Mutation Analysis of SLC26A4 for Pendred Syndrome and Nonsyndromic Hearing Loss by High-Resolution Melting  Neng Chen, Lisbeth Tranebjærg, Nanna Dahl Rendtorff, Iris Schrijver  The Journal of Molecular Diagnostics  Volume 13, Issue 4, Pages 416-426 (July 2011) DOI: 10.1016/j.jmoldx.2011.03.003 Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Agarose gel electrophoresis of PCR products of all the primer sets. All the PCR products were run on a 2% agarose gel to confirm the size and specificity of the amplicons. The name of the targeted amplicon is given above each panel. The reaction of the first and second lanes of each target amplicon contains gDNA template and no-template control, respectively. The size of the molecular standard (M) is given on the right side. The Journal of Molecular Diagnostics 2011 13, 416-426DOI: (10.1016/j.jmoldx.2011.03.003) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 HRM-normalized melting curves and difference plots of variants detected in exon 10. HRM analysis of exon 10 indicated that there were seven normalized melting curves (A) and difference plots (B) that were different from the reference (Ref; green) samples. To correlate each normalized melting curve and difference plot with its corresponding variant, each variant is also illustrated separately. For example, C represents the normalized melting curve for c.1198delT. The difference plot (D) confirms that c.1198delT generates melting curves (red) different from the Ref samples. E and F: p.Glu384Gly/p.Thr416Pro. G and H: p.Glu384Gly. I and J: p.Thr416Pro. K and L: p.Thr416Pro/p.Ala406 (no change in amino acid). M and N: p.Thr416Pro/p.Thr416Pro. O and P: p.Arg409His/p.Arg409His. Some variant curves were not automatically detected by the software but instead were identified by visual inspection and were manually colored. Variants with more than one sample tested have multiple curves in the graph (D, J, L, and N). The Journal of Molecular Diagnostics 2011 13, 416-426DOI: (10.1016/j.jmoldx.2011.03.003) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Example of HRM mutation analysis in three patient samples. One reference and three patient samples were tested on the prespotted plate. Well position I was used for reference gDNA (Ref), and positions II, III, and IV were used for patients P1, P2, and P3, respectively. Four positive controls (top) were evaluated in this run: Ctrl 1 [p.Met1Thr (c.2T>C)] for amplicon ex 2-1, Ctrl 2 [p.Ile300Leu (c.898A>C)] for ex 7, Ctrl 3 (c.919-2A>G/c.919-2A>G) for ex 8, and Ctrl 4 [p.Thr416Pro (c.1246A>C)] for ex 10. gDNA from each of the positive controls was added only to the well that amplifies the sequence containing the variant (ie, well position V of that amplicon). All the wells at position VI contained the no-template control. The normalized melting curves for the positive controls are in the top panels. For patient samples, only the amplicons with altered normalized melting curves are depicted. The difference plot of the amplicon for ex 10 is included to confirm the difference observed on the normalized melting curves for P1 and Ctrl 4. The Journal of Molecular Diagnostics 2011 13, 416-426DOI: (10.1016/j.jmoldx.2011.03.003) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions