Abstract: This invention provides methods for massive parallel nucleic acid analysis. A closed complex of nucleic acid template, nucleotide and polymerase can be formed during polymerase reaction, absent divalent metal ion. This is used to trap the nucleotide complementary to the next template nucleotide in the closed complex. Detection of the trapped nucleotide allows determination of the sequence of this next correct nucleotide. In this way, sequential nucleotides of a nucleic acid template can be identified, effectively determining the sequence. This method is applied to sequence multiple templates in parallel, particularly if they are immobilized on a solid support.

Abstract: The invention relates to particles for use in a scintillation proximity assays. In particular, the invention relates to particles for use in a scintillation proximity assay for detecting nitric oxide synthase activity in a sample. The invention also relates to a method of measuring nitric oxide synthase activity in a sample using these particles in a scintillation proximity assay. The invention further relates to methods using these particles to detect the presence of modulators of nitric oxide synthase activity.

Abstract: A method of characterizing an analyte sample is provided that includes the steps of: (a) anchoring the analyte to a nucleic acid template of known sequence; (b) conducting a DNA polymerase reaction that includes the reaction of a template, a non-hydrolyzable primer, at least one terminal phosphate-labeled nucleotide, DNA polymerase, and an enzyme having 3??5? exonuclease activity which reaction results in the production of labeled polyphosphate; (c) permitting the labeled polyphosphate to react with a phosphatase to produce a detectable species characteristic of the sample; (d) detecting the detectable species. The method may include the step of characterizing the nucleic acid sample based on the detection. Also provided are methods of analyzing multiple analytes in a sample, and kits for characterizing analyte samples.

Abstract: The invention provides a novel, non-destructive and dynamic process for determining the cell cycle position of living cells. The invention also provides DNA constructs, and cell lines containing such constructs, that exhibit activation and deactivation of a detectable reporter molecule in a cell cycle specific manner. The invention thus allows greater precision in determining cell cycle phase status than existing techniques and further provides a method for continuous monitoring of cell cycle progression in individual cells.

Abstract: An assay is provided for nucleic acids that can be post-synthetically labeled, wherein modified nucleoside triphosphates are used that are more efficiently and specifically incorporated during nucleic acid synthesis than labeled nucleoside triphosphates. In a preferred embodiment, nucleoside ?-thiotriphosphates are utilized. Maleimide or iodoacetamide conjugating moieties can be attached post-synthetically. The conjugating moieties may include a reporter group. Also disclosed are new methods for detecting single nucleotide polymorphism.

Abstract: The present invention relates to improved methods of detecting a target using a labeled substrate or substrate analog. The methods comprise reacting the substrate or substrate analog in an enzyme-catalyzed reaction which produces a labeled moiety with independently detectable signal only when such substrate or substrate analog reacts. The present invention, in particular, describes methods of detecting a nucleic acid in a sample, based on the use of terminal-phosphate-labeled nucleotides as substrates for nucleic acid polymerases. The methods provided by this invention utilize a nucleoside polyphosphate, dideoxynucleoside polyphosphate, or deoxynucleoside polyphosphate analogue which has a colorimetric dye, chemiluminescent, or fluorescent moiety, a mass tag or an electrochemical tag attached to the terminal-phosphate. When a nucleic acid polymerase uses this analogue as a substrate, an enzyme-activatable label would be present on the inorganic polyphosphate by-product of phosphoryl transfer.

Abstract: The invention concerns scintillation proximity assays performed in multiwell plates where a charge coupled device is used to image the wells. Conventional phosphors emit blue light (350–450 nm) which is absorbed by yellow or brown assay components. This problem is addressed by the use of phosphors that emit radiation of longer wavelength (480–900 nm).

Abstract: The invention relates to a method for removing a positively charged substance from an aqueous liquid (I) by contacting the liquid with a cation-exchanger (1), possibly followed by a subsequent desorption of said substance. The cation-exchanger is selected to be capable of (a) binding to said substance by cation-exchange in an aqueous liquid reference (II) at an ionic strength corresponding to 0.3 M NaCl and (b) permitting a break through capacity for said substance 3200%, such as 3300% or 3500%, of the break-through capacity of said substance for a reference cation-exchanger (2) containing sulphopropyl groups —CH2CH2CH2SO2O—. The cation exchange ligands have an at least bimodal function by comprising a cation exchanging group and a separate hydrogen-bonding atom. The invention also relates to a method for testing the appropriateness of a cation-exchanger for removing a substance from a liquid and novel cation exchangers.

