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. 2018 Nov 21;8:17195. doi: 10.1038/s41598-018-35479-0

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© The Author(s) 2018

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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Step-by-step optimisation of Two-Step ORNi-PCR using KOD DNA polymerase. (Step I) Design of an ORN. (Step II) Determination of the optimal temperature. In this step, 1 μM ORN is used as a default. (Step III) Determination of the optimal ORN concentration. (Step IV) Practical application of Two-Step ORNi-PCR, such as detection of nucleotide mutations. Herein, we show an example using gDNA extracted from mammalian cells, for which 20 ng of gDNA and 30 cycles of denaturing and annealing plus elongation were used as a default. Other types of DNA (e.g., plasmids, fragments, cDNA) and α-type DNA polymerases (e.g., Pfu DNA polymerase) can be used for ORNi-PCR. Dependent on materials, the amount of DNA and the number of cycles of denaturing and annealing plus elongation may require modification.