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

Peptide nucleic acids on microarrays and other biosensors

“…Without the quenching effect, the fluorescence dye emits a signal, thus reporting the occurrence of hybridization. Molecular beacons represent a versatile tool in DNA diagnostics [24]. Although these molecules initially had a hairpin structure with a stem, it was later found, especially for PNA molecules, that a stem is not required for their functioning [25,26].…”

SNP genotyping using microsphere‐linked PNA and flow cytometric detection

“…This condition not only makes the assay highly reliable but also offers the possibility of single-tube multiplexing, where each same sample is typed simultaneously for two or more SNPs. The temperature optimum was found to be 60°C, which was 5°C below the expected optimum as determined by measuring melting temperatures between PNA and its complementary DNA (13). A possible explanation to this lowered melting temperature could be steric hindrance during the hybridization of the single-stranded DNA probes to the surface-bound PNA molecules.…”

“…They are easily manufactured and modified and have good hybridization properties compared with DNA oligos. PNA has been used in a handful of SNP genotyping assays (13)(14)(15)(16)(17)(18)(19) and as RNA capture probe coupled to the surface of microspheres (25).…”

“…For the present study we found that a 15-mer PNA complementary to the A allele and a 13-mer complementary to the C allele fulfilled these requirements. Both molecules should be able to discriminate between a perfectly matching and a mismatching probe above 60°C, and the matching probe should stay annealed until 72°C for the 15-mer and 76°C for the 13-mer (13). To analyze PNA-DNA interactions by flow cytometry, the PNA molecules had to be coupled to a platform with free accessibility in solution and with uniform size.…”

“…We recently reported that short PNA beacons can successfully replace DNA-based probes in two real-time polymerase chain reaction (PCR) genotyping assays (13). Other groups have reported PNA-based allele discrimination using mass spectroscopy (14 -16), quartz crystal microbalance biosensors (17), surface plasmon resonance (18), or fluorescence polarization (19).…”