Digital PCR Applications: Hemato-Oncology Research

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Blood-based biomarkers play an essential role in elucidating the molecular basis of diseases and help enable research into the clinical management of disease. Measurable residual disease (MRD, also called minimal residual disease and molecular residual disease) describes the proportion of cancer cells that remain after treatment, which are typically undetectable by standard imaging and other techniques. Whereas biomarkers are typically identified through genome-wide screens, subsequent confirmation and further research is best served by fast, robust, and higher-throughput techniques such as qPCR and dPCR.

Digital PCR (dPCR) is a valuable tool to complement other molecular techniques across a wide range of applications, including hemato-oncology research. dPCR divides the reaction into many smaller micro-reactions, each of which is read individually and interpreted as positive or negative based on the presence or absence of fluorescence (endpoint detection). Separating the targets into micro-reactions permits direct quantification of the target sequence (through Poisson statistics) without the need for reference standards or a standard curve. dPCR excels at providing highly precise quantification of rare variants, and compartmentalization of the reaction renders dPCR less sensitive to PCR inhibitors, mismatched assay efficiencies, and inter-assay competition.

Learn more about the use of dPCR in hemato-oncology in our technical note.

Frequently asked questions

NGS and microarray offer a breadth of targets for initial discovery, whereas dPCR and qPCR are better suited for quantification of a few targets. Unlike qPCR, dPCR provides absolute quantification of targets without a standard curve. The higher accuracy, precision, and sensitivity of dPCR enables reliable quantification of low-abundance variants even in a high-wild-type background.

dPCR complements qPCR and NGS in hemato-oncology research. Whereas the wide dynamic range and throughput of qPCR is ideal for quantification of higher-frequency variants, dPCR excels at quantifying low-abundance targets. dPCR can also aid in longitudinal surveillance of select markers with shorter turnaround time and lower cost after the biomarker of interest is identified via NGS. Furthermore, dPCR is more tolerant of assays with varying efficiencies and inter-assay competition, which provides greater flexibility for multiplexing of variant and wild type or multiple variants in the same reaction.

Because the reaction is compartmentalized, dPCR is more tolerant of PCR inhibitors than qPCR. Nucleic acid extraction techniques vary in their ability to remove common PCR inhibitors that are present in blood and bone marrow samples. Some inhibitors co-purify with nucleic acids and can be difficult or impossible to remove completely. Whether your sample contains inhibitors or is limited, the high sensitivity and high precision of dPCR can enable more robust quantification of mutant allele frequencies than other molecular techniques.