Detection of PCR products using PNA strand invasion

The unique ability of homopyrimidine peptide nucleic acid (PNA) to strand i nvade homopurine sites of duplex DNA offers a potential alternative to exis ting techniques for rapid detection of PCR products. From gel shift studies , PNA was found to specifically strand invade homopurine sites that had bee n incorporated into an amplicon during the PCR cycle. This was achieved by adding a homopyrimidine sequence to the 5'-terminus of a PCR primer. The po sition of the strand invasion sites at the termini of the DNA duplex offers kinetic advantages for PNA strand invasion, since the termini of DNA duple xes are known to be unwound. This unwound state was demonstrated using a no vel assay that determined single-stranded regions within the amplicon. The presence of the PNA moiety in strand invasion complexes was confirmed by a novel electroblot, an Enzyme Linked Nucleic Acid assay and by an increase i n stability as demonstrated by T-m studies with the Idaho RapidCycler. Sinc e the strand invasion sites can be controlled through selection of the homo purine sequence there is great flexibility for designing strand invasion mo tifs unique to a particular PCR amplicon, thus providing a huge potential f or differentiating and detecting multiplex PCR products. (C) 2000 Academic Press.