Detection of genetic variation using dual-labeled peptide nucleic acid (PNA) probe-based melting point analysis

Thermal denaturation of probe-target hybrid is highly reproducible, and which makes probe melting point analysis reliable in the detection of mutations, polymorphisms and epigenetic differences in DNA. To improve resolution of these detections, we used dual-labeled (quencher and fluorescence), full base of peptide nucleic acid (PNA) probe for fluorescence probe based melting point analysis. Because of their uncharged nature and peptide bond-linked backbone, PNA probes have more favorable hybridization properties, which make a large difference in the melting temperature between specific hybridization and partial hybridization.

Here, we have shown that full base dual-labeled PNA is apt material for fluorescence probe-based melting point analysis with large difference in the melting temperature between full specific hybridization and that of partial hybridization, including insertion and deletion. In case of narrowly distributed mutations, PNA probe effectively detects three mutations in a single reaction tube with three probes. Moreover, we successfully diagnose virus analogues with amplification and melting temperature signal. Lastly, Melting temperature of PNA oligomer can be easily adjusted just by adding gamma-modified PNA probe.

The PNA probes offer advantage of improved flexibility in probe design, which could be used in various applications in mutation detection among a wide range of spectrums.