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Fluorescence-Based Single-Tube Assays to Rapidly Detect Human Gene Mutations

agulation factor II (prothrombin) gene. The mutation, a G to A substitution at nucleotide position 20210, is associated with elevated plasma prothrombin levels and an increased risk of deep vein thrombosis.5,6 The mutation is also being evaluated clinically as a risk factor for cardiovascular disease.7

The HFE (C282Y) Mx4000 molecular beacon allelic discrimination kitff, is used to detect a G to A substitution at the nucleotide position 845 of the HFE gene.8 This mutation, which changes cysteine to tyrosine at amino acid codon 282, is found in homozygous form in more than 80% of Caucasian patients suffering from hereditary haemochromatosis. One of the most common human genetic diseases, it characterized by an iron overload that eventually results in tissue damage and death to the patient. The estimated frequencies of the disease varies from 1 in 200 to 1 in 400 in individuals of Northern European descent.9

Assay Validation

Fig.4

The assay specificity of the three kits was determined by real-time PCR analysis using the ABI Prism 7700 sequence detector. First, validated genotype-specific human genomic DNA was used as PCR template, and the expected results were obtained by molecular beacon analysis (assay details in kits manual). Examples of the results for CFTR (DF508) allelic discrimination are shown in Figure 2 and Figure 3. An example of the results for factor II (G20210A) allelic discrimination is shown in Figure 4. Second, 50 human genomic DNA, previously genotype-characterized by PCR/RFLP analysis for the factor II and the HFE mutations, were tested, and results of the molecular beacon analysis were in complete agreement with tho
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