Simultaneous Genotyping of α-Thalassemia Deletional and Nondeletional Mutations by Real-Time PCR–Based Multicolor Melting Curve Analysis Qiuying Huang,

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Simultaneous Genotyping of α-Thalassemia Deletional and Nondeletional Mutations by Real-Time PCR–Based Multicolor Melting Curve Analysis Qiuying Huang, Xudong Wang, Ning Tang, Tizhen Yan, Ping Chen, Qingge Li The Journal of Molecular Diagnostics Volume 19, Issue 4, Pages 567-574 (July 2017) DOI: 10.1016/j.jmoldx.2017.04.003 Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Location of the primers and probes used in the multicolor melting curve analysis assay. Schematic representation of the α-globin gene cluster, indicating the extent of the four deletions, position of the three nondeletional mutations, and relative positions of the primers and probes (except for the ABL-F and ABL-R internal positive control primers and probe P1, which are located on a different chromosome). Locations of X, Y, and Z sequence homology boxes and hypervariable regions are also shown. The Journal of Molecular Diagnostics 2017 19, 567-574DOI: (10.1016/j.jmoldx.2017.04.003) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Melting curve results from genomic DNA samples with various α-globin genotypes. Melting curves and corresponding genotypes of the four deletions and three nondeletional mutations and the wild-type (WT) allele are shown by their corresponding probes. Gray lines indicate the no template control. −dF/dT, negative derivative of fluorescence with respect to temperature versus temperature. The Journal of Molecular Diagnostics 2017 19, 567-574DOI: (10.1016/j.jmoldx.2017.04.003) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Schematic illustration of the melting profiles of five genotypes that contained the HKαα allele (A) and the exact peak height ratios of HKαα/αα, HKαα/-α3.7, and -α3.7/αα in the multicolor melting curve analysis assay (B). Rm1 denotes the height of the -α3.7 allele melting peak and Rm2 the height of the wild-type (WT) allele melting peak, both of which were automatically obtained by the software of SLAN-96 real-time PCR system. Dotted lines indicate positions of the melting peaks. CS, Constant Spring; −dF/dT, negative derivative of fluorescence with respect to temperature versus temperature; QS, Quong Sze; WS, Westmead. The Journal of Molecular Diagnostics 2017 19, 567-574DOI: (10.1016/j.jmoldx.2017.04.003) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions