This invention provides methods of monitoring the expression levels of a multiplicity of genes. The methods involve hybridizing a nucleic acid sample to a high density array of oligonucleotide probes where the high density array contains oligonucleotide probes complementary to subsequences of target nucleic acids in the nucleic acid sample. In one embodiment, the method involves providing a pool of target nucleic acids comprising RNA transcripts of one or more target genes, or nucleic acids derived from the RNA transcripts, hybridizing said pool of nucleic acids to an array of oligonucleotide probes immobilized on surface, where the array comprising more than 100 different oligonucleotides and each different oligonucleotide is localized in a predetermined region of the surface, the density of the different oligonucleotides is greater than about 60 different oligonucleotides per 1 cm2, and the oligonucleotide probes are complementary to the RNA transcripts or nucleic acids derived from the RNA transcripts; and quantifying the hybridized nucleic acids in the array.

Expression monitoring by hybridization to high density oligonucleotide arrays

A highly selective, colorimetric polynucleotide detection method based on mercaptoalkyloligonucleotide-modified gold nanoparticle probes is reported. Introduction of a single-stranded target oligonucleotide (30 bases) into a solution containing the appropriate probes resulted in the formation of a polymeric network of nanoparticles with a concomitant red-to-pinkish/purple color change. Hybridization was facilitated by freezing and thawing of the solutions, and the denaturation of these hybrid materials showed transition temperatures over a narrow range that allowed differentiation of a variety of imperfect targets. Transfer of the hybridization mixture to a reverse-phase silica plate resulted in a blue color upon drying that could be detected visually. The unoptimized system can detect about 10 femtomoles of an oligonucleotide.

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Selective Colorimetric Detection of Polynucleotides Based on the Distance-Dependent Optical Properties of Gold Nanoparticles

A synthetic strategy for the creation of large scale chemical diversity. Solid-phase chemistry, photolabile protecting groups, and photolithography are used to achieve light-directed spatially-addressable parallel chemical synthesis. Binary masking techniques are utilized in one embodiment. A reactor system, photoremovable protective groups, and improved data collection and handling techniques are also disclosed. A technique for screening linker molecules is also provided.

Arrays of materials attached to a substrate

We designed a panel of four 16S rRNA-targeted oligonucleotide probes specific for bacteria of the phylum cytophaga-flavobacter-bacteroides (CFB). Probes CF319a and CF319b are targeted to members of the flavobacteria-cytophaga group and the genus Porphyromonas, whereas probe BAC303 has a target region characteristic for the genera Prevotella and Bacteroides within the bacteroides group. The probe FFE8b was developed for species-specific hybridizations with Flavobacterium ferrugineum. All probes were designed by computer-assisted sequence analysis and compared to all currently accessible 16S and 23S rRNA sequences. The oligonucleotides were further evaluated by whole-cell and non-radioactive dot-blot hybridization against reference strains of the CFB phylum and other major lineages of Bacteria. The newly developed probes were used together with other higher-order probes to analyse the structure and community composition in complex environments. In activated sludge samples, members of the flavobacteria-cytophaga group were revealed by in situ hybridization as important constituents of sludge flocs and characteristic colonizers of filamentous bacteria. By application of fluorescent probe BAC303, members of the genera Bacteroides and Prevotella could be visualized without prior cultivation as an important part of the human faecal microflora.

Application of a suite of 16S rRNA-specific oligonucleotide probes designed to investigate bacteria of the phylum cytophaga-flavobacter-bacteroides in the natural environment.

A method and apparatus for preparation of a substrate containing a plurality of sequences. Photoremovable groups are attached to a surface of a substrate. Selected regions of the substrate are exposed to light so as to activate the selected areas. A monomer, also containing a photoremovable group, is provided to the substrate to bind at the selected areas. The process is repeated using a variety of monomers such as amino acids until sequences of a desired length are obtained. Detection methods and apparatus are also disclosed.

