Fiber-optic 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. Modifications 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 process to the sensing surface of fused silica fibers. The gene for a chimeric protein composed of the IgG-binding beta 2 subdomain of streptococcal protein G fused with the DNA binding domain of yeast GCN4 was constructed, and this PG/GCN4 protein was overexpressed in Escherichia coli. The purified protein was noncovalently bound to IgG-modified fibers utilizing strong and specific interactions between the protein G beta 2 domain and goat IgG that had been covalently immobilized on the fiber surface. Nanomolar concentrations of amplified DNA labeled with the fluorophore tetramethylrhodamine and the AP-1 consensus nucleotide sequence recognized by GCN4 (5'-ATGACTCAT) were rapidly and selectively bound within the evanescent zone of multimode laser-illuminated fibers. Signal from unincorporated fluorescent PCR primer was negligible. Individual fibers could be used for multiple sequential assays, since the 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.
Mauro
J M
JM
GEO-CENTERS, INC., Fort Washington, Maryland 20744, USA. mmauro@cbmse.nrl.navy.mil
Cao
L K
LK
Kondracki
L M
LM
Walz
S E
SE
Campbell
J R
JR
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005336:Fiber Optic Technology; D005656:Fungal Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D055100:Optical Fibers; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013050:Spectrometry, Fluorescence / Q000379:methods
D017931:DNA Primers; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D011494:Protein Kinases