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Detect Single Nucleotide Sequence Changes with Mx4000 Molecular Beacon,,,Allelic Discrimination Kits

lele is labeled with the fluorophore tetrachlorofluorescein (TET), and the molecular beacon specific to the mutant allele is labeled with the fluorophore 6-carboxyfluorescein (FAM). DABCYL is used as the quencher on both molecular beacons. By using two different molecular beacons in each PCR reaction, three possible allelic combinations (genotypes) of two sequence variants can be distinguished simultaneously by the type of fluorescence detected: TET fluorescence indicates homozygosity for the wild-type allele, FAM fluorescence indicates homozygosity for the mutant allele, and both TET and FAM fluorescence together indicates heterozygosity (Figure 2).

Fig.2

Screening is performed with PCR mixtures that consist of a DNA template, both allele-specific molecular beacons, PCR primers, dNTPs, Taq2000 DNA polymerase, and the optimized PCR buffer. Amplification is carried out in a spectrofluorometric thermal cycler. The alleles in a DNA sample are determined by comparing the endpoint fluorescence value (Figure 3) and/or the threshold cycle (Ct) value (Figure 4 and Figure 5) of the DNA sample to the endpoint fluorescence values and/or Ct values of the three genotype-specific DNA controls.

Fig.3

Fig.4

Fig.5

Applications of the Allelic Discrimination Kits

Three kits (CCR2-64I allelic discrimination kit, CCR5-D32 allelic discrimination kit, and SDF-1-3A allelic discrimination kit) are used to detect three common sequence variants that occur in the human CCR2, CCR5, and SDF-1 genes. These variant
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