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

as the quencher on both molecular beacons.

Fig.2

The assay is performed in a single PCR tube that contains a DNA template, both allele-specific molecular beacons and other PCR reagents. We carried out the amplification in the ABI Prism 7700 sequence detector. The genotype of a DNA sample is determined by measuring the endpoint fluorescence value (endpoint analysis, Figure 2) and/or the threshold cycle (Ct) value (real-time analysis) of the sample. Monitoring PCR reactions in real time produces a fluorescent signal during each cycle. Molecular beacon fluorescence is reported during each annealing step when the molecular beacon is bound to its complementary target (Figure 3 and Figure 4).

Results are displayed as an amplification plot that reflects the change in fluorescence during cycling. The threshold cycle is the cycle at which the fluorescent signal is first detectable above background. For the three assays described in the article, a Ct value of 20 to 32 indicates the presence of a specific allele and a Ct value of 40 indicates the absence of a specific allele. The maximum Ct value is 40 as PCR proceeds to 40 cycles.

The Specific Kits

The CFTR (DF508) Mx4000 molecular beacon allelic discrimination kitff, is designed to rapidly detect/screen the most common mutation that causes cystic fibrosis (CF). This mutation, a three-base-pair deletion, within exon 10 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, accounts for approximately 70% of all mutant CF alleles.3,4 The Factor II (G20210A) Mx4000 molecular beacon allelic discrimination kitff, is used to detect a common genetic variant in the co
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