Nonvolatile copolymer compositions for fabricating gel element microarrays.

By modifying polymer compositions and cross-linking reagents, we have developed a simple yet effective manufacturing strategy for copolymerized three-dimensional gel element arrays. A new gel-forming monomer, 2-(hydroxyethyl) methacrylamide (HEMAA), was used. HEMAA possesses low volatility and improves the stability of copolymerized gel element arrays to on-chip thermal cycling procedures relative to previously used monomers. Probe immobilization efficiency within the new polymer was 55%, equivalent to that obtained with acrylamide (AA) and methacrylamide (MA) monomers. Nonspecific binding of single-stranded targets was equivalent for all monomers. Increasing cross-linker chain length improved hybridization kinetics and end-point signal intensities relative to N,N-methylenebisacrylamide (Bis). The new copolymer formulation was successfully applied to a model orthopox array. Because HEMAA greatly simplifies gel element array manufacture, we expect it (in combination with new cross-linkers described here) to find widespread application in microarray science.

Unidirectional excitation of surface plasmons by slanted gratings.

Surface plasmon excitation by normally incident light on surface-relief metallic diffraction gratings is studied numerically. Predominantly unidirectional excitation is achieved with a grating of either a slanted lamellar or an inclined sinusoidal groove profile, both having shallow depths. Maps of Poynting vector illustrate that the energy flow turns from normal incidence in the far-field region to a pattern almost parallel to the grating surface in the required direction of excitation of a single SPP wave.

Diagnostic oligonucleotide microarray fingerprinting of Bacillus isolates.

A genome-independent microarray and new statistical techniques were used to genotype Bacillus strains and quantitatively compare DNA fingerprints with the known taxonomy of the genus. A synthetic DNA standard was used to understand process level variability and lead to recommended standard operating procedures for microbial forensics and clinical diagnostics.

Suspension array analysis of 16S rRNA from Fe- and SO(4)2- reducing bacteria in uranium-contaminated sediments undergoing bioremediation.

A 16S rRNA-targeted tunable bead array was developed and used in a retrospective analysis of metal- and sulfate-reducing bacteria in contaminated subsurface sediments undergoing in situ U(VI) bioremediation. Total RNA was extracted from subsurface sediments and interrogated directly, without a PCR step. Bead array validation studies with total RNA derived from 24 isolates indicated that the behavior and response of the 16S rRNA-targeted oligonucleotide probes could not be predicted based on the primary nucleic acid sequence. Likewise, signal intensity (absolute or normalized) could not be used to assess the abundance of one organism (or rRNA) relative to the abundance of another organism (or rRNA). Nevertheless, the microbial community structure and dynamics through time and space and as measured by the rRNA-targeted bead array were consistent with previous data acquired at the site, where indigenous sulfate- and iron-reducing bacteria and near neighbors of Desulfotomaculum were the organisms that were most responsive to a change in injected acetate concentrations. Bead array data were best interpreted by analyzing the relative changes in the probe responses for spatially and temporally related samples and by considering only the response of one probe to itself in relation to a background (reference) environmental sample. By limiting the interpretation of the data in this manner and placing it in the context of supporting geochemical and microbiological analyses, we concluded that ecologically relevant and meaningful information can be derived from direct microarray analysis of rRNA in uncharacterized environmental samples, even with the current analytical uncertainty surrounding the behavior of individual probes on tunable bead arrays.

Evaluation of gel-pad oligonucleotide microarray technology by using artificial neural networks.

Past studies have suggested that thermal dissociation analysis of nucleic acids hybridized to DNA microarrays would improve discrimination among duplex types by scanning through a broad range of stringency conditions. To more fully constrain the utility of this approach using a previously described gel-pad microarray format, artificial neural networks (NNs) were trained to recognize noisy or low-quality data, as might derive from nonspecific fluorescence, poor hybridization, or compromised data collection. The NNs were trained to classify dissociation profiles (melts) into groups based on selected characteristics (e.g., initial signal intensity, area under the curve) using a data set of 21,044 profiles derived from 186 probes hybridized to a study set of RNA extracted from 32 microbes common to the human oral cavity. Three melt profile groups were identified: one group consisted mostly of ideal melt profiles; another group consisted mostly of poor melt profiles; and, the remainder were difficult to classify. Screening of melting profiles of perfect-match hybrids revealed inconsistencies in the form of melting profiles even for identical probes on the same microarray hybridized to same target rRNA. Approximately 18% of perfect-match duplex types were correctly classified as poor. Experimental variability and deviation from ideal melt behavior were shown to be attributable primarily to a method of local background subtraction that was very sensitive to displacement of the grid frames used for image capture (both determined by the image analysis system) and duplexes with low binding constants. Additional results showed that long RNA fragments limit the discriminating power among duplex types.

Differential theory: application to highly conducting gratings.

The recently developed fast Fourier factorization method, which has greatly improved the application range of the differential theory of gratings, suffers from numerical instability when applied to metallic gratings with very low losses. This occurs when the real part of the refractive index is small, in particular, smaller than 0.1-0.2, for example, when silver and gold gratings are analyzed in the infrared region. This failure can be attributed to Li's "inverse rule" [L. Li, J. Opt. Soc. Am. A 13, 1870 (1996)] as shown by studying the condition number of matrices that have to be inverted. Two ways of overcoming the difficulty are explored: first, an additional truncation of the matrices containing the coefficients of the differential system, which reduces the numerical problems in some cases, and second, an introduction of lossier material inside the bulk, thus leaving only a thin layer of the highly conducting metal. If the layer is sufficiently thick, this does not change the optical properties of the system but significantly improves the convergence of the differential theory, including the rigorous coupled-wave method, for various types of grating profiles.

Radical-generating coordination complexes as tools for rapid and effective fragmentation and fluorescent labeling of nucleic acids for microchip hybridization.

DNA microchip technology is a rapid, high-throughput method for nucleic acid hybridization reactions. This technology requires random fragmentation and fluorescent labeling of target nucleic acids prior to hybridization. Radical-generating coordination complexes, such as 1,10-phenanthroline-Cu(II) (OP-Cu) and Fe(II)-EDTA (Fe-EDTA), have been commonly used as sequence nonspecific "chemical nucleases" to introduce single-strand breaks in nucleic acids. Here we describe a new method based on these radical-generating complexes for random fragmentation and labeling of both single- and double-stranded forms of RNA and DNA. Nucleic acids labeled with the OP-Cu and the Fe-EDTA protocols revealed high hybridization specificity in hybridization with DNA microchips containing oligonucleotide probes selected for identification of 16S rRNA sequences of the Bacillus group microorganisms. We also demonstrated cDNA- and cRNA-labeling and fragmentation with this method. Both the OP-Cu and Fe-EDTA fragmentation and labeling procedures are quick and inexpensive compared to other commonly used methods. A column-based version of the described method does not require centrifugation and therefore is promising for the automation of sample preparations in DNA microchip technology as well as in other nucleic acid hybridization studies.

SuUR protein binds to the boundary regions separating forum domains in Drosophila melanogaster.

Forum domains are 50-150 kb DNA fragments that are released during spontaneous fragmentation of chromosomes. They are separated by islands of putative heterochromatin boundary regions. The SuUR protein, which is involved in the control of chromosome organization, is localized exclusively in heterochromatin and often colocalizes on chromosomes with Polycomb group proteins. To test whether the SuUR protein is associated with boundary regions, we used gel retardation assays and found that the SuUR protein binds specifically to boundary regions and that boundary regions are under-replicated. These results suggest that the regular distribution of boundary regions in chromosomes may represent the dispersion of sites designed for chromosomal silencing.