FIBEROPTIC FLUOROMETRIC SENSING OF POLYMERASE CHAIN REACTION-AMPLIFIED DNA USING AN IMMOBILIZED DNA CAPTURE PROTEIN

A fiber-optic assay for amplified DNA products has been developed. Mod ifications of the DNA capture strategy described previously by Kemp et al. [Proc. Natl. Acad. Sci. USA 86, 2423-2427 (1989)] were made that allowed selective binding of DNA labeled during the amplification proc ess to the sensing surface of fused silica fibers. The gene for a chim eric protein composed of the IgG-binding beta 2 subdomain of streptoco ccal protein G fused with the DNA binding domain of yeast GCN4 was con structed, and this PG/GCN4 protein was overexpressed in Escherichia co li. The purified protein was noncovalently bound to IgG-modified fiber s utilizing strong and specific interactions between the protein G bet a 2 domain and goat IgG that had been covalently immobilized on the fi ber surface. Nanomolar concentrations of amplified DNA labeled with th e fluorophore tetramethylrhodamine and the AP-1 consensus nucleotide s equence recognized by GCN4 (5'-ATGACTCAT) were rapidly and selectively bound within the evanescent zone of multimode laser-illuminated fiber s. Signal from unincorporated fluorescent PCR primer was negligible, I ndividual fibers could be used for multiple sequential assays, since t he fluorescent double-stranded DNA was rapidly and completely stripped from their surfaces with high salt solutions, leaving the IgG-PG/GCN4 DNA binding complex intact to accept another PCR sample. (C) 1996 Aca demic Press, Inc.