Rapid Single-Tube Screening of the C282Y Hemochromatosis Mutation by Real-Time Multiplex Allele-specific PCR without Fluorescent Probes

Abstract

An accurate determination of the major HFE mutation (C282Y), which is associated with hereditary hemochromatosis, is important in diagnosis and risk assessment for this disease. We report a single-tube high-throughput PCR method for the detection of C282Y. We combined three previously described principles: allele-specific PCR, mutagenically separated PCR, and amplicon identification by specific dissociation curves. PCR amplification was performed with fluorescence detection or conventional thermocycler using the same primers, reactant constituents, and cycling protocol. Primer cross-reactions were prevented by deliberate primer:primer and primer:template mismatches. PCR products were identified by their characteristic melting temperatures based on SYBR Green I fluorescence. For each of the 256 random and 17 known HFE C282Y samples, mutant homozygous, wild-type, and heterozygous samples were unequivocally distinguished. This homogeneous assay is rapid, reproducible, does not require fluorescent oligonucleotide probes, and correctly identifies HFE genotypes.