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Solid-Phase Amplification for Detection of C282Y and H63D Hemochromatosis (HFE) Gene Mutations

¹ Cooperative Research Centre for Diagnostic Technologies, Queensland University of Technology, GPO Box 2434, Brisbane, Qld 4001, Australia.
² Molecular Genetics Laboratory, Queensland Health Pathology Service, Royal Brisbane Hospitals Complex, Herston, Brisbane, Qld 4006, Australia.
³ School of Pharmacy and Medical Sciences, University of South Australia, GPO Box 2471, Adelaide, SA 5000, Australia.

^aCurrent address: Institute for Molecular Bioscience, University of Queensland, Brisbane, Qld 4072, Australia.^b Author for correspondence. Fax 617-3864-1534; e-mail p.morris{at}qut.edu.au.

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.

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