Very large scale immobilized polymer synthesis

A modified polynucleotide containing at least one cleavable or abasic site as shown below. ##STR1## DNA 1 is a first segment of DNA; DNA 2 is a second segment of DNA; and R m is C 1 to C 16 alkylene or an oxytheylene oligomer --(CH 2 CH 2 O) z -- where z is an interger in the range of 1 to 16 inclusive, and R n is selected from the group consisting of ##STR2## Such polynucleotides are useful in solid phase hybridizations because they permit the release of a label from the solid support after the hybridization reaction.

Oligonucleotides with selectably cleavable and/or abasic sites

Linear or branched oligonucleotide multimers useful as amplifiers in biochemical assays which comprise (1) at least one first single-stranded oligonucleotide unit that is complementary to a single-stranded oligonucleotide sequence of interest, and (2) a multiplicity of second single-stranded oligonucleotide units that are complementary to a single-stranded labeled oligonucleotide. Amplified sandwich nucleic acid hybridizations and immunoassays using the multimers are exemplified.

Nucleic acid multimers and amplified nucleic acid hybridization assays using same

Novel N-4 modified pyrimidine analogs are provided having the structure (I) or (II) ##STR1## wherein: R 1 is selected from the group consisting of hydrogen, acid-sensitive, base-stable blocking groups and acyl capping groups; R 2 is lower alkyl; R 3 is C 1 -C 12 alkylene containing 0 to 6 linkages selected from the group consisting of --0--, --S-- and --NH--; R 4 is ##STR2## in which R 9 is hydrogen or an optionally substituted aliphatic group; R 10 and R 11 are hydrocarbyl or together form a mono- or polyheterocyclic ring; R 5 is hydrogen or lower alkyl; R 6 is selected from the group consisting of hydrogen, methyl, bromo and iodo; R 7 is C 1 -C 12 alkylene containing 0 to 6 linkages selected from the group consisting of --O--, --S-- and --NR 12 -- wherein R 12 is hydrogen or lower alkyl; and R 8 is a protecting group that can be removed and replaced by reduction. Synthetic methods for preparing the compounds are provided as well, as are methods for making and using polynucleotide probes containing the novel pyrimidine analogs.

N-4 modified pyrimidine deoxynucleotides and oligonucleotide probes synthesized therewith

Novel photolabile photochemical reagents are disclosed. The reagents are useful in a variety of biochemical and chemical contexts, including nucleic hybridization assays and chemical phosphorylation of hydroxyl-containing compounds. The reagents are particularly useful for introducing cleavable sites into oligonucleotide or polynucleotide chains, i.e., sites which are cleavable upon photolysis. The reagents are also useful in both 5'- and 3'-phosphorylation of oligonucleotide or polynucleotide chains.

Photolabile reagents for incorporation into oligonucleotide chains

N4-[N-(6-trifluoroacetylamidocaproyl)-2-aminoethyl]-5'-O-dimethoxy trityl -5-methyl-2'-deoxycytidine-3'-N,N-diisopropyl-methylphosphoramidite++ + has been synthesized. This N4-alkylamino deoxycytidine derivative has been incorporated into oligonucleotide probes during chemical DNA synthesis. Subsequent to deprotection and purification, fluorescent (fluorescein, Texas Red and rhodamine), chemiluminescent (isoluminol), and enzyme (horseradish peroxidase, alkaline phosphatase) labels have been specifically incorporated. Detection limits of the labels and labeled probes were assessed. Also, the detection limits and nonspecific binding of the labeled probes in sandwich hybridization assays were determined. The enzyme modified oligonucleotides were found to be significantly better labeling materials than the fluorescent or chemiluminescent derivatives, providing sensitivities comparable to 32P-labeled probes.

A comparison of non-radioisotopic hybridization assay methods using fluorescent, chemiluminescent and enzyme labeled synthetic oligodeoxyribonucleotide probes.

Thomas Horn

Papers

Oligonucleotides with selectably cleavable and/or abasic sites

Nucleic acid multimers and amplified nucleic acid hybridization assays using same

N-4 modified pyrimidine deoxynucleotides and oligonucleotide probes synthesized therewith

Photolabile reagents for incorporation into oligonucleotide chains

A comparison of non-radioisotopic hybridization assay methods using fluorescent, chemiluminescent and enzyme labeled synthetic oligodeoxyribonucleotide probes.