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. 2020 Sep 25;10:15745. doi: 10.1038/s41598-020-72834-6

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Selection of a protocol for isolation of EV-associated DNA (EV-DNA). (A) Experiment workflow; (B) Recovery of BRAF^V600E-positive extracellular vesicles (EVs) from healthy donor plasma samples with ultracentrifugation (UC), chemical precipitation (CP), peptide affinity (PA) isolation and immunoaffinity (IA) isolation. Following DNA extraction, both mutant and wild type BRAF and KRAS genes were detected by allele-specific quantitative PCR (AS-QPCR). Results are representative of four independent experiments. (C) Western blot analysis of exosome markers Alix, Tsg101 and GAPDH after UC, CP, PA and IA isolation; blot is representative of three independent experiments. The original full-length blot is available as supplementary information (Supplementary Figure 6). (D) Quantitation of DNA extracted from UC- CP-, PA- and IA-derived pellets by fluorimetric assay.