Hereditary hemochromatosis is one of the most common hereditary disorders in Caucasians.The disease is linked to two single-nucleotide polymorphisms (SNPs) in the HFE gene.The two point mutations result in a change of Cys to Tyr at position 282 and His to Asp at position 63 in the resultant protein.We have developed a single-nucleotide extension (SNE) assay for hereditary hemochromatosis genetic testing, which employs capillary electrophoresis to simultaneously detect the SNE products generated from the two SNP sites. An upstream or a downstream primer adjacent to the possible mutation site is designed and extended one nucleotide further at the 3' end, complementary to the nucleotide at the possible mutation site.The extended nucleotide is one of four fluorescently labeled dideoxynucleotide triphosphates that also act as terminators. Analysis of the extended products by laser-induced fluorescence capillary electrophoresis (LIF-CE) directly reflects the identity of the possible mutation site. Using one primer upstream or downstream from the possible mutation site, three genotypes at one mutation site can be distinguished. Using both upstream and downstream primers provides a second level of specificity and increases the accuracy of the genetic test. The protocol can also be applied to the study of other SNP analyses and to simultaneous detection of multiple mutation sites.