Sequence-specific detection of PCR-amplified DNA by restriction enzyme release of hybrids.

We have developed a method to detect PCR amplicons that contain a restriction enzyme site. The method is called Polymerase Chain Reaction-Hybridization and Enzymatic Release Assay (PCR-HERA). The PCR product is hybridized to a synthetic oligonucleotide that is immobilized on a microtitre plate. Specific cleavage of the resulting hybrid by a restriction endonuclease liberates a fluorescein labeled arm of the immobilized DNA. We demonstrate the use of PCR-HERA to detect species-specific sequences of extra-chromosomal, ribosomal DNA, of Entamoeba histolytica. This highly specific assay does not require any washing steps and can be easily automated.