Abstract
DNA is widely used as a biomarker of contamination, infection, or disease, which has stimulated the development of a wide palette of detection and quantification methods. Even though several analytical approaches based on isothermal amplification have been proposed, DNA is still mainly detected and quantified by quantitative PCR (qPCR). However, for some analyses (e.g., in cancer research) qPCR may suffer from limitations arising from competitions between highly similar template DNAs, the presence of inhibitors, or suboptimal primer design. Nevertheless, digitalizing the analysis (i.e., individualizing DNA molecules into compartments prior to amplifying them in situ) allows to address most of these issues. By its capacity to generate and manipulate millions of highly similar picoliter volume water-in-oil droplets, microfluidics offers both the required miniaturization and parallelization capacity, and led to the introduction of digital droplet PCR (ddPCR). This chapter aims at introducing the reader to the basic principles behind ddPCR while also providing the key guidelines to fabricate, set up, and use his/her own ddPCR platform. We further provide procedures to detect and quantify DNA either purified in solution or directly from individualized cells. This approach not only gives access to DNA absolute concentration with unrivaled sensitivity, but it may also be the starting point of more complex in vitro analytical pipelines discussed at the end of the chapter.
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Acknowledgments
This work of the Interdisciplinary Thematic Institute “IMCBio,” as part of the ITI 2021-2028 program of the University of Strasbourg, CNRS, and Inserm, was supported by IdEx Unistra (ANR-10-IDEX-0002), by SFRI-STRAT’US project (ANR-20-SFRI0012) and EUR IMCBio (ANR-17-EURE-0023) under the framework of the French Investments for the Future Program as well as from the previous LabEx NetRNA (ANR-10-LABX-0036). This work was also supported by the “Centre National de la Recherche Scientifique” (CNRS), the “Université de Strasbourg,” and its Initiative of Excellence (IdEx).
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Baudrey, S., Cubi, R., Ryckelynck, M. (2022). Droplet-Based Microfluidic Chip Design, Fabrication, and Use for Ultrahigh-Throughput DNA Analysis and Quantification. In: Caballero, D., Kundu, S.C., Reis, R.L. (eds) Microfluidics and Biosensors in Cancer Research. Advances in Experimental Medicine and Biology, vol 1379. Springer, Cham. https://doi.org/10.1007/978-3-031-04039-9_18
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DOI: https://doi.org/10.1007/978-3-031-04039-9_18
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