Abstract: The invention relates to methods and compositions utilizing diphtheria toxin for screening purposes. The invention is particularly useful in screening for modulators of IgE synthesis, secretion and switch rearrangement.

Abstract: Nature evolves biological molecules such as proteins through iterated rounds of diversification, selection, and amplification. The present invention provides methods, compositions, and systems for synthesizing, selecting, amplifying, and evolving non-natural molecules based on nucleic acid templates. The sequence of a nucleic acid template is used to direct the synthesis of non-natural molecules such as unnatural polymers and small molecules. Using this method combinatorial libraries of these molecules can be prepared and screened. Upon selection of a molecule, its encoding nucleic acid template may be amplified and/or evolved to yield the same molecule or related molecules for re-screening. The inventive methods and compositions of the present invention allow for the amplification and evolution of non-natural molecules in a manner analogous to the amplification of natural biopolymer such as polynucleotides and protein.

Abstract: The disclosure provides a method of sequencing a nucleic acid molecule that contains two or more target regions to be sequenced (such as, for example, barcodes). The invention is advantageous for sequencing by synthesis two or more target regions whose combined lengths plus the length of any intermediate sequence exceeds the available read length on a given sequencing platform. The methods of the invention utilize nucleic acid constructs containing at least the following elements: a complement of a first universal primer, a first target sequence, an optional polynucleotide spacer, a complement of a second universal primer, and a second target sequence. A first round of sequencing by synthesis is performed to sequence the first target sequence by elongating the first universal primer. Once the sequence of the first target region is obtained, and before the complement of the second primer is reached, the first round of sequencing is terminated.

Abstract: A method and device for digital multiplex PCR assays employ a microfluidic chip for performing real-time, continuous flow PCR within microchannels of the chip. A stream of sample material is introduced into each microchannel and alternating boluses of assay-specific reagents and buffer are introduced into the stream to form sequentially configured test boluses. A PCR procedure is performed on the test boluses followed by a thermal melt procedure. During the thermal melt procedure, fluorescent output is detected and fluorescence vs temperature data is collected and compared to expected normal correlations. The results, positive or negative, are converted to digital format, with positive results designated as “1” and negative results designated as “0”, or vice versa.

Abstract: We disclose quality controls methods that allow quick and accurate verification of a test oligonucleotide deposited on a solid support. It is especially useful for the verification of oligonucleotides representing alleles of a multi-allelic locus. It employs single base extension, with labeled dideoxynucleotides, to locate and verify the identity of test oligonucleotides. This approach involves synthesizing a complement probe oligonucleotide for each oligonucleotide being tested. Probe oligonucleotides are optionally grouped. They are then hybridized to test oligonucleotides, and the hybridized pair is subject to single base extension and detection. It requires the presence of one unique base, either in the last two bases at the free hanging end of the test oligonucleotide—as opposed to the end anchored to the solid support surface, or in the last two bases at one end of the probe oligonucleotide.

Abstract: Reference membrane components are either pre-labeled or labeled during assays for purposes of normalizing signals associated with binding or functional assays employing G-protein coupled receptor microarrays. A reference component may be included in a membrane in which the target GPCR is embedded or may be present in another membrane printed in conjunction with the target membrane on a microspot. Or, a GPCR microarray may be pre-labeled by incorporating a label on an exposed substrate in a defect in the printed microspot.

Abstract: Methods for identifying modified proteases with modified substrate specificity or other properties are provided. The methods screen candidate and modified proteases by contacting them with a substrate, such as a serpin, an alpha macroglobulins or a p35 family protein or modified serpins and modified p35 family members or modified alpha macroglobulins, that, upon cleavage of the substrate, traps the protease by forming a stable complex. Also provided are modified proteases.

