Multiplex analysis of the most common mutations related to hereditary haemochromatosis: two methods combining specific amplification with capillary electrophoresis.

We present the first application of a multiplex multicolour assay for the simultaneous detection of three of the most frequent mutations related to hereditary haemochromatosis (C282Y, H63D and S65C), using fluorescent detection and capillary electrophoresis. We describe two methods: the first is based on a single base extension assay, resulting in a single base difference of the extended products; and the second is a competitive allele-specific polymerase chain reaction (PCR), based on competition between allele-specific primers. Specificity of the latter primers is enhanced with a mismatch at the antepenultimate nucleotide. Primers are designed to amplify products of different sizes and with different fluorescent dyes in order to accurately distinguish all possible combinations of genotypes (homozygous and heterozygous for each mutation) in a multiplex PCR analysis. An advantage of the present approach is that capillary electrophoresis analysis of the amplified products enables easy, rapid, unambiguous and high resolution discrimination between wild-type and mutant alleles, although different mutations may be present in the multiplex analysis. This will facilitate automated genotyping for routine molecular diagnostics and large-scale genetic studies.