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Genotyping Single Nucleotide Polymorphisms with Molecular Beacons

elicited strong fluorescence, whereas the mismatched probe-target hybrid had significantly lower fluorescence; hence, a clear discrimination was seen between the targets containing a single nucleotide difference.

Distinguishing Three Genotypes

Figure 2 illustrates the results in a plot of threshold cycle (Ct) values where molecular beacons were used to analyze six samples and one no-template control (each sample was assayed in triplicate). Ct is defined as the cycle at which fluorescence is determined to be above background while the reaction is in the exponential phase. Samples that showed no detectable increase in fluorescence throughout the reaction (Ct=40) were considered negative. Two individuals (A/A homozygous on SNP11 shown as black closed diamonds) produced detectable fluorescence beginning at about cycle 29 (average Ct standard deviation=29.10.17) with molecular beacon SNP11A but showed no detectable fluorescence above the background (Ct=40) with SNP11G. On the other hand, two individuals (G/G homozygous on SNP11 shown as black open diamonds) had no increased fluorescence (Ct=40) with molecular beacon SNP11A, and both showed detectable fluorescence above cycle 28 (average Ct STD =28.50.30) with molecular beacon SNP11G. For the two heterozygous individuals (A/G heterozygous on SNP11 shown as green solid stars), similar Cts were seen with both molecular beacons: one had Ct values at close to 35 (average Ct STD=34.90.48), and another had Ct values at around 31 for both molecular beacons (average Ct
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