Genotyping of Human Platelet Antigens 1 to 6 and 15 by High-Resolution Amplicon Melting and Conventional Hybridization Probes Michael Liew, Lesa Nelson,

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Presentation transcript:

Genotyping of Human Platelet Antigens 1 to 6 and 15 by High-Resolution Amplicon Melting and Conventional Hybridization Probes Michael Liew, Lesa Nelson, Rebecca Margraf, Sheri Mitchell, Maria Erali, Rong Mao, Elaine Lyon, Carl Wittwer The Journal of Molecular Diagnostics Volume 8, Issue 1, Pages 97-104 (February 2006) DOI: 10.2353/jmoldx.2006.050053 Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Melting curves of HPAs 1 to 5 and 15 from HybProbe assays performed on the LightCycler. Each HPA locus (1 to 5 and 15) is displayed on separate graphs (A–F). The genotype of each curve is indicated with arrows. The thin gray line without an arrow on each graph represents the no template control for each reaction. The Journal of Molecular Diagnostics 2006 8, 97-104DOI: (10.2353/jmoldx.2006.050053) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 HPA 6 melting curves from the HybProbe assay performed on the LightCycler. There are six melting curves shown, each representing a homozygous genotype at HPA 6 (nucleotide 1544) and the adjacent 3′ nucleotide (nucleotide 1545). The genotype of each curve is labeled with an arrow. The thin gray line without an arrow on each graph represents the no template control for each reaction. The first letter is the nucleotide at position 1544; the second letter is the nucleotide at position 1545. The Journal of Molecular Diagnostics 2006 8, 97-104DOI: (10.2353/jmoldx.2006.050053) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Derivative curves of high-resolution melting data from an HPA 1 and 5 duplex assay. Three samples are shown, which represent the six possible genotypes. Each genotype is labeled with an arrow. Sample 1 (thin black line) is HPA 5 b/b and HPA 1 a/b, sample 2 (thick gray line) is HPA 5 a/b and HPA 1 a/a, and sample 3 (thick black line) is HPA 5 a/a and HPA 1 b/b. The Journal of Molecular Diagnostics 2006 8, 97-104DOI: (10.2353/jmoldx.2006.050053) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Normalized curves of high-resolution melting data from HPAs 1 to 5 and 15. Each HPA locus (1 to 5 and 15) is represented by one graph (A–F). Three samples are shown on each graph, which represent the three possible genotypes: a/a (thick black line), a/b (thin gray line), and b/b (thick gray line). Each genotype is labeled with an arrow. The Journal of Molecular Diagnostics 2006 8, 97-104DOI: (10.2353/jmoldx.2006.050053) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 Derivative curves of high-resolution melting data using an unlabeled oligonucleotide probe to genotype HPA 6. The first graph represents the homozygous genotypes (A). There are six melting curves shown, each representing HPA 6 (nucleotide 1544) and the adjacent 3′ nucleotide (nucleotide 1545). The genotype of each curve is labeled with an arrow. The first letter is the nucleotide at position 1544; the second letter is the nucleotide at position 1545. The second graph shows two heterozygous genotypes generated by mixing the appropriate homozygous samples together (B). These heterozygotes are the most likely combinations based on the nucleotide frequencies. The Journal of Molecular Diagnostics 2006 8, 97-104DOI: (10.2353/jmoldx.2006.050053) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions