Identification of high-stringency DNA hairpin probes by partial gene folding

Hairpin DNA sequences are widely used as probes for oligonucleotides in a broad range of assays, often as “molecular beacons”. A potential disadvantage of the standard methodology for molecular beacon design is the need to add several self-complementary bases to each end of the probe, since these do not correspond to the target sequence. We describe a conceptually new method of hairpin DNA probe identification, in which a secondary structure prediction algorithm is employed to identify oligonucleotide sequences within an expressed gene having the requisite hairpin structure. Intuitively, such probes should have significantly improved performance over “traditional” hairpin probes, because they are fully complementary with the target. We present experimental evidence verifying this hypothesis for a series of hairpin probes targeting the pag gene of Bacillus anthracis.