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

The present invention provides method and apparatus for sequencing, fingerprinting and mapping biological macromolecules, typically biological polymers. The methods make use of a plurality of sequence specific recognition reagents which can also be used for classification of biological samples, and to characterize their sources.

Method of detecting nucleic acids

A method and device for forming large arrays of polymers on a substrate ( 401 ). According to a preferred aspect of the invention, the substrate is contacted by a channel block ( 407 ) having channels ( 409 ) therein. Selected reagents are delivered through the channels, the substrate is rotated by a rotating stage ( 403 ), and the process is repeated to form arrays of polymers on the substrate. The method may be combined with light-directed methodolgies.

Combinatorial strategies for polymer synthesis

Disclosed are compositions and a method for of amplifying nucleic acid sequences useful for detecting the presence of molecules of interest. The method is useful for detecting specific nucleic acids in a sample with high specificity and sensitivity. The method also has an inherently low level of background signal. A preferred form of the method consists of a DNA ligation operation, an amplification operation, and a detection operation. The DNA ligation operation circularizes a specially designed nucleic acid probe molecule. This operation is dependent on hybridization of the probe to a target sequence and forms circular probe molecules in proportion to the amount of target sequence present in a sample. The amplification operation is rolling circle replication of the circularized probe. A single round of amplification using rolling circle replication results in a large amplification of the circularized probe sequences. Following rolling circle replication, the amplified probe sequences are detected and quantified using any of the conventional detection systems for nucleic acids such as detection of fluorescent labels, enzyme-linked detection systems, antibody-mediated label detection, and detection of radioactive labels. Because, the amplified product is directly proportional to the amount of target sequence present in a sample, quantitative measurements reliably represent the amount of a target sequence in a sample. Major advantages of this method are that the ligation step can be manipulated to obtain allelic discrimination, the DNA replication step is isothermal, and signals are strictly quantitative because the amplification reaction is linear and is catalyzed by a highly processive enzyme. In multiplex assays, the primer oligonucleotide used for the DNA polymerase reaction can be the same for all probes. Also described are modes of the method in which additional amplification is obtained using a cascade of strand displacement reactions.

Rolling circle replication reporter systems

Methods and reagents are provided for synthesizing polynucleotides containing modified deoxyribose residues. Monomeric reagents having the structural formula (I) ##STR1## wherein R 1 , R 2 , R 3 , R 4 and R 5 are as defined herein, are used to create polynucleotides having nonnucleotidic moieties -A-Z-(R 9 ) n at the 1 position of selected deoxyribose units. The polynucleotides so provided are useful in a variety of hybridization assay formats.

Polynucleotide reagents containing modified deoxyribose moieties and associated methods of synthesis and use

Novel reagents 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 and/or abasic sites into oligonucleotide or polynucleotide chains.

Multifunctional nucleic acid monomer

Comb-type branched polynucleotides are used as amplification multimers in nucleic acid hybridization assays.

Nucleic acid hybridization assays employing large comb-type branched polynucleotides

Methods and compositions are provided for rapid detection of nucleic acid sequences. The method employs two reagent sets. The first set is a labeling set comprising: (1) a first nucleic acid sequence probe having an analyte complementary region and a first recognition sequence region and (2) a labeled sequence complementary to the first recognition sequence region. The second set is a capturing set comprising: (1) a second nucleic acid sequence probe having an analyte complementary region and a second recognition sequence region, (2) a specific binding pair member conjugated to a sequence complementary to the second recognition sequence, and (3) a separating means to which is bound a complementary specific binding pair member. The sample and probes are combined under annealing conditions, followed by addition of the other reagents, separation of the bound label from the supernatant and detection of the label in either phase. The invention also encompasses nucleic acid probes formed from one or more modified derivatizable nucleotides.

Solution phase nucleic acid sandwich assay and polynucleotide probes useful therein

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.

Thomas Horn

Papers

Polynucleotide reagents containing modified deoxyribose moieties and associated methods of synthesis and use

Multifunctional nucleic acid monomer

Nucleic acid hybridization assays employing large comb-type branched polynucleotides

Solution phase nucleic acid sandwich assay and polynucleotide probes useful therein

Modified N-4 nucleotides for use in amplified nucleic acid hybridization assays