Fluorescence-based detection of short DNA sequences under non-denaturing conditions

The ability of peptide nucleic acid (PNA) to open up duplex DNA in a highly sequence-specific manner makes it possible to detect short DNA sequences on the background of or within genomic DNA under non-denaturing conditions. To do so, chosen marker sites in double-stranded DNA are locally opened by a pair of PNA openers, thus transforming one strand within the target region (20–30 bp) into the single-stranded form. Onto this accessible DNA sequence a circular oligonucleotide probe is assembled, which serves as a template for rolling circle amplification (RCA). Both homogeneous and heterogeneous assay formats are investigated, as are different formats for fluorescence-based amplicon detection. Our recent data with immobilized analytes suggest that marker sequences in plasmid and bacterial chromosomal DNA can be successfully detected.

Different formats to detect the presence of short DNA marker sequences (20–30 bp) without denaturation of the DNA sample are presented. Duplex DNA is selectively opened at the marker site by a pair of peptide nucleic acid (PNA) openers, followed by hybridization and circularization of an oligonucleotide probe. Fluorescent detection is, for instance, carried out by rolling-circle amplification in the presence of a fluorophore-tagged decorator probe.Figure optionsDownload as PowerPoint slide