Digital PCR assays for cancer-associated gene variants

End-point, or conventional PCR, offers qualitative or semi-quantitative analysis, while dPCR offers absolute quantification. Digital PCR is faster, more precise and reproducible. It offers multiplexing capabilities, but it also requires a larger initial investment. 

If you’re ready to enter the world of dPCR, but don’t know where to start, this educational hub covers key topics designed for beginners.

Digital PCR provides a number of benefits, such as absolute target quantification without the need for standards or reference curves. It also has a high tolerance to PCR inhibitors, high precision for detecting small-fold changes, high sensitivity, low limit of detection and high reproducibility, especially across different laboratories.

Applications requiring the detection of small amounts of input nucleic acid or the finer resolution of target amounts, such as rare event detection, copy number variation analysis and gene expression analysis of the low-abundance transcripts, can significantly benefit from the partitioning effect of dPCR. Applications such as liquid biopsy, single-cell analysis, NGS library quantification, quantification of low viral and bacterial loads, analysis of gene editing events and GMO detection can also leverage the tremendous precision and high sensitivity that digital PCR offers compared to qPCR. For most researchers, dPCR represents a complementary approach to qPCR.

Our QIAcuity digital PCR systems have been developed on a 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 the ddPCR method. Besides, simultaneous reading of all partitions of the sample in nanoplates results in a 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 and imaging are integrated into a fully automated instrument, delivering results in under two hours vs. more than four hours required by some other digital PCR systems. Moreover, scalable configurations and higher multiplexing capability make QIAcuity digital PCR easy to adapt to all kinds of labs and application requirements. Using the QIAcuity Software Suite, digital PCR experiments, samples and reaction mixes can be defined, assigned to nanoplates and transferred to the QIAcuity instrument. After the run, data can be analyzed, reports can be created and data can be exported for remote analysis.

Transitioning into a simple and rapid digital PCR workflow is easy with our informative hub.

dPCR LNA Mutation Assays offer significant advantages to cancer researchers working on precise and sensitive mutation detection. These assays are specifically designed for use with the QIAcuity Digital PCR System and are enhanced with Locked Nucleic Acid (LNA) technology. This enhancement greatly improves the specificity and sensitivity of mutation detection, making it possible to identify DNA sequence mutations at very low abundance, with a sensitivity as fine as 0.1% in a single nanoplate well.

The key benefits of dPCR LNA Mutation Assays for cancer researchers include:

• High precision and sensitivity: The use of duplex, hydrolysis probe-based assays allows for highly precise detection of mutations. The presence of both mutant and wild-type probes in the same reaction ensures that researchers can detect and quantify minor genetic variations with great accuracy, crucial for studies in heterogeneous cancer samples where only a few cells may carry the mutation.
• Enhanced specificity: The integration of LNA into the probes increases the binding affinity and specificity towards the target sequences, minimizing the risk of non-specific bindings and improving the overall reliability of the assays.
• Multiplexing capability: Each assay is capable of detecting mutations using two different fluorescent dye combinations, allowing for the simultaneous analysis of mutant and wild-type alleles within the same reaction. This multiplexing ability is particularly useful in applications requiring the analysis of multiple targets, such as assessing co-occurring mutations in cancer.
• Flexibility in sample analysis: By dividing the reaction across multiple wells, even greater sensitivity can be achieved, facilitating the detection of extremely rare mutations. This is especially valuable in cancer research, where detecting low-frequency mutations can inform prognosis and treatment strategies.
• Streamlined workflow: Supplied in a single-tube format with ready-to-use primer pairs and probes, these assays simplify the experimental setup, enabling efficient and straightforward integration into existing research workflows.