Abstract: The invention provides methods, kits, and compositions comprising novel mutant loxP sites. Such sites are particularly useful for procedures requiring multiple site-specific recombination reactions, including deletions or insertions of multiple genes or other sequences in the same organism, staged insertions or deletions of genes of the same organism at different times, assembly of large polynucleotide constructs by serial site-specific recombination, and the like. In one aspect, compositions of the invention includes particular mutant spacer oligonucleotides of loxP recombination elements, the recombination elements themselves, and pairs of non-promiscuous mutant loxP sites.

Abstract: The invention provides a method of generating nested sets of double stranded DNA (dsDNA) circles that may be used as size ladders in nucleic acid separations and as templates in DNA sequencing operations. In one aspect, the invention provides methods for generating nested sets of double stranded DNA circles in a self-sustaining enzymatic reaction comprising the activities of at least one endonuclease, at least one single stranded exonuclease, and at least one ligase. In another embodiment, such nested sets are generated from linear dsDNA fragments having ligatable terminators that are self-ligated to form corresponding dsDNA circles.

Abstract: A photoactivation apparatus (200) separately delivers light to a plurality of wells of a multiwell plate (110). The device-includes a plurality of light emitting diodes (210) attached to a first surface of a substrate (220) such that when the photoactivation apparatus is positioned on the multiwell plate each of the light emitting diodes delivers light to one corresponding well. The light emitting diodes (210) may extend at least partially into the wells of the multiwell plate. A heat sink (250) and fan may be provided on the device to dissipate heat generated by the light emitting diodes. The components are preferably enclosed in a conveniently-sized housing (270). The housing may include one or more grips and air vents. The apparatus includes an interlocking lip (230) defining a recess which is sized to receive at least a portion of the multiwell plate. A power supply connected to the apparatus and configured to provide an equal voltage across each of the light emitting diodes.

Abstract: The invention provides methods for sorting polynucleotides from a population based on predetermined sequence characteristics. In one aspect, the method of the invention is carried out by extending a primer annealed polynucleotides having predetermined sequence characteristics to incorporate a predetermined terminator having a capture moiety, capturing polynucleotides having extended primers by a capture agent that specifically binds to the capture moiety, and melting the captured polynucleotides from the extended primers to form a subpopulation of polynucleotides having the predetermined sequence characteristics. In another aspect, the method of the invention is carried out on a population of tagged polynucleotides so that after a subpopulation is selected, the members of the subpopulation may be simultaneously analyzed using the unique tags on the polynucleotides to convey analytical information to a hybridization array for a readout.

Abstract: The activity of a cell surface protease, particularly an ADAM, is determined in a rapid and sensitive assay employing a whole cell system. The assays are effective to identify effector molecules that affect the activity of a cell surface protease directly or indirectly, and to screen for therapeutic agents that modulate those effector molecules. The assays can also be used to screen for therapeutic agents that modulate the activity of a cell surface protease associated with a disease or medical condition. Kits comprising at least one component of the present assays are also provided.

Abstract: Probe sets for the multiplexed detection of the binding of, or interaction between, one or more ligands and target antiligands are provided. Detection involves the release of identifying tags as a consequence of target recognition. The probe sets include electrophoretic tag probes or e-tag probes, comprising a detection region and a mobility-defining region called the mobility modifier, both linked to a target-binding moiety. The probes comprise interactive functionalities adjacent the cleaved portion positioned in the probes such that the interactive functionality does not form part of the e-tag reporters. Target antiligands are contacted with a set of e-tag probes and the contacted antiligands are treated with a selected cleaving agent resulting in a mixture of e-tag reporters and uncleaved and/or partially cleaved e-tag probes. The mixture is exposed to a capture agent effective to bind to uncleaved or partially cleaved e-tag probes, followed by electrophoretic separation.

