High-performance multiplex SNP analysis of three hemochromatosis-related mutations with capillary array electrophoresis microplates

An assay is described for high-throughput single nucleotide polymorphism (S NP) genotyping on a microfabricated capillary array electrophoresis (CAE) m icrochip. The assay targets the three common variants at the HFE locus asso ciated with the genetic disease hereditary hemochromatosis (HHC). The assay employs allele-specific PCR (ASPCR) for the C282Y (845g->a), H63D (187c->g ), and S65C (193a->t) variants using fluorescently-labeled energy-transfer (ET) allele-specific primers. Using a 96-channel radial CAE microplate, the labeled ASPCR products generated from 96 samples in a reference Caucasian population are simultaneously separated with single-base-pair resolution an d genotyped in under 10 min. Detection is accomplished with a laser-excited rotary four-color fluorescence scanner. The allele-specific amplicons are differentiated on the basis of both their size and the color of the label e mission. This study is the first demonstration of the combined use of ASPCR with ET primers and microfabricated radial CAE microplates to perform mult iplex SNP analyses in a clinically relevant population.