Improved real-time detection of the H63D and S65C mutations associated with hereditary hemochromatosis using a SimpleProbe assay format.

Background: Genetic predisposition to hemochromatosis involves several different point mutations in the HFE gene. Routine testing for two such mutations (H63D and S65C) using real-time genotyping deserves special care, as these mutations are in close proximity (6 bp) of each other.

Methods: A novel assay was designed for these two mutations based on a SimpleProbe assay format that allows for more flexibility in assay design, as it requires only one detection probe instead of the two probes that are commonly used with a hybridization probe based assay.

Results: The SimpleProbe assay format yielded data that were easily interpreted without significant optimizing efforts.

Conclusions: The SimpleProbe assay format offers some unique advantages compared to hybridization probes and offers a robust and interesting alternative to hybridization probes in the detection of genetic variations. This is the first time that the use of this assay format is described in the clinical literature.