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.