Abstract: Methods of modifying a nucleophilic surface of a support are described. The methods involve reacting a multifunctional electrophilic reagent with nucleophilic groups on the surface of the support. The resulting electrophilic surface can be used for the covalent attachment of particles (e.g. beads) having nucleophilic functional groups. For example, nucleic acid templates with nucleophilic (e.g., amine) groups can be attached to a surface of the particles. The nucleophilic groups on the nucleic acid templates can then be used to attach the particles to the modified surface of the support. The resulting support-bound particles can be used to analyze (e.g., sequence) the nucleic acid templates on the particles.

Abstract: The invention relates to methods for pairwise sequencing of a double-stranded polynucleotide template, which methods result in the sequential determination of nucleotide sequences in two distinct and separate regions of the polynucleotide template. Using the methods of the invention it is possible to obtain two linked or paired reads of sequence information from each double-stranded template on a clustered array, rather than just a single sequencing read from one strand of the template.

Abstract: This invention relates to improved methods for sequencing and genotyping nucleic acid in a single molecule configuration. The method involves single molecule detection of fluorescent labeled PPi moieties released from NTPs as a polymerase extension product is created.

Abstract: The invention relates to methods and devices for analyzing single molecules, i.e. nucleic acids. Such single molecules may be derived from natural samples, such as cells, tissues, soil, air and water without separating or enriching individual components. In certain aspects of the invention, the methods and devices are useful in performing nucleic acid sequence analysis by probe hybridization.

Abstract: The present invention is directed to methods for identifying agents which affect cell state. The instant invention provides rapid and efficient methods for identifying agents which affect cell state. Methods are directed toward the screening of complex combinations of agents for their ability to affect cell state. In one embodiment, cells are incubated under suitable conditions and subjected to different agents. After an appropriate amount of time, the cells are assayed to determine what, if any, characteristics they possess. Cell characteristics can be organized in a manner such that different and novel cell states can be identified.

Abstract: The present invention is directed to methods and compositions for acquiring nucleotide sequence information of target sequences using adaptors interspersed in target polynucleotides. The sequence information can be new, e.g. sequencing unknown nucleic acids, re-sequencing, or genotyping. The invention preferably includes methods for inserting a plurality of adaptors at spaced locations within a target polynucleotide or a fragment of a polynucleotide. Such adaptors may serve as platforms for interrogating adjacent sequences using various sequencing chemistries, such as those that identify nucleotides by primer extension, probe ligation, and the like. Encompassed in the invention are methods and compositions for the insertion of known adaptor sequences into target sequences, such that there is an interruption of contiguous target sequence with the adaptors. By sequencing both “upstream” and “downstream” of the adaptors, identification of entire target sequences may be accomplished.

Abstract: The present invention is related to a method for a simultaneous determination of the relative amounts of more than one target polynucleotide sequence and nucleotide variations in said targets. The method is carried out by separating and recording single-stranded probes, which have hybridized to the targets and which are determined and distinguished by their defined properties including size and optional detectable label. The probes are complementary to a region in the target that has a sequence being contiguous to the nucleotide variations to be determined. After being hybridized with affinity-tagged targets, the probes are attached to a solid support and purified. The target probe hybrids are elongated using enzyme-assisted elongations. The elongated probes are recorded after release from the solid supports and the amount of each of the targets and their nucleotide variations and the ratio of modified and modified target polynucleotide sequences are calculated from the recorded results.

Abstract: Methods for determining invertebrate- and insect-specific, such as mosquito-specific, residues of acetylcholinesterases are provided herein. The methods can be used to design pesticides and insecticides that are specific for the invertebrate or insect (e.g., mosquito) enzymes, resulting in reduced toxicity concerns for mammals. Compositions for inhibiting invertebrate and insect (e.g., mosquito) acetylcholinesterases and methods for preparing the same are also provided.

Abstract: Methods of achieving realistic hit rates and reducing or eliminating false positive rates in medicinal compound high throughput screening are provided. The methods of the invention include chromatographic resolution, for example by liquid or gas chromatography of biological substrates and/or substrate products, followed by sensitive with mass spectrometry to measure biological activity screening to generate meaningful drug leads. The methods of the invention save significant method development and thus are directly applicable to the high throughput screening time scale while providing high accuracy and sensitivity.

