Nonisotopic DNA detection system employing elastase and a fluorogenic rhodamine substrate

An alternative fluorescence-based method has been developed for the direct detection of small quantities of DNA in solution. In this system, a serine protease (elastase) is coupled to a DNA oligonucleotide through a disulfide linkage. A bis-(tetraalanine)-derivatized rhodamine molecule (BZTAlaR) has been synthesized for use as a substrate. BZTAlaR is nonfluorescent in its derivatized form and shows negligible hydrolysis in solution. Cleavage of the tetraalanyl groups from the rhodamine portion of the molecule restores its fluorescence. Hybridization of the elastase-oligonucleotide conjugate to its target, capture of the conjugate-target complex with streptavidin-coated magnetic beads, addition of substrate, and subsequent detection of the target by fluorescence are accomplished in solution