PNA openers as a tool for direct quantification of specific targets in duplex DNA

We report a new approach for target quantification directly within DNA dupl ex. Our assay is based on the formation of a new biomolecular structure, th e PD-loop. The approach takes advantage of a selective hybridization of a p robe to double-stranded DNA (dsDNA), which is locally opened by a pair of b is-PNA oligomers. To optimize the technique, several experimental formats a re tested with the use of PNA and oligonucleotide probes. The highest sensi tivity is achieved when the hybridized probe is extended and multiply label ed, with I-125-dCTP by DNA polymerase via strand displacement in the presen ce of single-strand binding (SSB) protein. In this case, the PNA-assisted p robe hybridization combined with the method of multiphoton detection (MPD) allows to monitor sub-attomolar amounts of the HIV-1 target on the backgrou nd of unrelated DNA at sub-nCi level of radioactivity. The developed robust methodology is highly discriminative to single mutations, thus being of pr actical use for DNA analysis.