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 C282Y and H63D mutations found in the hemochromatosis gene (HFE) by use of clinical samples.


METHODS
The method, termed solid-phase amplification (SPA), involves dual liquid- and solid-phase amplification of a target sequence by the use of two PCR primers, one of which is in two forms: the first is covalently immobilized to the wall of a microwell, and the second is free in solution. During allele-specific amplification, both the free and solid-phase amplicons are labeled by incorporation of digoxigenin (DIG)-dUTP. The amount of surface-bound amplicon is determined colorimetrically by the use of an alkaline phosphatase-anti-DIG-Fab conjugate and p-nitrophenyl phosphate.


RESULTS
Two different amplicon-labeling methods were evaluated. Analysis of 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 length polymorphism assay results.


CONCLUSIONS
The SPA assay has potential for medium-scale mutation detection, having the advantage of being manipulatively simple and immediately adaptable for use in clinical laboratories with existing ELISA instrumentation.
Turner
M S
MS
Cooperative Research Centre for Diagnostic Technologies, Queensland University of Technology, GPO Box 2434, Brisbane, Qld 4001, Australia.
Penning
S
S
Sharp
A
A
Hyland
V J
VJ
Harris
R
R
Morris
C P
CP
van Daal
A
A
D004587:Electrophoresis, Agar Gel; D006680:HLA Antigens / Q000235:genetics*; D006432:Hemochromatosis / Q000235:genetics*; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I / Q000235:genetics*; D006801:Humans; D008565:Membrane Proteins; D020125:Mutation, Missense; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity
C103673:HFE protein, human; D006680:HLA Antigens; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I; D008565:Membrane Proteins