Nonisotopic DNA detection system employing elastase and fluorogenic rhodamine substrate.

Abstract

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. Hybridization is rapid and specific, with over 90% of a target sequence successfully hybridized and captured. This method exhibits low background and an amplified fluorescent signal over time, resulting in a current detection limit of 0.49 fmol of elastase alone, or 2.64 fmol of conjugate, within 2 h.