Abstract: Devices and methods are provided using microfluidic devices for manipulating small volumes and determining a variety of chemical and physical events. The devices rely upon an opening to the atmosphere of a small volume in a zone, where a sample is placed in the zone where evaporation can occur. The zone is maintained in contact with a liquid medium that serves to replenish the liquid in the zone and maintain the composition of the mixture in the zone substantially constant. The diffusion of components in the zone is restricted during the course of the determination by the liquid flux into the zone.

Abstract: Methods and compositions are provided for amplifying tandem repeats, particularly for detecting binding events, using a nucleic acid reagent comprising two features, one having a tandem repeat region and the other having an extendable 3′ terminus. When the two oligonucleotides are hybridized in the presence of a DNA polymerase and NTPs for replicating the tandem repeat region, repetitive extension of the extendable 3′ terminus along the tandem repeat region is obtained. By having labeled NTPs, the amplification can be detected as indicative of the binding event. Kits are provided for performing the method.

Abstract: A method is disclosed for detecting the presence of a difference between two related nucleic acid sequences. In the method a complex is formed comprising both strands of each sequence. Each member of at least one pair of non-complementary strands within the complex have labels. The association of the labels as part of the complex is determined as an indication of the presence of a difference between the two related sequences. The complex generally comprises a Holliday junction. In one aspect a medium suspected of containing said two related nucleic acid sequences is treated to provide partial duplexes having non-complementary tailed portions at one end. The double stranded portions of the partial duplexes are identical except for said difference.

Abstract: A method for detection of nucleic acid targets using as reagents: (1) a stem loop probe having a long strand for hybridizing to a target, a short strand, usually with a free 3′-end for extension, hybridized to a portion of the long strand and a linker forming a loop and joining the short and long strands; and (2) a hybridizing reagent that hybridizes to the short strand when released by the target. The target is detected by extension of one or both the probe or hybridizing reagent along each other. A circular hybridizing reagent can be employed with DNA polymerase for concatenated extension of the probe 3′-end or extension of the 3′-end of the probe along a hybridizing reagent having a promoter sequence for forming transcripts of at least a portion of the long strand or the hybridizing reagent.

Abstract: Methods are provided for the determination of specific nucleotide sequences, particularly single nucleotide polymorphisms, by using probes comprising a first strand complementary to the target sequence, a shorter second strand forming a stem and a linker adjacent the double stranded stem connecting the two strands, whereby binding of target to the probe under conditions that do not cause melting of the double stranded stem in the absence of target results in the dissociation of the second strand from the first strand. The ss second strand is then detected as exemplified by using a FRET pair, where dissociation of the stem results in separation of the FRET pair and increase in fluorescence. Amplification of the target sequence may be employed prior to combination with the probe. The method finds particular application with complex nucleic acid mixtures.

Abstract: A method is disclosed for detecting the presence of a difference between two related nucleic acid sequences. In the method a complex is formed comprising both strands of each sequence. Each member of at least one pair of non-complementary strands within the complex have labels. The association of the labels as part of the complex is determined as an indication of the presence of a difference between the two related sequences. The complex generally comprises a Holliday junction. In one aspect a medium suspected of containing said two related nucleic acid sequences is treated to provide partial duplexes having non-complementary tailed portions at one end. The double stranded portions of the partial duplexes are identical except for said difference.

Abstract: Devices and methods are provided using microfluidic devices for manipulating small volumes and determining a variety of chemical and physical events. The devices rely upon an opening to the atmosphere of a small volume in a zone, where a sample is placed in the zone where evaporation can occur. The zone is maintained in contact with a liquid medium that serves to replenish the liquid in the zone and maintain the composition of the mixture in the zone substantially constant. The diffusion of components in the zone is restricted during the course of the determination by the liquid flux into the zone.

Abstract: Devices and methods are provided using microfluidic devices for manipulating small volumes and determining a variety of chemical and physical events. The devices rely upon an opening to the atmosphere of a small volume in a zone, where a sample is placed in the zone where evaporation can occur. The zone is maintained in contact with a liquid medium that serves to replenish the liquid in the zone and maintain the composition of the mixture in the zone substantially constant. The diffusion of components in the zone is restricted during the course of the determination by the liquid flux into the zone.

Abstract: Methods and compositions are provided for nucleic acid analysis. The method employs a primer and a probe that bind to a target nucleic acid sequence, where the primer has an effector agent, which causes cleavage of a bond when the primer and the probe are bound to the same target molecule. The primer and probe have arms that do not bind to the target, hybridize with each other and comprise the effector and cleavable bond, where the probe has a tag defining the probe that is released upon bond cleavage.

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: Devices and methods are provided using microfluidic devices for manipulating small volumes and determining a variety of chemical and physical events. The devices rely upon an opening to the atmosphere of a small volume in a zone, where a sample is placed in the zone where evaporation can occur. The zone is maintained in contact with a liquid medium that serves to replenish the liquid in the zone and maintain the composition of the mixture in the zone substantially constant. The diffusion of components in the zone is restricted during the course of the determination by the liquid flux into the zone.

