Abstract: An apparatus and system are provided for simultaneously analyzing a plurality of analytes anchored to microparticles. Microparticles each having a uniform population of a single kind of analyte attached are disposed as a substantially immobilized planar array inside of a flow chamber where steps of an analytical process are carried out by delivering a sequence of processing reagents to the microparticles by a fluidic system under microprocessor control. In response to such process steps, an optical signal is generated at the surface of each microparticle which is characteristic of the interaction between the analyte carried by the microparticle and the delivered processing reagent. The plurality of analytes are simultaneously analyzed by collecting and recording images of the optical signals generated by all the microparticles in the planar array.

Abstract: Multiplexed determinations of large numbers of events are achieved in an accurate and simple manner by using a multitude of primer reagents in combination with different capture reagents that serve for sequestering all the reagents at a single site, followed by independent release of subsets of the primer reagents using differential release conditions. Also included as part of the primer reagents may be identifiers, which serve to identify a particular characteristic. The method is illustrated using primers with sequences for initiation of chain extension that are joined to or serve as a capture sequence, and where the extended primer has an identifier. After extending the primer, the extended primers are sequestered via the capture sequence onto a sequestering agent, sequentially released and the released extended primers analyzed to provide multiplexed determinations.

Abstract: The invention provides a method of tracking, identifying, and/or sorting classes or subpopulations of molecules by the use of oligonucleotide tags. Oligonucleotide tags of the invention comprise oligonucleotides selected from a minimally cross-hybridizing set. Preferably, such oligonucleotides each consist of a plurality of subunits 3 to 9 nucleotides in length. A subunit of a minimally cross-hybridizing set forms a duplex or triplex having two or more mismatches with the complement of any other subunit of the same set. The number of oligonucleotide tags available in a particular embodiment depends on the number of subunits per tag and on the length of the subunit. An important aspect of the invention is the use of the oligonucleotide tags for sorting polynucleotides by specifically hybridizing tags attached to the polynucleotides to their complements on solid phase supports.

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: The invention provides a method of tracking, identifying, and/or sorting classes or subpopulations of molecules by the use of oligonucleotide tags. Oligonucleotide tags of the invention comprise oligonucleotides selected from a minimally cross-hybridizing set. Preferably, such oligonucleotides each consist of a plurality of subunits 3 to 9 nucleotides in length. A subunit of a minimally cross-hybridizing set forms a duplex or triplex having two or more mismatches with the complement of any other subunit of the same set. The number of oligonucleotide tags available in a particular embodiment depends on the number of subunits per tag and on the length of the subunit. An important aspect of the invention is the use of the oligonucleotide tags for sorting polynucleotides by specifically hybridizing tags attached to the polynucleotides to their complements on solid phase supports.

Abstract: The invention provides a method and materials for monitoring and isolating differentially expressed genes. In accordance with the method of the invention, differently labeled populations of DNAs from sources to be compared are competitively hybridized with reference DNA cloned on solid phase supports, e.g. microparticles, to provide a differential expression library which, in the preferred embodiment, may be manipulated by fluorescence-activated cell sorting (FACS). Monitoring the relative signal intensity of the different fluoresent labels on the microparticles permits quantitative analysis of expression levels relative to the reference DNA. Populations of microparticles having relative signal intensities of interest can be isolated by FACS and the attached DNAs identified by sequencing, such as with massively parallel signature sequencing (MPSS), or with conventional DNA sequencing protocols.

Abstract: The invention provides a method of tracking, identifying, and/or sorting classes or subpopulations of molecules by the use of oligonucleotide tags. Oligonucleotide tags of the invention comprise oligonucleotides selected from a minimally cross-hybridizing set. Preferably, such oligonucleotides each consist of a plurality of subunits 3 to 9 nucleotides in length. A subunit of a minimally cross-hybridizing set forms a duplex or triplex having two or more mismatches with the complement of any other subunit of the same set The number of oligonucleotide tags available in a particular embodiment depends on the number of subunits per tag and on the length of the subunit. An important aspect of the invention is the use of the oligonucleotide tags for sorting polynucleotides by specifically hybridizing tags attached to the polynucleotides to their complements on solid phase supports.