Abstract: The present invention is a method of producing a chromatographic separation matrix, wherein porous polymeric particles comprised of two layers with different properties are prepared in a two-phase system by (a) providing porous polymeric particles with reactive groups on their surfaces; (b) washing said particles with a first solvent and draining the particles to enclose a first phase; (c) wetting the outer layer of the particles by adding a second solvent, which is essentially insoluble in the first solvent; (d) reacting the reactive groups in the outer layer by adding a reagent, which is essentially non-reactive in the first solvent; and (e) coupling of chromatographic binding groups to the reactive groups in the inner layer. The invention also encompasses a porous polymer particle suitable for use as a chromatographic separation matrix and a process of separation, wherein a matrix according to the invention is used.

Abstract: A method for the production of a cross-linked support matrix that in form of a bed will permit liquid flow velocities above 5 cm/h, preferably above 50 cm/h, to pass through the bed. The method is characterized in that it comprises the steps: (a) providing a starting support matrix that has pendent unsaturated groups, and (b) subjecting said starting support matrix to electron beam or gamma-ray irradiation. The use of a cross-linked support matrix produced by a method comprising the steps of: (a) providing a starting support matrix that has pendent unsaturated groups, and (b) subjecting said starting support matrix to electron beam or gamma-ray irradiation as a support matrix in liquid chromatography, cell culturing, step-wise solid phase synthesis of organic compounds, running catalytic reactions by the use of a solid phase bound catalyst.

Abstract: The present invention relates to the preparation of a metal chelating separation medium, which comprises providing a compound defined by formula: R—O—CO—(CH2)n—CHNH2—CO—OH wherein R is a selectively cleavable group; and n is an integer of 1–4; reacting it with ethyl alcohol; derivatisation; cleavage of R; immobilisation of the resulting compound to a base matrix; and deprotection of the carboxylic acid. In one embodiment, the compound provided in the first step is ethyl ester of aspartic acid or of glutamic acid, and the derivatisation agent is bromo-acetic acid ethyl acetate.

Abstract: Disclosed are cyanine dyes having meso-substituents in the polymethine chain linking the heterocyclic ring systems. The dyes are of formula (1): in which groups R3 and R4 are attached to the Z1 ring structure and groups R5 and R6 are attached to the Z2 ring structure, and n=1, 2 or 3; Z1 and Z2 independently represent the atoms necessary to complete one ring, or two fused ring aromatic or heteroaromatic systems, each ring having five or six atoms selected from carbon atoms and optionally no more than two atoms selected from oxygen, nitrogen and sulphur; X and Y are the same or different and are selected from oxygen, sulphur, —CH?CH— and the group: at least one of groups R1, R2, R3, R4, R5, R6 (and R8 and R9 if present) is the group -E-F where E is a spacer group and F is a target bonding group; one of groups R7 is selected from —CN, —Cl, —F, —CF3 and —C(O)R10 wherein R10 is selected from H, C1–C6 alkyl and aryl.

Abstract: The present invention relates a method of electrophoretic separation of protein and/or peptide components of a sample in a convection stabilized medium. More specifically, the method comprises the steps to contact the sample with the separation medium; to apply a voltage across said medium; and to observe the results by analysis of one or more sections of the separation medium. In the present method, a disulphide-comprising compound is added before or during the procedure to make an excess of reactive disulphide groups accessible to react with the cysteine groups of the proteins and/or peptides all through the separation procedure. The present invention also relates to electrophoretic separation media that comprises reactive disulphide groups, such as polyacrylamide gels, and the use of a solution that comprises reactive disulphide groups to pretreat an electrophoretic separation medium.

