Mediator Probe-based Multiplex Digital Methylation-Specific PCR for Sensitive Detection and Methylation Analysis of Biomarker Panels

Introduction:: Liquid biopsy applications, such as the analysis of cell-free tumor DNA, have gained significant attention in the field of cancer diagnostics due to its non-invasive nature and the ability to provide a comprehensive body tissues sampling. DNA methylation has been widely studied as a means of cancer detection [1]. Advances in microfluidics and nucleic acid detection technologies enable digital PCR to be used in analyzing liquid biopsy with superior analytical sensitivity and specificity. Although digital methylation-specific PCR (dMSP) is a useful method for detecting DNA methylation, its multiplexing capacity is limited by the number of available fluorophores and considerable cost. In this work, we present a novel platform for mediator probe-based multiplex digital methylation-specific PCR (MPM-dMSP), which combines a flap endonuclease activity-based high-degree multiplex technology and a probe-based digital melting curve analysis, to achieve quantitative and simultaneous identification of eight DNA methylation biomarkers.


Materials and Methods:: The microfluidic device, consisting of glass and PDMS, is fabricated using ultra-thin soft lithography technique [2]. Each device contains four independent but identical 10,400-nanowell modules. To perform digital PCR, we vacuum-load the sample into a microfluidic chip and pressure-load partitioning oil such that each nanowell contains no more than one copy of targets (Fig. 1A). The PCR reaction mixture contains eight pairs of tagged PCR primers, universal primer, a cocktail of eight unique mediator primers attached to eight target-specific mediator probes, molecular beacon reporters, and Platinum Taq polymerase. During MPM-dMSP, Taq polymerase first extends the target-specific primers and subsequently cleaves the flap sequence of mediator probes, generating target-specific mediator primers [3], through its 5’ flap endonuclease activity. Mediator primers then hybridize to the corresponding universal molecular beacon reporters and get extended, producing target-specific two-dimensional combinations of fluorescent color and probe melting temperature (Fig. 1B). Homo-Tag Assisted Non-Dimer System (HANDS) is employed to reduce primer-dimer formation [4]. For data acquisition, we employed a custom-built multicolor thermal system, consisting of fluorescence imaging components and a flatbed heater (Fig. 2A), to perform thermal cycling and digital melting. Acquired melt images are analyzed using a customized MATLAB algorithm to extract and analyze the fluorescence intensity of each nanowell on the microfluidic device at each temperature for melt curve analysis.


Results, Conclusions, and Discussions: We demonstrated the ability of MPM-dMSP to simultaneously detect eight targets with only two fluorescent channels (Fig. 2B). Multiplex target detection by a single universal molecular beacon significantly reduces the complexity of the assay as well as time and labor costs. We also analytically validated MPM-dMSP by spiking in a known number of synthetic targets representative of bisulfite converted fully methylated sequences of eight genes in a biomarker panel of interest. Probe-based digital melting curve analysis enables accurate identification of the biomarkers (Fig. 2B-2C). MPM-dMSP demonstrated single-copy sensitivities as the detected numbers of positive wells match with the expected numbers. Representative results of four biomarkers detected in the red channel are shown in Fig. 2D.

Conclusion: In conclusion, this study presents a novel MPM-dMSP platform, which combines flap endonuclease activity-based high-degree multiplex technology with a probe-based digital melting curve analysis. MPM-dMSP enables multiplex discrimination and methylation analysis of biomarker panels. We demonstrated the multiplex detection abilities of MPM-dMSP by detecting a panel of eight epigenetic biomarkers at single-copy sensitivities. Overall, our MPM-dMSP provides a simple method for quantitative and simultaneous determination of DNA methylation patterns for biomarker panels.


Acknowledgements (Optional): :

References (Optional): : [1] Danilova, Ludmila, et al. Epigenetics (2022) 17(2).

[2] O’Keefe, Christine, et al. Science Advances (2018) 4.

[3] Huang, Qiuying, et al. Proceedings of the National Academy of Sciences (2022) 119(9).

[4] Brownie, Jannine, et al. Nucleic Acids Research (1997) 25(16).