratories measured four blinded
samples containing different quantities of a <i>KRAS</i> fragment encoding G12D, an important genetic marker for guiding
therapy of certain cancers. This marker is challenging to quantify
reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence
of competing wild type sequences and the need for calibration. Using
dPCR, 18 laboratories were able to quantify the G12D marker within
12% of each other in all samples. Three laboratories appeared to measure
consistently outlying results; however, proper application of a follow-up
analysis recommendation rectified their data. Our findings show that
dPCR has demonstrable reproducibility across a large number of laboratories
without calibration. This could enable the reproducible application
of molecular stratification to guide therapy and, potentially, for
molecular diagnostics
Multiplexed Target Detection Using DNA-Binding Dye Chemistry in Droplet Digital PCR
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 āsecond generationā 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