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. 2020 Apr 10;10:6213. doi: 10.1038/s41598-020-63256-5

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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 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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Minigene assays. (A) Scheme of the different minigene constructs for the exon 12, 14, 15–16 and exon 17 genomic regions. (B,C) PCR products of the minigene constructs obtained using SA and SD primers inside pSPL3 vector. MW: DNA Molecular Weight VI (Roche). V1 and V2 denote the artificial exons of the pSPL3 vector. The sizes of the PCR products are shown on the right. In B) lanes 1 and 2 show the PCR products of the constructs carrying exon 12 wild type (lane 1) and c.1107G > T (Glu369Asp) (lane 2), exon 15–16 wild type (lane 3), c.1282 -1G > A (lane 4), exon 17 wild type (lane 5) and c.1601C > G (Ser534*)(lane 6). Ex12, Ex15, Ex16, Ex17 and Ex16* of EYA4 gene denote exons 12, 15, 16, 17 and 16 lacking the first 68 pb, respectively. In C) lanes 1 and 2 show the PCR products of the empty vector (263 bp) and the RT negative control, respectively. Lane 3 shows a dramatic reduction in the intensity of the 352pb band and the presence of supernumerary bands.