qPCR and dPCR: Advantages and Best Uses

The amplification of DNA using PCR is a bedrock lab technique today, and innovations continue to expand its use through research labs to clinics. Quantitative PCR (qPCR) allows a relative quantification of target DNA, and a reliable, established method of testing for the presence or absence of particular sequences (e.g., most PCR-based coronavirus tests use qPCR). Another form of PCR, digital PCR (dPCR) or droplet digital PCR (ddPCR), uses similar chemistry to detect DNA sequences but performs it in many tiny volumes or droplets, using the power of mathematics to improve the signal-to-noise ratio.

There are many ways in which qPCR and dPCR are alike and different, but several important qualities stand out as more notable when talking to PCR experts. This may change as time goes on because PCR technologies continue to evolve. This article discusses some important differences between the advantages and best uses in today’s qPCR and dPCR technologies.

Sensitivity, and absolute vs relative quantitation

For years quantitative PCR has been the gold standard for many applications. However, dPCR is capable of providing more sensitive measurements, and absolute quantification of target DNA (vs qPCR’s relative quantification). “Using qPCR, scientists can only determine a nucleic acid concentration in a sample by comparing the result to a standard curve,” explains Marwan Alsarraj, BioPharma Segment Manager, Digital Biology Group at Bio-Rad Laboratories. “ddPCR, on the other hand, quantifies nucleic acids directly, making the tool more accurate and therefore more sensitive to small nucleic acid concentrations.”

This makes dPCR well-suited to applications where the target exists within complex samples, such as analyzing circulating tumor DNA from liquid biopsies. “As target molecules become rarer, it becomes increasingly more challenging to detect and precisely quantify those molecules,” says Paul Hung, Senior Director and General Manager dPCR, Thermo Fisher Scientific. “Since dPCR naturally dilutes molecules across many reactions, sensitivity can be improved when a given target exists in a high concentration of non-target molecules.” Thermo Fisher recently launched the Applied Biosystems QuantStudio Absolute Q Digital PCR System, a hands-off dPCR instrument with a qPCR-like workflow and 2-hour run time.

Increasing use of dPCR in labs and clinics

Research labs and clinics are both benefiting from the increased sensitivity of dPCR. “As cell and gene therapy research and development continues to evolve, developers are increasingly turning to dPCR to precisely measure different targets including engineered viral vectors, gene-editing events, plasmids, and complete capsids,” says Hung.

Clinical fields such as oncology and organ transplantation also benefit from dPCR’s ability to detect rare DNA, and to quantify small but significant fold-changes in rare DNA. For example, dPCR is used to detect circulating tumor DNA in people with cancer, and evaluate the correct dosages for cell and gene therapies. “We expect ddPCR to become more commonplace in the clinic as precision medicine becomes more mainstream, [because] ddPCR can detect minute changes in tumor load in response to cancer treatment,” says Alsarraj. “For example, serial monitoring with ddPCR can enable oncologists to identify patients at risk of relapse following treatment.”

Dynamic range and flexibility

Although dPCR might excel in sensitivity, qPCR is still the best choice for many other applications. “We recommend our customers use qPCR in gene expression because of its wide dynamic range; its ability to quantify different expression levels, discriminate splice variants and multiplex; and its capability for high-throughput applications and automation,” says Alsarraj. Indeed, it is better known in labs and in a regulatory context, and widely used in screening for genes, health conditions, or infectious diseases.

When analyzing large numbers of samples, using qPCR may also be less expensive than dPCR. “qPCR is an inherently flexible and cost-effective technology for many research needs, enabling researchers to select consumables that fit their specific needs including numerous reaction chemistries, assay options, and easily interchangeable plastics,” says Hung. “In the biopharma industry, qPCR is used in the manufacturing process for monoclonal antibodies, vaccines, cell and gene therapy, and biosimilars as a tool for quality control and genetic analysis.”

With constant innovation and its widespread use, qPCR applications will only broaden. “Scientists continually identify new ways to use qPCR, as it has expanded from gene expression to in-process testing for manufacturing and diagnostics,” says Alsarraj. “We expect that innovation to continue as the technology’s core applications mature.”

Advances in multiplexing

Though qPCR used to have the advantage in multiplexing, this is less true today. Digital PCR is quickly developing higher multiplexing capabilities—for example, platforms such as the naica^® system from Stilla Technologies can run 30-plex or greater dPCR reactions. “Some customers can run the same assays on the naica^® system as they do on qPCR instruments, but they run their samples just one time and get absolute quantitation, rather than relying on standard curves and having to run their samples in replicate,” says Matthew Grow, VP of Global Marketing and Commercial Operations at Stilla Technologies. “Digital PCR on the naica^® system provides researchers the ability to see the amplification and see the assay efficiency, right down to the level of the single droplet and each PCR reaction happening in parallel within their precious samples.”

With dPCR enabling the detection of rare gene expression or cancer-related copy number variation events, greater multiplexing is likely to reveal more avenues for future use. “Multiplexed dPCR [is] an excellent diagnostic complementary to current next-generation sequencing techniques, and could give hospital labs the opportunity to run more in-house patient testing, rather than having to work with outsourced or centralized labs,” notes Grow.

qPCR and dPCR are likely to continue existing in a complementary relationship. “We have seen the importance of qPCR for monitoring and managing the COVID-19 pandemic, and it will remain an essential tool for clinical testing labs, assay developers, and the biotech industry,” says Hung. “As dPCR technology becomes more accessible with simplified workflows and faster times to answer, it will gain adoption for use cases that require robust, reliable, and precise quantification.”