Abstract: The present invention includes a method of analyzing one or more molecular components in a mixture of components. In the method, a mixture of molecular components, i.e., an analyte mixture, is separated on a capillary liquid chromatography column. The component-containing eluate from the column is deposited as a series of discrete, defined-volume microdrops, along a region of an adsorbent collection layer. During the chromatographic separation, the column eluate may also be monitored to detect the presence of separated components in the eluate. The one or more components deposited in the collection layer are then analyzed by selected analytical techniques. In related aspect, the invention includes a method of collecting one or more molecular components derived from a mixture of components, a blotter apparatus useful in the above method, and a system for analyzing one or more molecular components in a mixture of components.

Abstract: The invention provides a method for constructing a high resolution physical map of a polynucleotide. In accordance with the invention, the polynucleotide is digested successively with at least two different restriction endonucleases and the ends of the restriction fragments are sequenced after each digestion. In this manner, restriction fragments having sequenced ends are produced that can be aligned by their sequences to give a physical map of the polynucleotide. Preferably, restriction fragment ends are sequenced by massively parallel signature sequencing (MPSS), or a like parallel sequencing technique.

Abstract: Disclosed are methods for detecting or quantifying one or more target polynucleotide sequences in a sample. In one aspect, a sample is contacted with first and second probe pair that are capable of hybridizing to a selected target sequence and a corresponding complementary sequence, respectively. Probe cleavage and ligation results in the formation of ligation products which can be generated in an exponential fashion when the target sequence and/or complement are present in the sample. In another embodiment, a single probe pair can be used to form ligation product in a linear fashion from a complementary template. Reagents and kits are also disclosed.

Abstract: A method for obtaining sequence information from a plurality of target polynucleotides in a sample is disclosed. In one embodiment, the method involves contacting a plurality of different sequence primers with a polynucleotide sample under conditions effective for the primers to anneal to primer-complementary regions in one or more target polynucleotides, to form one or more target-primer hybrid(s). Each different-sequence primer contains (i) a target binding segment and (ii) a tag segment having a nucleotide sequence that uniquely identifies the target binding segment. The hybrid(s) are contacted with a labeled nucleotide terminator in the presence of a primer-extending reagent, under conditions effective to append the base to an end of the annealed primer in the hybrid only when the base is complementary to a base in the target polynucleotide that is immediately adjacent to the end of the annealed primer, to form an extended hybrid mixture.

Abstract: Long wavelength, narrow emission bandwidth fluorecein dyes are provided for detecting spacially overlapping target substances. The dyes comprise 4,7-dichlorofluoresceins, and particularly 2′,4′,5′,7′-tetrachloro-4,7-dicloro-5- (and 6-) carboxyfluoresceins. Methods and kits for using the dyes in DNA analysis are provided.

Abstract: Disclosed is a method for determining the number of repeat units in a repeat region of a target nucleic acid. In a first aspect, the method of the invention includes the steps of annealing a primer to a target nucleic acid; performing a first primer extension reaction using a first primer extension reagent; separating the target-primer hybrid and unreacted first primer extension reagent; performing a second primer extension reaction using a second primer extension reagent, wherein at least one of the first or second primer extension reagents includes an extendible nucleotide having a label attached thereto; separating the target-primer hybrid from unreacted second primer extension reagent; measuring a signal produced by the label; treating the label so as to render the label undetectable; and repeating the above steps until the signal is substantially less than a signal detected in a previous cycle.

Abstract: The present invention includes, in one aspect, a kit comprising one or more sets of oligonucleotide probes whose perfectly matched duplexes with complementary sequences have similar or substantially the same free energy of duplex formation.

Abstract: Provided is a method of nucleic acid amplification. In one embodiment, the method comprises performing nucleic acid amplification on a target polynucleotide using a nucleic acid polymerase having 5′ to 3′ nuclease activity, a primer capable of hybridizing to the target polynucleotide, and an oligonucleotide probe under amplification conditions such that the probe hybridizes to the target polynucleotide 3′ relative to the primer and the probe does not hybridize with itself to form a hairpin structure. The oligonucleotide probe has at one end a fluorescent reporter and at the other end a quencher that quenches the fluorescence of the reporter molecule when both the fluorescent reporter and quencher are attached to the probe. Digestion of the oligonucleotide probe by the polymerase during amplification is effective to separate the reporter from the quencher, whereby a fluorescence signal of the reporter is increased.

Abstract: A method is provided for assessing the toxicity of a compound in a test organism by measuring gene expression profiles of selected tissues. Gene expression profiles are measured by massively parallel signature sequencing of cDNA libraries constructed from mRNA extracted from the selected tissues. Gene expression profiles provide extensive information on the effects of administering a compound to a test organism in both acute toxicity tests and in prolonged and chronic toxicity tests.

Abstract: Disclosed are an apparatus, system, and method for two dimensional electrophoresis of analytes of interest, particularly polypeptides. The apparatus includes a sample separation cavity comprising (1) an electrophoresis region located along an upper portion of the cavity for performing charge and/or size-based electrophoresis in a first dimension along the upper portion, and (2) below the electrophoresis region, a second electrophoresis region for performing electrophoresis in a second dimension in a direction substantially perpendicular to the first dimension. In one preferred embodiment, the second electrophoresis region contains an isoelectric focusing region containing a continuous pKa gradient immobilized on at least one of the major opposing surfaces of the cavity.