Abstract: In one aspect, the present invention relates to a method for obtaining structural information about an encoded molecule. The encoded molecule may be produced by a reaction of a plurality of chemical entities and may be capable of being connected to an identifier oligonucleotide containing codons informative of the identity of the chemical entities which have participated in the formation of the encoded molecule. In a certain embodiment, primers are designed complementary to the codons appearing on the identifier oligonucleotide, and the presence, absence or relative abundance of a codon is evaluated by mixing a primer with the identifier oligonucleotide in the presence of a polymerase and substrate (deoxy)ribonucleotide triphosphates and measuring the extension reaction. In another aspect, the invention provides a method for selecting compounds which binds to a target.

Abstract: The present invention provides a method of screening a library of molecules to identify and/or select one or more members thereof which are candidate binding partners for one or more ligands comprising: a) contacting an expression library in solution with one or more ligands; b) capturing ligands which have become bound to one or more members of the expression library onto a solid phase; and c) detecting the presence of a ligand, thereby detecting the presence of one or more members of the expression library which are candidate binding partners for the ligand.

Abstract: This invention provides methods for 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 labeled nucleotide complementary to the next template nucleotide in the closed complex. Detection of the label allows determination of the identity of this next correct nucleotide. Identification can be either in place, as part of the complex, or as the dye is eluted from the complex when the reaction cycle is completed by the addition of divalent metal ion. In this way, sequential nucleotides of a DNA can be identified, effectively determining the DNA sequence. This method can be applied to nucleic acid single molecules or to collections of identical or nearly identical sequence such as PCR products or clones. Multiple templates can be sequenced in parallel, particularly if they are immobilized on a solid support.

Abstract: The invention provides methods and devices for high throughput single molecule sequencing of a plurality of target nucleic acids using a universal primer. Devices of the invention comprise a plurality of oligonucleotides, each having the same sequence, bound to a solid support, and ligated to a plurality of target nucleic acids.

Abstract: Nucleic acid arrays and methods of using nucleic acid arrays are disclosed.

Abstract: A method for targeted and comprehensive sequencing using high-density oligonucleotide array, comprising the steps of hybridizing nucleic acid from an investigative species with high-density oligonucleotide arrays of a related species, identifying the oligonucleotide probes that generate high hybridization signals, using the probes sequences to make PCR primers, amplifying heterologous genes by PCR with the gene specific PCR primers and an anchoring primer, and sequencing the PCR products.

Abstract: The methods and compositions of the invention find use in the clinical diagnosis of cholestasis related syndromes, particularly PFIC types 1, 2, and 3; BRIC types 1 and 2; Alagille syndrome, and alpha 1-antitrypsin deficiency. The compositions of the invention include isolated nucleic acid molecules and oligonucleotide pairs suitable for use in amplifying regions of cholestasis related genes. Compositions of the invention include a cholestasis related gene resequencing microarray suitable for determining the nucleotide sequence of a region of a cholestasis related gene. Knowledge of the nucleotide sequence of one or more regions of a patient's cholestasis related gene allows diagnosis of the patient's syndrome.

Abstract: Provided are devices comprising one or more nanoscale pores for use in, inter alia, analyzing various biological molecules. Also provided are methods for the fabrication of nanoscale pores in solid-state substrates, methods for functionalizing nanopores in solid-state substrates, and methods for sequencing polymers using devices containing nanoscale pores.

Abstract: Disclosed are methods for identifying molecular interactions between proteins and DNA sequences in vitro. All of the methods of the invention employ known or suspected DNA-binding proteins and genomic DNA from a stable library. Interacting molecules direct the expression of a reporter gene, the expression of which is then assayed. Also disclosed are genetic constructs useful in practicing the methods of the invention.

Abstract: The present invention includes composition and methods for making and using a combinatorial library having two or more beads, wherein attached to each bead is a unique nucleic acid aptamer that have disposed thereon a unique sequence. The library aptamers may be attached covalently to the one or more beads, which may be polystyrene beads. The aptamers may include phosphorothioate, phosphorodithioate and/or methylphosphonate linkages and may be single or double stranded DNA, RNA or even PNAs.