Abstract: Kits for the multiplexed detection of the binding of, or interaction between, one or more ligands and target antiligands are provided. Detection involves the release of identifying tags as a consequence of target recognition. The kits include sets of electrophoretic tag probes or e-tag probes, a capture agent and optionally a cleaving agent. The e-tag probes comprise a detection region and a mobility-defining region called the mobility modifier, both linked to a target-binding moiety. In using the kits, target antiligands are contacted with a set of e-tag probes and the contacted antiligands are treated with a selected cleaving agent resulting in a mixture of e-tag reporters and uncleaved and/or partially cleaved e-tag probes. The mixture is exposed to a capture agent effective to bind to uncleaved or partially cleaved e-tag probes, followed by electrophoretic separation. In a multiplexed assay, different released e-tag reporters may be separated and detected providing for target identification.

Abstract: Devices and methods are disclosed for moving charged molecules through a medium by the application of a plurality of electrical fields of sufficient strength and applied for sufficient amounts of time so as to move the charged molecules through the medium. The devices although preferably small in size, preferably generate large numbers (100 or more) of electrical fields to a movement area which preferably contains a liquid buffered or gel medium. Mixtures of charged molecules are pulled through the gel by the force of the electrical fields. The fields are preferably activated simultaneously or sequentially one after another at various speeds to create complex force field distributions or moving field waves along the separation medium. Charged molecules capable of moving quickly through the gel will be moved along by the faster moving field waves and be separated from slower moving molecules.

Abstract: Kits for the multiplexed detection of known, selected nucleotide target sequences are provided. Detection involves the release of identifying tags as a consequence of target recognition. The kits include sets of electrophoretic tag probes or e-tag probes, capture agent and optionally a nuclease. The e-tag probes comprise a detection region and a mobility-defining region called the mobility modifier, both linked to a target-binding moiety. In using the kits, the target-binding moiety of the e-tag probes hybridizes to complementary target sequences followed by nuclease cleavage of the e-tag probes and release of detectable e-tags or e-tag reporters. The mixture is exposed to a capture agent which binds uncleaved and/or partially cleaved e-tag probes, followed by electrophoretic separation. In a multiplexed assay, different released e-tag reporters may be separated and detected providing for target identification.

Abstract: The present invention is directed to methods and compositions for determining the presence, absence, and/or amounts of one or more membrane-associated analytes in a sample. In accordance with the invention, binding compounds derivatized with releasable molecular tags specifically bind to selected membrane-associated analytes, after which the molecular tags are released upon activation of cleavage moieties, or sensitizers, anchored in the same membrane as the membrane-associated analytes. The released molecular tags are then identified by their distinct separation and detection characteristics.

Abstract: A microfluidic device having integrated components for conducting chemical operations. Depending upon the desired application, the components include electrodes for manipulating charged entities, heaters, electrochemical detectors, sensors for temperature, pH, fluid flow, and other useful components. The device may be fabricated from a plastic substrate such as, for example, a substantially saturated norbornene based polymer. The components are integrated into the device by adhering an electrically conductive film to the substrate. The film may be made of metal or an electrically conducting ink and is applied to the device through metal deposition, printing, or other methods for applying films. Methods for reducing bubble formation during electrokinetic separation and methods for heating material in a microfluidic device are also disclosed.

Abstract: Probe sets for the multiplexed detection of known, selected nucleotide target sequences are provided. Detection involves the release of identifying tags as a consequence of target recognition. The probe sets include electrophoretic tag probes or “e-tag probes”, comprising a detection region and a mobility-defining region called the mobility modifier, both linked to a target-binding moiety. The target-binding moiety of the e-tag probes hybridizes to complementary target sequences followed by nuclease cleavage of the e-tag probes and release of detectable e-tags or e-tag reporters. The mixture is exposed to a capture agent which binds uncleaved and/or partially cleaved e-tag probes, followed by electrophoretic separation. In a multiplexed assay, different released e-tag reporters may be separated and detected providing for target identification.

