When to go digital in PCR
What is digital PCR?
Digital PCR (dPCR) is the latest standard for absolute quantification of nucleic acids without the need for a DNA calibrator. Unparalleled accuracy and precision are achieved through the partitioning or dividing of bulk PCR reactions into a large number (up to 26,000) of discrete reactions at nanolitre volumes before undergoing PCR amplification to the endpoint.
Similar to quantitative real-time PCR (RT-qPCR/qPCR), intercalating fluorescent dyes or hydrolysis probes are used in dPCR. After amplification, reactions containing target molecule(s) fluoresce and are counted as 1s, while partitions with no target are counted as 0, giving a binary or ‘digital’ readout. The ratio of positively to negatively fluorescing partitions is calculated, and Poisson statistics applied to determine the absolute concentration of the target present in the initial sample.
Where does dPCR overcome challenges of qPCR?
Absolute quantification: Using standard curves to plot unknown sample fluorescence and derive a ‘relative quantification’ result has made qPCR one of the most popular tools in the molecular biologists’ arsenal. However, this process is still affected by qPCR’s key limitations, namely the reliance on assay efficiency and susceptibility to PCR inhibitors. As described above, dPCR offers true ‘absolute quantification’ of nucleic acids with the ability to distinguish 10% differences in DNA concentration with 95% CI. This level of accuracy is an advantage when validating reference materials, viral titers and molecular QC. Furthermore, studies in copy number variation and gene expression are greatly simplified due to the ease of doing calculations with an absolute copy number output.
Inhibitor tolerance: PCR inhibitors making their way into your samples are an unavoidable part of lab experiments. They are introduced from sample material, nucleic acid preparation and even some nucleic acid purification methods. Inhibitors disrupt PCR amplification, leading to inaccurate data interpretation, decreased sensitivity and potentially outright failure of the PCR reaction. In dPCR, sample partitioning effectively dilutes inhibitors, minimising their effect. Moreover, a yes/no endpoint PCR that does not rely on reaction kinetics helps maintain the count’s accuracy. This is evident amid the COVID-19 pandemic, where dPCR has consistently overcome sample inhibition to determine positive calls, where qPCR has returned false negatives. Likewise, in environmental samples such as soil and water, pathogen detection can be done without compromise.
Increased sensitivity: Signal-to-noise ratios of rare targets increase significantly in dPCR partitions, making detection much easier than conventional qPCR, where low-abundance molecules are difficult to detect amid much higher background levels. This is especially pertinent to applications requiring increased precision, such as the detection of rare mutations in liquid biopsies and minimal residual disease monitoring, monitoring environmental samples and evaluating CAR-T transduction for downstream cell therapy. Where starting material is limited, the sensitivity of dPCR even at low copy numbers may overcome the need for pre-amplification.
Introducing the new QIAcuity™: fully integrated nanoplate digital PCR system
Despite the clear benefits of the technology, dPCR has not been widely adopted so far. Some of the most prevalent challenges currently limiting accessibility are low-throughput instrumentation, overly complex workflows and limited multiplexing capabilities. QIAGEN®’s new dPCR system, the QIAcuity, is an integrated platform that carries out sample partitioning, thermocycling and imaging of the dPCR reactions in microfluidic nanoplates. The overall result is a system that delivers results in under 2 hours, 5-colour multiplexing capabilities and a simple workflow upstream of walkaway operation that mimics qPCR. Once completed, the easy-to-use software analyses the data and presents the final output in copies per microliter.
Find out more at https://www.qiagen.com/dpcr.
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