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 rest riction enzyme site. The method is called Polymerase Chain Reaction-Hy bridization and Enzymatic Release Assay (PCR-HERA). The PCR product is hybridized to a synthetic oligonucleotide that is immobilized on a mi crotitre plate. Specific cleavage of the resulting hybrid by a restric tion endonuclease liberates a fluorescein labeled arm of the immobiliz ed DNA. We demonstrate the use of PCR-HERA to detect species-specific sequences of extra-chromosomal, ribosomal DNA, of Entamoeba histolytic a. This highly specific assay does not require any washing steps and c an be easily automated. (C) 1996 Academic Press Limited