Abstract
Background: Hemochromatosis is a common genetic disease, affecting one in every 200 individuals in the United States. A PCR assay was designed using fluorescent melting curve analysis to simultaneously detect the G845→A (C282Y) and C187→G (H63D) mutations. The G845→A and C187→G loci are distinguished by color, and mutant alleles are distinguished from wild type by probe melting temperature (Tm).
Methods and Results: The probe sets used two fluorophore pairs, fluorescein with LCRed 640 for G845→A and fluorescein with LCRed 705 for C187→G. The probes, complementary to the mutant allele, dissociate from the product at specific Tms. Wild-type alleles form mismatches with the probes, reducing the Tms by 6°C (G845→A) and 10°C (C187→G). One of 133 samples had a Tm shift 4°C less than the wild-type Tm for the G845→A locus. Sequencing confirmed the sample to be homozygous for G845→A and heterozygous for a C→A substitution at position 842 (C842→A), substituting lysine for threonine.
Conclusions: Multiplexing by color and Tm allows for simultaneous genotyping of each mutation. A novel base-pair alteration was detected in cis with a G845→A mutation.
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Phillips, M., Meadows, C.A., Huang, M.Y. et al. Simultaneous Detection of C282Y and H63D Hemochromatosis Mutations by Dual-color Probes. Molecular Diagnosis 5, 107–116 (2000). https://doi.org/10.1007/BF03262029
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DOI: https://doi.org/10.1007/BF03262029