Abstract: A method and system for polynucleotide synthesis are provided which employ solid phase synthesis on a nonswellable porous polystyrene support by phosphoramidite or hydrogen phosphonate chemistries. The polystyrene support gives rise to fewer tritylated failure sequences caused by chain growth from extraneous support sites, and allows lower amounts of monomer reactants to be used to achieve equal or better coupling efficiencies as those achieveable with CPG. The method and system also employ nucleoside intermediates whose exocyclic amines are protected by base-labile groups which permit simultaneous cleavage and deprotection of the completed polynucleotide chain in the presence of the solid phase support. This latter feature allows practical automation of both the synthesis and purification of polynucleotides.

Abstract: An improvement in adaptor-based sequence analysis is provided that addresses the problems created by self-ligation of target polynucleotides that have complementary ends. The improvement includes preparation of target polynucleotides with dephosphorylated 5′ strands and the use of adaptors having a 3′ blocking group. In a preferred embodiment, adaptors are ligated to target polynucleotides by a single strand, 3′ blocking groups are removed, the adjacent 5′ hydroxyl of the target polynucleotide is phosphorylated, and the ligation of the adaptor is completed by treatment with a ligase.

Abstract: The invention provides 1,4-naphthoquinone compounds and a method for inhibiting tumor cell growth in a subject by administering such compounds. The compounds are represented by the structures: where R1 is lower alkyl, halogenated lower alkyl, phenyl, benzyl, phenethyl, or —(CH2)mCOOX, where m is 2 or 3 and X is H, methyl, or ethyl; R2 is halo or NHY, where Y is hydrogen, loweralkyl, halogenated lower alkyl, hydroxylated lower alkyl, lower dialkylaminoalkyl, phenyl, benzyl, or phenethyl; R3 is lower alkyl, halogenated lower alkyl, phenyl, benzyl, phenethyl, or —(CH2)mCOOX, where m and X are as defined for R1 above; and R4 is hydrogen, lower alkyl, lower aminoalkyl, halogenated lower alkyl, phenyl, benzyl, or phenethyl.

Abstract: Modified oligonucleotides 3′-NHP(O)(O−)O-5′ phosphoramidates were synthesized on a solid phase support. The phosphoramidate analogs were found to have significantly increased resistance toward phosphodiesterase digestion. Thermal dissociation experiments demonstrated that these compounds form more stable duplexes than phosphodiesters with complementary DNA and particularly RNA strands. Further, the phosphoramidate analogs can also form stable triplexes with double-stranded DNA target, where under similar conditions parent phosphodiester compounds failed to do so.

Abstract: The invention is directed to a method and device for simultaneously testing a sample for the presence, absence, and/or amounts of one or more a plurality of selected analytes. The invention includes, in one aspect, a device for detecting or quantitating a plurality of different analytes in a liquid sample. The device includes a substrate which defines a sample-distribution network having (i) a sample inlet, (ii) one or more detection chambers, and (iii) channel means providing a dead-end fluid connection between each of the chambers and the inlet. Each chamber may include an analyte-specific reagent effective to react with a selected analyte that may be present in the sample, and detection means for detecting the signal. Also disclosed are methods utilizing the device.

Abstract: The invention is directed to a method and device for simultaneously testing a sample for the presence, absence, and/or amounts of one or more a plurality of selected analytes. The invention includes, in one aspect, a device for detecting or quantitating a plurality of different analytes in a liquid sample. The device includes a substrate which defines a sample-distribution network having (i) a sample inlet, (ii) one or more detection chambers, and (iii) channel means providing a dead-end fluid connection between each of the chambers and the inlet. Each chamber may include an analyte-specific reagent effective to react with a selected analyte that may be present in the sample, and detection means for detecting the signal. Also disclosed are methods utilizing the device.

Abstract: Long wavelength, narrow emission bandwidth fluorecein dyes are provided for detecting spacially overlapping target substances. The dyes comprise 4,7-dichlorofluoresceins, and particularly 2',4',5', 7'-tetrachloro-4,7-dichloro-5- (and 6-) carboxyfluoresceins. Methods and kits for using the dyes in DNA analysis are provided.

Abstract: Compounds referred to herein as oligonucleotide clamps are provided that stably bind to target polynucleotides in a sequence-specific manner. The oligonucleotide clamps comprise one or more oligonucleotide moieties capable of specifically binding to a target polynucleotide and one or more pairs of binding moieties covalently linked to the oligonudeotide moieties. In accordance with the invention, upon annealing of the oligonucleotide moieties to the target polynucleotide, the binding moieties of a pair are brought into juxtaposition so that they form a stable covalent or non-covalent inkage or complex. The interaction of the binding moieties of the one or more pairs effectively clamps the specifically annealed oligonucleotide moieties to the target polynucleotide.

Abstract: Disclosed are an apparatus, system, and method for two dimensional electrophoresis of analytes of interest, particularly polypeptides. The apparatus includes a sample separation cavity comprising (1) an electrophoresis region located along an upper portion of the cavity for performing charge and/or size-based electrophoresis in a first dimension along the upper portion, and (2) below the first said electrophoresis region, a second electrophoresis region for performing electrophoresis in a second dimension in a direction substantially perpendicular to the first dimension. In one preferred embodiment, the second electrophoresis region contains an isoelectric focusing region containing a continuous pKa gradient immobilized on at least one of the major opposing surfaces of the cavity.

Abstract: Compounds having a certain benzoxazine ring system and their precursors are disclosed which are useful for enhancing synaptic responses mediated by AMPA receptors. Also disclosed are methods for preparing such compounds, and methods for their use in treating subjects suffering from impaired nervous or intellectual functioning due to deficiencies in the number of excitatory synapses or in the number of AMPA receptors. The invention compounds can also be used for the treatment of non-impaired subjects for enhancing performance in sensory-motor and cognitive tasks which depend on brain networks utilizing AMPA receptors and for improving memory encoding.