Multiplex detection of single-nucleotide variations using molecular beacons

We demonstrate that single-nucleotide differences in a DNA sequence can be detected in homogeneous assays using molecular beacons. In this method, the region surroundings the site of a sequence variation is amplified in a pol ymerase chain reaction and the identity of the Variant nucleotide is determ ined by observing which of four differently colored molecular beacons binds to the amplification product. Each of the molecular beacons is perfectly c omplementary to one variant of the target sequence and each is labeled with a different fluorophore. To demonstrate the specificity of these assays, w e prepared four template DNAs that only differed from one another by the id entity of the nucleotide at one position. Four amplification reactions were prepared, each containing all four molecular beacons, but each initiated w ith only one of the four template DNAs. The results show that in each react ion a fluorogenic response was elicited from the molecular beacon that was perfectly complementary to the amplified DNA, but not from the three molecu lar beacons whose probe sequence mismatched the target sequence. The color of the fluorescence that appeared in each tube during the course of the amp lification indicated which nucleotide was present at the site of variation. These results demonstrate the extraordinary specificity of molecular beaco ns. Furthermore, the results illustrate how the ability to label molecular beacons with differently colored fluorophores enables simple multiplex assa ys to be carried out for genetic analysis. (C) 1999 Elsevier Science B.V. A ll rights reserved.