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: 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 a method of tracking, identifying, and/or sorting classes or subpopulations of molecules by the use of oligonucleotide tags. Oligonucleotide tags of the invention comprise oligonucleotides selected from a minimally cross-hybridizing set. Preferably, such oligonucleotides each consist of a plurality of subunits 3 to 9 nucleotides in length. A subunit of a minimally cross-hybridizing set forms a duplex or triplex having two or more mismatches with the complement of any other subunit of the same set. The number of oligonucleotide tags available in a particular embodiment depends on the number of subunits per tag and on the length of the subunit. An important aspect of the invention is the use of the oligonucleotide tags for sorting polynucleotides by specifically hybridizing tags attached to the polynucleotides to their complements on solid phase supports.

Abstract: The invention provides a method of tracking, identifying, and/or sorting classes or subpopulations of molecules by the use of oligonucleotide tags. Oligonucleotide tags of the invention comprise oligonucleotides selected from a minimally cross-hybridizing set. Preferably, such oligonucleotides each consist of a plurality of subunits 3 to 9 nucleotides in length. A subunit of a minimally cross-hybridizing set forms a duplex or triplex having two or more mismatches with the complement of any other subunit of the same set. The number of oligonucleotide tags available in a particular embodiment depends on the number of subunits per tag and on the length of the subunit. An important aspect of the invention is the use of the oligonucleotide tags for sorting polynucleotides by specifically hybridizing tags attached to the polynucleotides to their complements on solid phase supports.

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 provides a method of tracking, identifying, and/or sorting classes or subpopulations of molecules by the use of oligonucleotide tags. Oligonucleotide tags of the invention comprise oligonucleotides selected from a minimally cross-hybridizing set. Preferably, such oligonucleotides each consist of a plurality of subunits 3 to 9 nucleotides in length. A subunit of a minimally cross-hybridizing set forms a duplex or triplex having two or more mismatches with the complement of any other subunit of the same set. The number of oligonucleotide tags available in a particular embodiment depends on the number of subunits per tag and on the length of the subunit.An important aspect of the invention is the use of the oligonucleotide tags for sorting polynucleotides by specifically hybridizing tags attached to the polynucleotides to their complements on solid phase supports.

Abstract: The invention provides a method and materials for sorting polynucleotides with oligonucleotide tags. Oligonucleotide tags of the invention are capable of hybridizing to complementary oligomeric compounds consisting of subunits having enhanced binding strength and specificity as compared to natural oligonucleotides. Such complementary oligomeric compounds are referred to herein as "tag complements." Subunits of tag complements may consist of monomers of non-natural nucleotide analogs, referred to herein as "antisense monomers" or they may comprise oligomers having lengths in the range of 3 to 6 nucleotides or analogs thereof, including antisense monomers, the oligomers being selected from a minimally cross-hybridizing set. In such a set, a duplex made up of an oligomer of the set and the complement of any other oligomer of the set contains at least two mismatches.

Abstract: The invention provides a computerized method, associated apparatus, and computer program product for determining a set of non-hybridizing oligonucleotides. The invention represents a first oligonucleotide in the computer's memory, generates a set of oligonucleotides, including the first oligonucleotide, that meet a specified condition that determines whether the generated oligonucleotides will not hybridize with the first oligonucleotide. The invention also examines each of the generated oligonucleotides in the set to remove oligonucleotides from the set that hybridize with other nucleotides in the set. Thus, the invention develops a minimally cross-hybridizing set of oligonucleotides that can be used for tracking, identifying, and/or sorting classes or subpopulations of molecules by the user of oligonucleotide tags.

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: The invention provides a method of nucleic acid sequence analysis based on the ligation of one or more sets of encoded adaptors to the terminus of a target polynucleotide. Encoded adaptors whose protruding strands form perfectly matched duplexes with the complementary protruding strands of the target polynucleotide are ligated, and the identity of the nucleotides in the protruding strands is determined by an oligonucleotide tag carried by the encoded adaptor. Such determination, or "decoding" is carried out by specifically hybridizing a labeled tag complement to its corresponding tag on the ligated adaptor.

Abstract: A method of purifying a hydrophobically substituted polynucleotide by reverse phase HPLC is described. The hydrophobic substituent may be removed from the polynucleotide under non-acidic conditions; the purification method is thus especially useful for acid sensitive polynucleotide analogs.