Abstract: The present invention relates to a method of producing one or more macroporous cross-linked polymer particles, which comprises polymerization and cross-linking of divinyl ether monomers in an inert solvent, in which method the polymerization is free radical initiated. The invention also relates to particles so produced, which are useful as separation medium e.g. in RPC or, after suitable derivatization, in other chromatographic methods.

Abstract: Methods for preparing nanoscale reactions using nucleic acids or proteins are presented. Nucleic acids are captured saturably, yet reversibly, on the internal surface of the reaction chamber, typically a capillary. Excess nucleic acid is removed and the reaction is performed directly within the capillary. Proteins are captured specifically and saturably on the modified inner surface of the reaction chamber, typically a capillary. Excess protein is removed and the reaction is performed directly within the capillary. Devices for effecting the methods of the invention and a system designed advantageously to utilize the methods for high throughput reactions involving nucleic acids or proteins are also provided.

Abstract: In a method of identifying an unknown nucleotide sequence using base addition, a sequence of bases is obtained from a template, a base in the sequence is identified as an unknown base, an “unknown” indicator is included in the sequence, and an output sequence is generated containing the unknown base indicator. The sequence of bases is obtained from the template by evaluation of a reporter and assigning the bases in accordance therewith. A determination is made as to whether the reporter is from a preceding cycle of base determination, and if the reporter is from a preceding cycle of base determination, the base assignation is discarded.

Abstract: The invention provides fluorescence-based assays for determining the phosphorylating or the dephosphorylating activity of an enzyme acting on a substrate molecule. The substrate comprises at least one moiety that is capable of being phosphorylated or dephosphorylated by an enzyme to yield a product, the substrate being labelled with a fluorescent dye, and which is capable of exhibiting change in fluorescence intensity and fluorescence lifetime upon phosphorylation of the dye-labelled substrate or upon dephosphorylation of the dye-labelled phosphorylated substrate. In preferred embodiments, the substrate is labelled with a fluorescent dye selected from the acridone and the quinacridone classes of dyes. Also provided is a method is provided for screening for a test agent whose effect on the phosphorylating or dephosphorylating activity of an enzyme is to be determined.

Abstract: The present invention relates generally to the use of terminal-phosphate-labeled nucleotides having three or more phosphates as substrates for nucleic acid polymerases and their use in DNA amplification. The labels employed are chemiluminescent, fluorescent, electrochemical and chromogenic moieties as well as mass tags and include those that are directly detectable, detectable after enzyme activation or feed into other processes to generate a different signal. The signal generated from the attached dyes may also be used to quantify the amount of amplification. Further provided are stabilizers that enhance the stability of terminal-phosphate labeled nucleoside polyphosphates in aqueous solutions and are useful for reducing non-enzymatic hydrolysis of these nucleotides, hence decrease background.

Abstract: Oligonucleotide tagged nucleoside triphosphates, OTNTPs, which are substrates for polymerases and or terminal nucleotidyl transferases are provided as well as methods of making these OTNTPs. Further provided are OTNTPs with fluorescent dyes including energy transfer dyes, attached to the oligonucleotide chain, OTNTPs with unnatural bases incorporated in the oligonucleotide sequence and methods for incorporating these OTNTPs in DNA or RNA. Also provided are methods for using the oligonucleotides on OTNTPs for amplifying the oligo sequence on the OTNTP using an amplification method described above. Further provided are reactive bifunctional amidites, methods of making these compounds and methods for detecting single nucleotide polymorphisms using the above OTNTPs. Methods for detecting differential gene expression using the OTNTPs and methods of separating specifically modified DNA or RNA using the OTNTPs are also provided.

Abstract: Disclosed is a method for the measurement of a cellular process, or for the measurement of the effect of a test compound on a cellular process, in one or more different populations of cells. The method comprises providing separate samples of one or more different populations of cells adhering to support particles, the support particles comprising a scintillant substance and being adapted for cell growth. In one embodiment, different samples of cells are introduced into separate reaction vessels in a fluid medium, together with a reagent labelled with a radioisotope, in the presence or the absence of the test compound, under conditions so as to cause a portion of said radiolabelled reagent to become associated with the cells. In another embodiment, multiparameter analysis may be performed to determine the effect of a test compound on a cellular process using two or more different cell populations present in the same well.