Abstract: Improved sealing for microstructures in microfluidic devices having a plurality of units is provided by providing collars surrounding the openings to the microstructures, such as reservoirs. The collars are protrusions extending from the surface of the devices and the internal walls of the collars generally aligned with the internal walls of the microstructure. Conformable and/or adhesive lids are employed for sealing the microstructures.

Abstract: A method for performing an electrophoretic separation on a microfluidic device includes applying an electric field to electrokinetically move a sample along a channel towards a location. The sample may be concentrated by application of the electric field. The method further comprises optically monitoring the location for at least a portion of the sample. Once the sample is detected, the electric field is automatically changed to further manipulate the sample. The sample may be spatially separated along a separation channel or channel portion. The closed-loop control of electric fields may be performed using a control unit adapted to apply a voltage potential between electrodes and an optical detector such as an LIF detector.

Abstract: Devices are provided comprising reflecting channels for directing horizontally a vertical light beam through a multiplicity of streams. The devices provide particular applications in microfluidics, where streams of narrow cross-sectional dimensions are involved. Desirably, channels having 45° walls are provided for directing the beams and where the streams are divided by walls, the walls are at substantially 90°. Methods of fabrication of the devices are provided.

Abstract: A system for material excitation in microfluidic devices is described. Aspects of the invention resemble a submerged periscope when in use. They allow for light to be redirected along a more advantageous trajectory as does the maritime device. A prism with a reflecting surface may be used to direct a laser beam along one or more microfluidic trenches or channels. Alternatively, a reflecting surface may be provided in connection with a simple support. Directing a beam along multiple paths is preferably accomplished by scanning a single laser across or around the reflecting surface provided. Provision may be made for at least a portion of the submersible used to function as an electrode to assist in electrokinetically driving fluids and/or ions within the microfluidic device.

Abstract: A mutiplexed enzyme assay method and substrate set for performing the method are disclosed. The method includes performing a plurality of enzyme reactions in the presence of a plurality of enzymes substrates, under conditions effective to convert an enzyme substrate to a corresponding product, where the product of each substrate and the substrate have different separation characteristics from each other and from the other substrates and their corresponding products. After performing the reactions, which may be carried out in separate or combined reactions, the substrates and products in said reactions are separated in a single separation medium. For each separated product and substrate, a separation characteristic effective to identify that product and substrate and a signal related to the amount of the product and substrate is detected. From this, one can determine the amount of substrate converted to the corresponding product in each of the reactions.

Abstract: An optical detection and orientation device is provided comprising a housing having an excitation light source, an optical element for reflecting the excitation light to an aspherical lens and transmitting light emitted by a fluorophore excited by said excitation light, a focusing lens for focusing the emitted light onto the entry of an optical fiber, which serves as a confocal aperture, and a moveable carrier for accurately moving said housing over a small area in relation to a channel in a microfluidic device. The optical detection and orientation device finds use in detecting fluorophores in the channel during operations involving fluorescent signals.

Abstract: A continuous form microstructure array device is constructed as a flexible elongate film laminate containing a plurality of microstructures or arrays of microstructures arranged serially lengthwise along the laminate. The laminate can be continuously drawn from a supply roll or stack, advanced within an analytic device and, when analysis is complete, taken up in a storage roll or stack. The device provides for high throughput microfluidic processing.

Abstract: Microfluidic devices are provided having units of 8-fold symmetry comprising 8 assay units, where a reservoir provides a common component to 8 assay units. The units can be compactly formed in a substrate to provide the ability to perform a large number of assays within a small area. The microfluidic devices find use in operations, such as assays, DNA sequence detection, etc. Various formats can be used to have the microfluidic device interrelate with microtiter well plates. Methods are provided for monitoring the flow rates/velocities of assay components and streams for comparison of results in different assay units and/or to modify the conditions to change the flow rates in particular channels.