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. 2019 Jun 20;9:8853. doi: 10.1038/s41598-019-45356-z

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© The Author(s) 2019

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Optimisation of the fluorescence signal and assay parameters. (a) Schematic depicting the method by which PG (green) intercalates with dsDNA (blue) to emit a fluorescent signal. Digestion of this PG:DNA complex by nucleases (red) disrupts the duplex structure and causes a concomitant reduction (grey) in signal that can be measured in real-time. (b) Calibration curve indicating the fluorescent signal emitted by increasing concentrations of 80 bp dsDNA and 13.1 kb plasmid DNA. (c) Standard curve composed of 80, 60, 40 and 20 bp sequences. The point shown at 0 on the x-axis is an 80-nt ssDNA oligomer to represent the end product of complete resection. (d) DNase I titration on an 80 bp dsDNA substrate. Dotted grey lines represent controls containing standard duplexes of intermediate sizes. (e) Extracted maximum gradient from (d) to determine the reaction rate at increasing concentrations of DNase I. (f) Maximum rate analysis of resection per nM DNase I per second. Error bars represent SEM; n = 3 in all cases.