An Allele-Specific PCR System for Rapid Detection and Discrimination of the CYP2C19∗4A, ∗4B, and ∗17 Alleles Stuart A. Scott, Qian Tan, Usman Baber,

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An Allele-Specific PCR System for Rapid Detection and Discrimination of the CYP2C19∗4A, ∗4B, and ∗17 Alleles Stuart A. Scott, Qian Tan, Usman Baber, Yao Yang, Suparna Martis, Jeffrey Bander, Ruth Kornreich, Jean-Sébastien Hulot, Robert J. Desnick The Journal of Molecular Diagnostics Volume 15, Issue 6, Pages 783-789 (November 2013) DOI: 10.1016/j.jmoldx.2013.06.004 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Illustration of the CYP2C19∗17 and CYP2C19∗4 variant allele locations and primers used in the ASP assay. ASP products are 865 bp, and the internal amplification control product (CYP2C19 exon 4) is 539 bp. Black numbered boxes represent CYP2C19 exons. ASP, allele-specific PCR. The Journal of Molecular Diagnostics 2013 15, 783-789DOI: (10.1016/j.jmoldx.2013.06.004) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 ASP system for detection of CYP2C19∗4A, CYP2C19∗4B, and CYP2C19∗17. Illustration of the primers and amplification strategy to detect and discriminate between the CYP2C19 wild-type (ie, non-∗4 or -∗17 alleles) (A), ∗17 (B), ∗4A (C), and ∗4B (D) alleles. Horizontal lines between forward and reverse primers represent successful PCR amplification, and interrupted horizontal lines represent failed PCR amplification due to only unidirectional primer extension. ASP, allele-specific PCR; FWD, forward; MUT, mutated; REV, reverse; WT, wild-type. The Journal of Molecular Diagnostics 2013 15, 783-789DOI: (10.1016/j.jmoldx.2013.06.004) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Results from ASP detection of representative samples with known CYP2C19 genotypes confirmed by allele-specific cloning and sequencing and/or targeted genotyping. A: Sanger sequence confirmation of representative controls for the defining ∗17 (c.−806C>T) and ∗4 (c.1A>G) variants. Representative ASP results from the assay development cohort (B) and from the assay validation cohort (C), including the targeted genotyping results and revised genotype results after ASP. ASP, allele-specific PCR; WT, wild-type. The Journal of Molecular Diagnostics 2013 15, 783-789DOI: (10.1016/j.jmoldx.2013.06.004) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions