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Genotyping Single Nucleotide Polymorphisms with Molecular Beacons

tion, it adopts a hairpin structure that brings the fluorphore and quencher sufficiently close to each other to allow efficient quenching of the fluorophore. If, however, the molecular beacon is bound to its complementary target, the fluorophore and quencher are far enough apart that the fluorophore cannot be quenched and the molecular beacon fluoresces.

Custom-Designed Molecular Beacons

Hybridization of molecular beacons with their complementary target is highly specific; therefore, we designed molecular beacons to study one of the SNPs of our interest. Based on linkage and association studies in a large Arab family with 20 affected relatives, a diabetes susceptibility locus (IDDM17) maps to 10q25.1.4 IDDM 17 and its flanking markers are localized to a 120-kb region within a completely sequenced bacterial artificial chromosome (BAC) based on association studies of a number of SNPs. We genotyped a group of people in the family and determined the presence of three genotypes. The genotypes were complementary with probe-target homozygote (A/A), mismatched probe-target homozygote (G/G), and heterozygote (A/G) for the SNP.

The fluorescence of the molecular beacon was monitored in real time while the PCR reaction was taking place. The fluorescent signal was monitored and reported during each anneal-extend step when the molecular beacon was bound to its complementary target. Results were displayed on an amplification plot, which reflected the change in fluorescence during cycling. Figure 1 shows the dissociation curves of hybrids between molecular beacons and A/A homozygote (green dots) or G/G homozygote (black circles). Fluorescence was plotted as the derivative value on the Y-axis. In the temperature interval from 62C to 72C, the perfectly complementary probe-target hybrid
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