The covalent attachment of disulfide-modified oligonucleotides to a mercaptosilane-modified glass surface is described. This method provides an efficient and specific covalent attachment chemistry for immobilization of DNA probes onto a solid support. Glass slides were derivatized with 3-mercaptopropyl silane for attachment of 5-prime disulfide-modified oligonucleotides via disulfide bonds. An attachment density of approximately 3 ؋ 10 5 oligonucleotides/m 2 was observed. Oligonucleotides attached by this method provided a highly efficient substrate for nucleic acid hybridization and primer extension assays. In addition, we have demonstrated patterning of multiple DNA probes on a glass surface utilizing this attachment chemistry, which allows for array densities of at least 20,000 spots/cm 2 . © 1999 Academic Press Key Words: covalent immobilization; oligonucleotide; glass; disulfide bonds; DNA microarray.In recent years, high-density miniaturized oligonucleotide arrays have emerged as promising tools for assessing genomic data with a lower cost and higher throughput than the traditional gel-based methods. Such oligonucleotide arrays, or DNA chips, have been applied to genetic mutational scanning (1, 2), molecular bar coding (3), gene expression monitoring (4, 5), and sequencing (6 -8). The power of the DNA chips come from the highly parallel, addressable, miniaturized array format that provides significant advantages over traditional gel-based formats in terms of reagent cost, labor, speed, throughput, and operational simplicity. The development of efficient chemistries for the manufacture of spatially resolved, microscale DNA arrays on a solid-support is essential for the realization of the DNA chip technology potential. In most DNA chip applications, the DNA arrays are used to capture or analyze the target sequences and/or detection probes via hybridization reactions alone (1-7) or with subsequent primer extension reactions (8). The reliability and integrity of the hybridization reactions are highly dependent, in addition to the actual base composition of the arrayed oligonucleotides, on the quality and the characteristics of the DNA arrays. In developing a useful and reliable chemistry for producing DNA arrays, the accessibility and functionality of the surface-bound DNA, the density of attachment, the stability of the array, the reproducibility of the attachment chemistry, and the fidelity of the immobilized sequences are all critical.There have been numerous reports regarding immobilization (9 -26) or direct synthesis (27, 28) of oligonucleotides on solid supports, such as glass, silicon, membranes, and polystyrene. Parallel synthesis of oligonucleotides directly onto the solid support by photoactivatable chemistries (27) or standard phosphoramidite chemistries (28) have, thus far, been the most successful approach to manufacturing high-density DNA arrays. Patterning of presynthesized oligonucleotides, however, is preferred for many research applications and low-to moderate-density-array applications requi...

The fluorescence intensity of rhodamine B (RhB) was found to display a sublinear dependence on incident power when excited with the focused output of a cavity-dumped dye laser. This effect was found to be proportional to the amplitude of the emission spectrum at the incident wavelength and to be associated with a decrease in the time-zero anisotropy of RhB. The absence of changes in the intensity decay law or rotational correlation time indicates the absence of photochemical processes. These results are consistent with "light quenching" of RhB due to stimulated emission. In viscous solution the extent of depolarization of the emission was found to be in agreement with theoretical expressions which account for photoselective light quenching and for spatial inhomogeneities in the incident laser beam. The phenomenon of light quenching has numerous potential applications in biophysics, such as studies of the orientation and dynamics of fluorescent macromolecules.

Experimental studies have recently demonstrated that fluorescence emission can be quenched by laser light pulses from modern high repetition rate lasers, a phenomenon we call "light quenching." We now describe the theory of light quenching and some of its effects on the steady-state and time-resolved intensity and anisotropy decays of fluorophores. Light quenching can decrease or increase the steady-state or time-zero anisotropy. Remarkably, the light quenching can break the usual z axis symmetry of the excited-state population, and the emission polarization can range from -1 to +1 under selected conditions. The measured anisotropy (or polarization) depends upon whether the observation axis is parallel or perpendicular to the propagation direction of the light quenching beam. The effects of light quenching are different for a single pulse, which results in both excitation and quenching, as compared with a time-delayed quenching pulse. Time-delayed light quenching pulses can result in step-like changes in the time-dependent intensity or anisotropy and are predicted to cause oscillations in the frequency-domain intensity and anisotropy decays. The increasing availability of pulsed laser sources offers the opportunity for a new class of two-pulse or multiple-pulse experiments where the sample is prepared by an excitation pulse, the excited state population is modified by the quenching pulse(s), followed by time- or frequency-domain measurements of the resulting emission.

Experimental studies have recently demonstrated that fluorescence emission can be quenched by laser light pulses from modern high-repetition rate lasers, a phenomenon we call "light quenching." In this overview article, we describe the possible effects of light quenching on the steady-state and time-resolved intensity and anisotropy of fluorophores. One can imagine two classes of experiments. Light quenching can occur within the single excitation pulse, or light quenching can be accomplished with a second time-delayed quenching pulse. The extent of light quenching depends on the amplitude of the emission spectrum at the quenching wavelength. Different effects are expected for light quenching by a single laser beam (within a single laser pulse) or for a time-delayed quenching pulse. Depending upon the polarization of the light quenching beam, light quenching can decrease or increase the anisotropy. Remarkably, the light quenching can break the usual z-axis symmetry of the excited state population, and the measured anisotropy (or polarization) depends upon whether the observation axis is parallel or perpendicular to the propagation direction of the light quenching beam. The polarization can increase to unity under selected conditions. Quenching with time-delayed light pulses can result in step changes in the intensity or anisotropy, which is predicted to result in oscillations in the frequency-domain intensity and anisotropy decays. These predicted effects of light quenching, including oscillations in the frequency-domain data, were demonstrated to occur using selected fluorophores. The increasing availability and use of pulsed laser sources requires consideration of the possible effects of light quenching and offers the opportunity for a new class of two-pulse or multiple-pulse time-resolved experiments where the sample is prepared by the excitation pulse and subsequent quenching pulses to modify the excited state population, followed by time- or frequency-domain measurement of the optically prepared excited fluorophores.