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Digital PCR

Digital is the new absolute

Finding a needle in a haystack

Researchers performing sensitive applications such as copy number variation and rare target detection often find themselves struggling to identify faint genetic signals against a strong background, especially when it is a single positive lost in a dense pool of negatives. Finding the rare allele or mutant sequence is a typical needle in a haystack problem! By partitioning the sample into a large number of individual reactions, digital PCR makes it surprisingly easy to detect the single positive. It is in such instances where the lower limit of detection excludes standard real-time quantitative PCR as a viable solution.

At QIAGEN, our mission is to empower you with everything you need to overcome challenges in real-time and digital PCR research applications. In the end, finding a needle in a haystack might not remain as challenging as it sounds.

Digital PCR is a highly precise approach to sensitive and reproducible nucleic acid detection and quantification. Measurements are performed by dividing the sample into partitions, such that there is either zero or one target molecule present in any individual reaction. Each partition is analyzed after end-point PCR cycling for the presence or absence of a fluorescent signal, and the absolute number of molecules present in the sample is calculated. It does not rely on a standard curve for sample target quantification. Eliminating the reliance on standard curve reduces error and improves precision. 
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Realize the full potential of digital PCR

Find the top applications to be covered by our new assays.

"We have used digital PCR because we needed a very good method with good sensitivity. We used it because we are sure the sensitivity is better, and there is a good precision. "


Peter Böhm and Nina Kjersgaard,
Department of Clinical Genetics, Rigshospitalet, Copenhagen

"Digital PCR allows precise, reliable, inferences of methylation levels and allows us to take epigenetics from the laboratories into the future of precision medicine."

Dr. Rob Philibert,
CEO of Behavioral Diagnostics
  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
If your future is digital, why shouldn't your PCR be, too? Imagine keeping the familiarity and ease of qPCR but gaining the higher sensitivity and precision of digital PCR, without prolonging the time to scientific insight. The QIAcuity, QIAGEN's fully integrated nanoplate-based digital PCR system, has been designed with your research needs and the limitations of the currently available methods in mind. It's time to simplify the transition from qPCR and prepare your critical research applications for the changing landscape of digital PCR.

Advantages over droplet digital PCR technology

The nanoplate-based technology offers significant benefits over droplet digital PCR (ddPCR). These include:

• Fixed partitions prevent variation in size and coalescence
• Sealed nanoplates prevent well to well contamination
• Faster readout possible due to simultaneous reading of all partitions of a sample
• User-friendly, familiar plates are easy to pipet just like for qPCR
• Plates are amenable to front-end automation

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It is easy to use the QIAcuity
Transition into a simple and rapid digital PCR workflow.

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.

 

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