Short PNA molecular beacons for real-time PCR allelic discrimination of single nucleotide polymorphisms.

The typing of a single nucleotide polymorphism with DNA probes is sometimes problematic because of the limited discriminating power of long DNA probes. As an alternative to existing assays, we have developed a real-time PCR assay for the genotyping of single nucleotide polymorphisms using short peptide nucleic acid (PNA) molecular beacons. A single nucleotide polymorphism in exon 6 of the XPD gene was chosen as the model system. The genotyping experiments were performed in the ABI 7700 using beacons labeled with either fluorescein or JOE, and in the Lightcycler using a fluorescein labeled beacon. QSY-7 was used as the quencher in all the beacons. The result of the genotyping was the same on both instruments and was in agreement with a previously performed RFLP genotyping of 79 samples. The length of PNA molecular beacons is significantly shorter than that of TaqMan or Lightcycler probes, making probe design and genotype discrimination easier.