Promising primosomes

Recently, however, a group at Boston University has developed an alternative assay that may relieve these limitations(ChemBioChem 2001, 2, 133–139). Using molecules called peptide nucleic acids (PNAs) under native conditions, the group performed various assays that have traditionally required DNA denaturation. “When applied to native DNA, the assays prove to be much less noisy than in [the] case of denatured DNA,” remarks group co-leader Maxim Frank-Kamenetskii (see figure 1). The reduced noise is possible because PNAs, which bind tightly in a sequence-specific manner to the DNA backbone, open a designated site in the DNA and allow the binding of oligonucleotides and proteins. This permits extremely sensitive and accurate detection of specified DNA sequences and has important implications for basic research and diagnostics.

Using specific PNAs and oligonucleotides, the researchers opened linear double-stranded DNA and created a so-called artificial primosome. Thisprocedurepermitted the binding of T7 DNA polymerase, a highly processiveenzyme capable of strand-displacement replication. In the presence of deoxy- and dideoxynucleotides, primers are extended and form a sequence ladder comparable to that generated by the traditional Sanger method. Furthermore, this nondenaturing duplex DNA sequencing should allow scientists to read a specific DNA sequence in a mixture of unrelated DNA.Thiscapability has implications in biomedical diagnostics because specific sequences might be read using a patient’s genomic DNA as a template.

By incorporating and quantifying radiolabeled nucleotides at the open complex, researchers can also use the artificial primosome to detect subattomolar quantities of a specific sequence against a background of unrelated DNA, thereby eliminating isolation steps.The Boston team is optimistic that PNAs will also allow selective DNA isolation and identification. “We are developing various DNA detection assays based on the artificial primosome,” Frank-Kamenetskii says.