An artificial primosome: Design, function, and applications

Double-stranded (ds) DNA is capable of the sequence-specific accommodation of an additional oligodeoxyribonucleotide strand by the peptide nucleic aci d(PNA)-assisted formation of a so-called PD-loop. We demonstrate here that the PD-loop may function as an artificial primosome within linear, nonsuper coiled DNA duplexes. DNA polymerase with its strand displacement activity u ses this construct to initiate the primer extension reaction at a designate d dsDNA site. The primer is extended by several hundred nucleotides. The ef ficiency of dsDNA priming by the artificial primosome assembly is comparabl e to the single-stranded DNA priming used in various assays. The ability of the PD-loop structure to perform like an artificial primosome on linear ds DNA may find applications in biochemistry, molecular biology, and molecular biotechnology, as well as for DNA diagnostics. In particular, multiple lab els can be incorporated into a chosen dsDNA site resulting in: ultrasensiti ve direct quantification of specific sequences. Furthermore nondenaturing d sDNA sequencing proceeds from the, PD-loop. This approach opens the way to direct isothermal reading of the DNA I sequence against a background of-unr elated DNA, thereby eliminating the need for purification of the target DNA .