Abstract: The present invention relates to host cells that produce compounds that are characterized as benzylisoquinolines, as well as select precursors and intermediates thereof. The host cells comprise one, two or more heterologous coding sequences wherein each of the heterologous coding sequences encodes an enzyme involved in the metabolic pathway of a benzylisoquinoline, or its precursors or intermediates from a starting compound. The invention also relates to methods of producing the benzylisoquinoline, as well as select precursors and intermediates thereof by culturing the host cells under culture conditions that promote expression of the enzymes that produce the benzylisoquinoline or precursors or intermediates thereof.

Abstract: The present invention relates to a method of selecting a protein specifically binding to a specific ligand. A method of selecting from a diversifying library a protein specifically binding to a ligand of interest, which comprises: a step of allowing proteins existing in the above library to come into contact with the ligand of interest, so as to select proteins binding to the ligand of interest; a step of allowing the obtained proteins to come into contact with a control ligand, so as to determine the presence or absence of the binding ability of the above proteins to the control ligand; and a step of selecting proteins that are determined not to have the binding ability to the control ligand.

Abstract: The present invention relates to optical confinements, methods of preparing and methods of using them for analyzing molecules and/or monitoring chemical reactions. The apparatus and methods embodied in the present invention are particularly useful for high-throughput and low-cost single-molecular analysis.

Abstract: The present invention relates to optical confinements, methods of preparing and methods of using them for analyzing molecules and/or monitoring chemical reactions. The apparatus and methods embodied in the present invention are particularly useful for high-throughput and low-cost single-molecular analysis.

Abstract: The invention concerns a method for identifying a ligand capable of selectively modulating a functional cascade involving a target, and uses thereof for high-throughput screening of molecules of interest, in particular therapeutic (medicine). Said method comprises at least the following steps: a) identifying an antibody or an antibody fragment comprising at least one of the variable domains of an immunoglobulin chain, capable of binding to said target and modulating said functional cascade involving said target; b) screening from a bank of molecules, ligands modulating the bond between said target and the antibody or antibody fragment identified in a); and c) identifying among said modulating ligands obtained in b), those capable of modulating said functional cascade.

Abstract: In a method and apparatus for identifying and distinguishing fungal species, a hyperspectral imaging scanner is used to acquire hyperspectral image data for radiation obtained from a sample area in which at least one unknown fungal species is present. A computer compares the acquired hyperspectral image data with spectral signature data stored in a digital library, which includes spectral signature data for each one of a group of known fungal species, and identifies the fungal species, based on the result of such comparison. The spectral signature data stored in the digital library take into account, for each fungal species, spectral variations that can occur due to at least one of environmental and temporal influences. The computer comparison includes a pixel-by-pixel analysis of the degree of difference between acquired hyperspectral image data and the spectral signature data, so that a spatial distribution of identified fungal species can be determined for a sample area.

Abstract: The invention provides a family of tethered nucleotide analogs useful in sequencing nucleic acids containing a homopolymer region comprising, for example, two or more base repeats, and to sequencing methods using such tethered nucleotide analogs.

Abstract: The invention relates to nucleic acid characterisation. In particular, the invention provides a method of sequencing and distinguishing between nucleic acid sequences originating from different sources on an array, the method comprising sequencing of a nucleic acid tag characteristic of the source of the nucleic acid sequences.

Abstract: Sets of genetic markers for specific tumor classes are described, as well as methods of identifying a biological sample based on these markers. Genes that are differentially expressed between different stages of colorectal tumors are identified. Genes that are differentially expressed between tumors that have metastasized to the regional lymph nodes and tumors that have not metastasized to the regional lymph nodes are identified. Changes in gene expression pattern between two samples or between a sample and a control may be used to determine tumor stage. Also described are diagnostic, prognostic, and therapeutic screening uses for these markers, as well as oligonucleotide arrays comprising these markers.