Fluorogenic polymerase, endonuclease, and ligase assays based on DNA substrates labeled with a single fluorophore

This paper describes the development of homogeneous, fluorogenic polymerase, restriction endonuclease, and ligase assays based on the use of DNA substrate molecules labeled with a single fluorophore. All three enzymatic assays are based on the same observed phenomenon whereby the fluorescence intensity of hairpin-type oligonucleotides with a 5′single-stranded extension, labeled with a single fluorophore, changes when the distance of the dye from the 3′ end of the molecule is altered as a result of the enzymatic transformation (i.e., polymerase extension, endonuclease hydrolysis, or ligation). The magnitudes of the observed fluorescence intensity changes range from 1.2-fold to 3.9-fold, and they are dependent on the type of dye used, its position within the substrate and product molecules, and the base composition surrounding the labeling site.