Latest – Webinars, events and stories
-
Digital PCR expert webinarJoin us on March 31 — Dr. Jim Huggett of the NML at LGC on the use of digital PCR for high accuracy molecular measurement. -
QIAcuity goes on(line)To keep you connected with the QIAcuity developments during the pandemic, we are incorporating interactive multi-session activities with live webinars and pre-recorded videos. Balance your quarantine playlist with some of these useful online content.
The power of digital PCR
-
Blue - Target
Red - Background (gDNA, cDNA; primers/probes; master mix) -
PCR reaction partitioning into thousands of individual reactions -
Green - Positive reactions
Blue - Negative reactions -
Absolute quantification
Realize the full potential of digital PCR
Find the top applications to be covered by our new assays.
Experts value the precision of digital PCR
Comparison of PCR techniques at a glance
| qPCR | dPCR | |
|---|---|---|
| Reliance on standard curve | Yes; relative quantification | no; absolute quantification |
| Precision | Low; detects mutation rate at >1% | High; detects mutation rate at ≥0.001% Increase signal-to-noise ratio |
| Amplification efficiency | Variable; quantification based on the analysis of the fluorescent signal at the exponential phase, prone to inhibitors | Unaffected; partitioning and endpoint fluorescence measurement of individual partitions |
| Inhibitor tolerance | Lower | Higher; small partition volume contributes to resilience to a large variety of inhibitors |
| Reliability and reproducibility | Lower; references or controls needed | Higher; reference/calibration-free |
Transforming the PCR experience
-
Scalability- Low- to high-throughput instrument configurations – 1-, 4- and 8-plate
- Up to 2 integrated thermocyclers
-
ThroughputSamples processed in a work day can increase from 96 to 1248 samples depending on the plate (24- and 96-well) and instrument (1-, 4- and 8-plate) configuration used -
Multiplexing- Up to 5 detection channels (2-plex and 5-plex; exclusive of reference)
-
Time to result- Simple and rapid plate-based workflow takes users from sample to result in under 2 hours
-
SensitivityFixed partition size and volume
Partition number and input volume per well depending on the plate configuration used - approx. 8,500 and 26,000; 10 and 30 µl reactions
Featured – Delve into digital
-
Are you digital-ready?Whether digital PCR is a totally alien concept or the next most important step in your research plan, we’ve designed this interesting quiz to test your digital future readiness. -
PCR at the crossroadsWhen it comes to molecular biology and genomics research involving nucleic acid quantification, scientists often find themselves standing at the crossroads. The choice of technology is application dependent. -
Expert webinar seriesOur digital PCR webinar topics range from an introduction to the technology to in-depth discussions on how the new nanoplate-based digital PCR solution is combining the familiarity of qPCR workflows with the power of partitioning to transform your research. -
The magic is insideWhat makes the next-gen digital PCR technology so promising? Why use it? Or, when is digital PCR the best choice? Well, the answer is in the details, and we’ve captured it all here.
FAQs about Digital PCR
What is digital PCR?
Digital PCR (dPCR) is a highly precise approach to nucleic acid quantification. It estimates the absolute number of target molecules through statistical methods rather than relying on the number of amplification cycles to determine the initial amount of template molecule in each sample.
How does it work?
You'll be relieved to know that the initial dPCR reaction is assembled using familiar assay components as there used in qPCR. By discretizing or partitioning each sample into a large number of individual and parallel reactions, you're left with one or more target molecules in some partitions whereas others may contain none. Partitioning can be achieved either by dividing the sample into microplates containing capillaries or channels, arrays of miniaturized chamber, or water-oil emulsions as droplets. Each partition undergoes PCR amplification to the endpoint. Partitions with and without amplified product are individually counted. Those containing amplified product and showing a fluorescent signal are designed as positive and scored as "1"; those with no amplified product and showing only a background signal are designated as negatives and scored as "0". Poisson statistical analysis is then applied to determine the absolute concentration of the target present in the initial sample, without relying on references or standards.
What are the major advantages over qPCR?
Quantitative PCR (qPCR) is well-established and preferred method of choice for relative measurement in routing applications requiring a broad dynamic range, high throughput, and rapid time to results in screening large numbers of samples. Absolute quantification with dPCR offers significant advantages over qPCR when it comes to quantifying rare targets in complex backgrounds and detecting small fold change differences with high sensitivity, superior precision, better reproducibility, and high multiplexing capabilities. Moreover, increased tolerance to inhibitors owing to partitioning, and non-reliance on amplification efficiency or standard curves of qPCR makes it a simple and affordable next-gen technology.
Which applications or assays can be performed using dPCR?
As mentioned above, applications such as rare mutation detection, copy number variation, NGS library quantification, low-level pathogen detection, viral load detection, and GMO detection can leverage the tremendous precision and high sensitivity that digital PCR offers compared to qPCR. For most researchers, dPCR represents a complementary approach to qPCR.
How will QIAGEN's digital PCR instrument prove to be any different from commercially available systems?
Our systems are developed as nanoplate-based technology, offering significant benefits over droplet digital PCR (ddPCR) technology. Fixed partitions integrated into the dPCR nanoplate prevent variation in size and coalescence as seen in ddPCR method. Besides, simultaneous reading of all partitions of sample results in faster readout. Nanoplates are not only user-friendly and easy to pipette just like qPCR but are also amenable to front-end automation. Most importantly, correctly sealed nanoplates prevent sample evaporation and well to well contamination.
Partitioning, thermocycling, imaging, and analysis are all integrated into a single fully automated instrument, delivering results in under two hours, vs. more than four hours for currently available digital PCR systems.