Reporter Emission Multiplexing increases multiplex capacity in digital PCR

Population-specific reporters enable the precise detection of additional DNA target sequences beyond the number of fluorescence detection channels

Emission multiplexing (REM) uses target sequence-independent reporters with fluorescence labeling that generate several distinct fluorescence signals of distinguishable intensity in one detection channel during a digital PCR (Calabrese et al.¹). This leads to a multiplication of the multiplex capacities beyond the number of detection channels.

Simultaneous detection of mutated DNA and wild-type DNA as gBlock DNA sequences in only one fluorescence channel using reporter emission multiplexing, at different concentrations (Naica®Prism6)

Simultaneous detection of mutated DNA and wild-type DNA as gBlock DNA sequences in only one fluorescence channel using reporter emission multiplexing, at different concentrations (QIAcuity)

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Since the reporter molecules used for this purpose are target sequence-independent, more complex modification layouts and/or configurations can be used to adjust the intensities of the fluorescence signals. These reporters are therefore population-specific (population-specific reporter/ PSR) and are activated by mediator sequences that are released during digital PCR by base-specific cleavage of a mediator probe (Schlenker et al.²). In this way, even point mutations (SNPs) can be distinguished highly specifically from wild-type DNA.

Without having to make any changes to the device hardware, reporter emission multiplexing increases the multiplex grades in digital PCR and maximizes specificity and sensitivity in the detection of DNA target sequences. Corresponding high-multiplex assays have already been developed for various devices (e.g. Naica® Prism6, Nio® und QIACuity®), making them ideal for digital PCR applications such as oncology, liquid biopsy, pathogen detection or plant breeding.

Hahn-Schickard has filed a patent application for Reporter Emission Multiplexing. If you are interested in this technology, please do not hesitate to contact us.

Literature

1. Reporter emission multiplexing in digital PCRs (REM-dPCRs). Silvia Calabrese, Anja M. Markl, Maximilian Neugebauer, Stefanie J. Krauth, Nadine Borst, Felix von Stetten and  Michael Lehnert. Analyst 2023, DOI:10.1039/D3AN00191A
2. Stringent Base Specific and Optimization-Free Multiplex Mediator Probe ddPCR for the Quantification of Point Mutations in Circulating Tumor DNA. F. Schlenker, E. Kipf, M. Deuter, I. Höffkes, M. Lehnert, R. Zengerle, F. von Stetten, F. Scherer, J. Wehrle, N. von Bubnoff, P. Juelg, T. Hutzenlaub, N. Borst. Cancers 2021. DOI: 10.3390/cancers13225742

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