A Melting Curve Analysis–Based PCR Assay for One-Step Genotyping of β- Thalassemia Mutations Fu Xiong, Qiuying Huang, Xiaoyun Chen, Yuqiu Zhou, Xinhua.

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A Melting Curve Analysis–Based PCR Assay for One-Step Genotyping of β- Thalassemia Mutations Fu Xiong, Qiuying Huang, Xiaoyun Chen, Yuqiu Zhou, Xinhua Zhang, Ren Cai, Yajun Chen, Jiansheng Xie, Shanwei Feng, Xiaofeng Wei, Qizhi Xiao, Tianlang Zhang, Shiqiang Luo, Xuehuang Yang, Ying Hao, Yanxia Qu, Qingge Li, Xiangmin Xu The Journal of Molecular Diagnostics Volume 13, Issue 4, Pages 427-435 (July 2011) DOI: 10.1016/j.jmoldx.2011.03.005 Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Proposed MeltPro HBB assay. A: Genotyping procedure. After DNA extraction, each sample tested is mixed with premixed PCR reagents in two separate tubes. PCR and the subsequent melting curve analysis were performed using a real-time PCR thermal cycler. Graphic output of genotyping results is produced. B: Principle of the MeltPro HBB assay. Asymmetric PCR is used to generate single-strand target sequences for hybridization using dual-labeled self-quenched probes, which are labeled at one of four different fluorophores at 5′-end and a quencher dye at 3′-end. One probe may detect one or several adjacent mutations, and it may also be possible for certain mutations to be detected by more than one probe. Examples shown are heterozygous (red line), wild-type (dashed green line), and homozygous mutant (dashed blue line) samples. The Journal of Molecular Diagnostics 2011 13, 427-435DOI: (10.1016/j.jmoldx.2011.03.005) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Identification of β-thalassemia genotypes using the MeltPro HBB assay. The negative derivative fluorescence over temperature versus temperature was plotted for each sample. Melting curves and corresponding genotypes (box) of the 24 mutations plus wild-type alleles are given according to tube 1 (A) and tube 2 (B). The four detection channels are shown by the corresponding fluorophores. Melting curves of HEX and CY5 channels in tube 1 and of HEX and ROX channels in tube 2 are separated into two panels. Black lines indicate no-template control. The Journal of Molecular Diagnostics 2011 13, 427-435DOI: (10.1016/j.jmoldx.2011.03.005) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Analytical sensitivity analysis of the MeltPro HBB assay. Tenfold serial dilutions of wild-type DNA template ranging from 100 ng to 10 pg in a 25-μL reaction were analyzed using four detection channels (indicated by the labeling fluorophores) in reaction tube 1 (A) and tube 2 (B). Top panels: Real-time PCR amplification curves. Middle panels: Linear relationship between the number of threshold cycle values (Cq) and the logarithm of the mass of genomic DNA. Bottom panels: Corresponding melting curves. Water was used as the no-template control (gray lines). The Journal of Molecular Diagnostics 2011 13, 427-435DOI: (10.1016/j.jmoldx.2011.03.005) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Comparison of the MeltPro HBB assay, RDB analysis, and sequencing insofar as time used for HBB variant genotyping. The Journal of Molecular Diagnostics 2011 13, 427-435DOI: (10.1016/j.jmoldx.2011.03.005) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions