An improved real time PCR method for simultaneous detection of C282Y and H63D mutations in the HFE gene associated with hereditary hemochromatosis.
HFE-linked hereditary hemochromatosis (HH) is one of the most common inherited diseases among individuals of Northern European ancestry. Two sites of point mutations in the HFE gene--C282Y and H63D--are associated with greater than 90% of HH cases. We have developed a sensitive real time PCR (TaqMan) 5'-nuclease assay for single nucleotide polymorphism (SNP) detection using novel DNA chemistry, and successfully applied this method to detect these mutations. Fluorogenic PCR probes, chemically modified with a minor groove binding agent to increase duplex stability, were used in single and multiplex probe closed tube formats. The probes were tested in two commercially available thermocycling fluorimeters (the Light Cycler and the ABI Prism 7700). Comparison of the results obtained from the analysis of 43 samples showed no discrepancies between our 5' nuclease assay and the restriction length polymorphism analysis, which is routinely used in hospitals. The reported real time PCR technology is ideal for the clinical setting as it is sensitive, eliminates the labor and supply costs of post-PCR steps, reduces the risk of crossover contamination, minimizes sources of error, and can be fully automated.
Walburger
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DK
Epoch Biosciences, Redmond, WA 98052, USA. dwalburger@epochpharm.com
Afonina
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Wydro
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D001483:Base Sequence; D003062:Codon / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics; D004355:Drug Stability; D005456:Fluorescent Dyes; D005470:Fluorometry; D006680:HLA Antigens / Q000235:genetics*; D006432:Hemochromatosis / Q000235:genetics* / Q000276:immunology*; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I / Q000235:genetics*; D006801:Humans; D008565:Membrane Proteins; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D013696:Temperature
D003062:Codon; D017931:DNA Primers; D015342:DNA Probes; D005456:Fluorescent Dyes; C103673:HFE protein, human; D006680:HLA Antigens; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I; D008565:Membrane Proteins