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Comparative analysis of the hemochromatosis-associated mutations C282Y, H63D and S65C in the  HFE  gene in 51 patients using three different methods is reported. One PCR-RFLP method was based on general primers, whereas another employed mutation-specific mismatched primers. The third method was a new PCR-based reverse hybridisation line probe assay (LiPA), comprising DNA amplification by general primers followed by a single step reverse hybridization to specific probes, immobilized on a nitrocellulose strip. Forty-eight (94%) of the 51 samples yielded identical results by all three methods. Three discrepant results were obtained, caused by polymorphisms in the primer binding region, resulting in no amplification at all or selective amplification, leading to misinterpretation of the  HFE  genotype by PCR-RFLP. The design of the assay and the stringency of the reaction conditions used are crucial to obtain a correct HFE genotype. PCR-LiPA offers an easy and reliable alternative to currently used conventional methods.

Genotyping of Hemochromatosis-Associated Mutations in the HFE Gene by PCR-RFLP and a Novel Revers Hybridization Method