( STD=31.0 0.44). The diff...)Genotyping Single Nucleotide Polymorphisms with Molecular Beacons

From the distribution of data, the three genotypes were easily distinguished from one another with either of the two molecular beacons. The advantage of having both molecular beacons is to clearly differentiate a homozygote from a heterozygote. For example, one heterozygote had Cts of approximately 31 with both molecular beacons, and the Ct values were not dramatically different from the complementary homozygous targets A/A of 29 Ct with probe A or G/G of 28 Ct with probe G. The perfectly matched homozygotes (A/A or G/G) became mismatched with the other probe (G or A), which resulted in a Ct of 40, so it was easily distinguished from heterozygous target (Ct=31). Negative control samples (shown as black closed dots) containing no template also generated no fluorescence (Ct=40). We blind-tested more samples and obtained very similar results with clear discrimination among different genotypes (data not shown). Results obtained from using molecular beacons were in complete agreement with results generated by conventional sequencing methods.

Conclusions

Stratagenes custom-designed molecular beacons are ideal for single nucleotide polymorphism genotyping. SNP genotyping requires only prior knowledge of a single nucleotide change within a target sequence and the design of a corresponding molecular beacon. With these requirements met, the subsequent molecular beacon procedure takes a quick 2 hours and is easily carried out in a 96-well format for large-scale screening. Additionally, using different molecular beacons labeled with different fluorophores to target different SNPs should make it possible to detect multiple polymorphisms⁵ simultaneously
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