Multicolor molecular beacons for allele discrimination

Molecular beacons are hairpin-shaped oligonucleotide probes that report the presence of specific nucleic acids in homogenous solutions. When they bind to their targets they undergo a conformationsl reorganization that restores the fluorescence of an internally quenched fluorophore. We found that their hairpin conformation enables the use of a wide variety of differently colored fluorophores. Using several molecular beacons, each designed to recognize a different target and each labeled with a different fluorophore, we demonstrate that multiple targets can be distinguished in the same solution, even if they differ from one another by as little as a single nucleotide. A comparison of “hairpin probes” with corresponding “linear probes” confirms that the presence of the hairpin stem in molecular beacons significantly enhances their specificity.