Rolling-Circle Amplifi cation of Duplex DNA Sequences assisted by PNA Openers

Peptide nucleic acid (PNA) oligomers can be employed as site-specifi c openers of the DNA double helix to locally expose a designated marker sequence inside duplex DNA. The opened DNA site is then hybridized to a circularizable oligonucleotide probe, which is subsequently closed by DNA ligase. This way, the marker sequence from the DNA duplex of interest can be isothermally amplifi ed by a variety of DNA polymerases via the rolling-circle amplifi cation (RCA) mechanism. An alternative strategy for the PNA-assisted RCA exploits a restriction enzyme (and an auxiliary linear oligonucleotide) to selectively introduce a nick within the exposed marker sequence. As a result, a single-stranded DNA segment with a free 3' end is obtained, which can serve as a primer in a subsequent RCA reaction, if hybridized to a circular probe oligonucleotide. Besides DNA polymerase, only one extra enzyme is required in these new promising RCA formats and the two examples presented here demonstrate their robust practical potential for DNA diagnostics.