Solid-phase amplification for detection of C282Y and H63D hemochromatosis (HFE) gene mutations

Background: There is a need for simple, rapid, and inexpensive methods for the detection of single-nucleotide polymorphisms. Our aim was to develop a single-tube ELISA-like PCR assay and evaluate it by detecting the common C2 82Y and H63D mutations found in the hemochromatosis gene (HFE) by use of cl inical samples. Methods: The method, termed solid-phase amplification (SPA), involves dual liquid- and solid-phase amplification of a target sequence by the use of tw o PCR primers, one of which is in two forms: the first is covalently immobi lized to the wall of a microwell, and the second is free in solution. Durin g allele-specific amplification, both the free and solid-phase amplicons ar e labeled by incorporation of digoxigenin (DIG)-dUTP. The amount of surface -bound amplicon is determined colorimetrically by the use of an alkaline ph osphatase-anti-DIG-Fab conjugate and p-nitrophenyl phosphate. Results: Two different amplicon-labeling methods were evaluated. Analysis o f 173 clinical samples for the C282Y and H63D HFE point mutations with SPA revealed that only one sample was incorrectly diagnosed, apparently because of operator error, when compared with conventional restriction fragment le ngth polymorphism assay results. Conclusions: The SPA assay has potential for medium-scale mutation detectio n, having the advantage of being manipulatively simple and immediately adap table for use in clinical laboratories with existing ELISA instrumentation. (C) 2001 American Association for Clinical Chemistry.