Abstract: The present invention relates to a method for selectively extending an oligonucleotide primer along a specific target polynucleotide sequence in a mixture of polynucleotides. Selective extension is achieved by controlling the concentration of the oligonucleotide primer by forming the oligonucleotide in situ by degrading the 3′-end of a modified oligonucleotide. The method comprises providing in combination the mixture of polynucleotides and the modified oligonucleotide having a 3′-end that is not extendable along any polynucleotide and extending the oligonucleotide primer selectively along the specific target polynucleotide sequence by controlling the degradation of the 3′-end of the modified oligonucleotide. In this way extension of the oligonucleotide primer along polynucleotides other than the specific target polynucleotide sequence is substantially reduced or avoided. In another aspect the invention is an improvement in a method for amplifying a target polynucleotide sequence.

Abstract: A method is disclosed for modifying an oligonucleotide, which method has application to the detection of a polynucleotide analyte. An oligonucleotide is reversibly hybridized with a polynucleotide, for example, a polynucleotide analyte, in the presence of a 5′-nuclease under isothermal conditions. The polynucleotide analyte serves as a recognition element to enable a 5′-nuclease to cleave the oligonucleotide to provide (i) a first fragment-that is substantially non-hybridizable to the polynucleotide analyte and (ii) a second fragment that lies 3′ of the first fragment (in the intact oligonucleotide) and is substantially hybridizable to the polynucleotide analyte. At least a 100-fold molar excess of the first fragment and/or the second fragment are obtained relative to the molar amount of the polynucleotide analyte. The presence of the first fragment and/or the second fragment is detected, the presence thereof indicating the presence of the polynucleotide analyte.

Abstract: The present invention relates to methods for immunoassay of analytes employing mutant glucose-6-phosphate dehydrogenase (G6PDH) enzymes as labels. In particular, the invention relates-to the use of conjugates of an analyte or analyte analog and a mutant NAD+ dependent G6PDH differing from any precursor G6PDH by the deletion, substitution, or insertion, or any combination thereof of at least one amino acid per subunit. The invention also involves the construction of several mutations in precursor glucose-6-phosphate dehydrogenase (G6PDH) enzymes. Typically, the mutations involve deletion or substitution of one or more lysine residues, or introduction of one or more cysteine residues by insertion of cysteine to precursor G6PDH or substitution of precursor G6PDH amino acids residues with cysteine.

Abstract: Disclosed are methods for detecting analytes with indicator systems which may undergo a molecular configurational change upon exposure to the analyte. The configurational change affects a detectable quality associated with the indicator system, thereby allowing detection of the presence or concentration of the analyte.

Abstract: Disclosed are methods for detecting analytes with indicator systems which may undergo a molecular configurational change upon exposure to the analyte. The configurational change affects a detectable quality associated with the indicator system, thereby allowing detection of the presence or concentration of the analyte.

Abstract: A method of measuring the amount of cyclosporin in a sample suspected of containing cyclosporin is disclosed. A method of inactivating interfering cross-reactive material in an assay for measuring the amount of cyclosporin in a sample suspected of containing cyclosporin is also disclosed. Compositions wherein cyclosporin is conjugated to an immunogenic carrier or a label, optionally through a linking group, at an alanine nitrogen atom of the cyclic backbone of cyclosporin are also disclosed. Compositions wherein atiocyclosporin is conjugated, optionally through a linking group, to an immunogenic carrier or a label are also disclosed. Where cyclosporin is conjugated to an immunogenic carrier, the conjugates may be used as immunogens for the preparation of antibodies which are capable of recognizing cyclosporin.

Abstract: Methods, compositions and kits are disclosed. The methods are directed to determining the presence or relative amounts of two or more components in a medium. A combination is provided comprising a medium suspected of containing the components and a label reagent for each of the components. The label reagent comprises a chemiluminescent composition that is activated by electromagnetic radiation. A first specific binding pair (sbp) member may be associated with the reagent depending on the components to be determined. Luminescence emitted by each of the chemiluminescent compositions upon activation is differentially detectable. Where a first sbp member is employed, it is capable of binding to the component or to a second sbp member to form a complex related to the amount of the component. At least one of the chemiluminescent compositions comprises a fluorescent energy acceptor. After the above are combined, the chemiluminescent compositions are activated.

Abstract: Methods are disclosed for determining an analyte in a medium suspected of containing the analyte. One method comprises treating a medium suspected of containing an analyte under conditions such that the analyte, if present, causes a photosensitizer and a chemiluminescent compound to come into close proximity. The photosensitizer generates singlet oxygen and activates the chemiluminescent compound when it is in close proximity. The activated chemiluminescent compound subsequently produces light. The amount of light produced is related to the amount of analyte in the medium. Preferably, at least one of the photosensitizer and chemiluminescent compound is associated with a surface which is usually a suspendible particle, and a specific binding pair member is bound thereto. Compositions and kits are also disclosed.

Abstract: Compositions are disclosed comprising (a) a metal chelate wherein the metal is selected from the group consisting of europium, terbium, dysprosium, samarium osmium and ruthenium in at least a hexacoordinated state and (b) a compound having a double bond substituted with two aryl groups, an oxygen atom and an atom selected from the group consisting of oxygen, sulfur and nitrogen wherein one of the aryl groups is electron donating with respect to the other. Such composition is preferably incorporated in a latex particulate material. Methods and kits are also disclosed for determining an analyte in a medium suspected of containing the analyte. The methods and kits employ as one component a composition as described above.