derived from DNA amplification carried out for pur-A fiber-optic assay for amplified DNA products has poses of pathogen screening, epidemiological studies, been developed. Modifications of the DNA capture genetic evaluation, and forensic investigation (1, 2). strategy described previously by Kemp et al. [Proc. The analysis strategy described by Kemp et al. (3,4) Natl. Acad. Sci. USA 86, 2423-2427 (1989)] were made employs a sequence-selective double-stranded DNA that allowed selective binding of DNA labeled during binding protein to immobilize DNA fragments bearing the amplification process to the sensing surface of unique PCR primer-derived nucleotide sequences. The fused silica fibers. The gene for a chimeric protein yeast transcriptional regulator protein GCN4, which composed of the IgG-binding b2 subdomain of streptoselectively binds the AP-1 consensus nucleotide secoccal protein G fused with the DNA binding domain quence (5-ATGACTCAT), has been used most often in of yeast GCN4 was constructed, and this PG/GCN4 prothis capture role in microtiter plate-based assays. In tein was overexpressed in Escherichia coli. The purithe assay scheme commonly employed, one of the two fied protein was noncovalently bound to IgG-modified target-specific single-stranded oligonucleotide primers fibers utilizing strong and specific interactions berequired for amplification is synthesized with an aptween the protein G b2 domain and goat IgG that had pended AP-1 sequence; the second required primer is been covalently immobilized on the fiber surface. Nalabeled with biotin. Using these modified primers, amnomolar concentrations of amplified DNA labeled with plified DNA labeled with both the AP-1 sequence and the fluorophore tetramethylrhodamine and the AP-1 biotin is obtained; this DNA is then selectively capconsensus nucleotide sequence recognized by GCN4
(5-ATGACTCAT) were rapidly and selectively bound tured by recombinant GCN4 preadsorbed on microtiter within the evanescent zone of multimode laser-illumiplate wells. After washing away excess biotinylated nated fibers. Signal from unincorporated fluorescent single-stranded PCR primer, selectively captured DNA PCR primer was negligible. Individual fibers could be is detected and quantitated using a streptavidin-enused for multiple sequential assays, since the fluoreszyme conjugate for color development. It has been sugcent double-stranded DNA was rapidly and completely gested, however, that detection of fluorescently labeled stripped from their surfaces with high salt solutions, DNA using this GCN4 capture strategy may be possible leaving the IgG-PG/GCN4 DNA binding complex by replacing biotinylated with fluorophore-labeled intact to accept another PCR sample. แญง 1996 Academic primers and by making appropriate modifications in Press, Inc.
signal readout technology (4).
Fluorescence-based fiber-optic immunoassay methods have been successful in detecting and quantitating Technology continues to evolve for rapid and efficient low concentrations of a number of substances (5-7). screening of polymerase chain reaction (PCR) products Development of comparable fiber-optic assays for fluorescent amplified DNA products seemed feasible based