Abstract: The invention relates to the analysis of complex protein mixtures such as entire proteomes, and in particular the rapid detection of previously unknown or unusually expressed proteins by common enzymatic digestion, subsequent chromatographic separation and analysis of the digestion peptides by mass spectrometry. The invention consists in subjecting fractions of the digestion peptides separated by liquid chromatography to analysis by mass spectrometry, at a time other than that of the chromatography, in a tandem time-of-flight mass spectrometer with ionization by matrix assisted laser desorption (MALDI). This method finds many times more proteins than are found through the procedures predominantly used until now of two-dimensional gel electrophoresis with subsequent time-of-flight mass spectrometry. It also removes the time pressure that dominates the real-time analysis of coupled LC-MS processes, and it allows measurements to be reduced to the interesting proteins by intermediate analysis.

Abstract: A multi-channel and multi-color bio-separation detection method and apparatus in which a single detector is coupled to a plurality of radiation sources, in a one detector/many radiation sources configuration. Each radiation source directs radiation at a detection zone of a single separation channel, and a single detector is applied to detect light emissions from the detection zones associated with several radiation sources. The radiation sources are activated to direct radiation at the detection zone in a predetermined sequence and further in a cyclic manner, with the detector output synchronized to the radiation sources by a controller. Bio-separation may be conducted simultaneously in all the channels in parallel, with detection time-staggered and/or time multiplexed with respect to the light sources. In one embodiment, low cost light emitting diodes may be used as radiation sources.

Abstract: pK-matched buffers, each containing two effective buffering components: one weak base and one weak acid which have similar pKa at 25° C. (within 0.3 pK units On agarose gels, the buffers in various concentrations were tested for separation of double-stranded DNA fragments with various DNA markers, agarose gel concentrations, and field strengths. Mobility was inversely proportional to the logarithm of molecular weight. The buffers provided high resolution without smearing at more dilute concentration than is possible with standard TAE (Tris/Acetate, pH 8.0) or TBE (Tris/Borate, pH 8.3) buffers. The buffers were also tested in 7M urea denaturing LongRanger™ sequencing gels and in non-denaturing polyacrylamide SSCP gels. The pK-matched buffers provide good separation and high resolution, at a broad range of potential pH values.

Abstract: A method is disclosed for the detection of a His-tagged target biomolecule while said molecule remains in the gel in which it has been separated from other constituents by electrophoresis. The method involves, while the separated His-tagged biomolecule remains in the gel, forming a chelate between the His-tagged molecule, a lanthanide metal ion which exhibits fluorescence, and a sensitizer which enhances the fluorescence of the ion. The gel is then exposed to a u.v. light source to thereby generate a fluorogenic signal indicating the presence of the chelate and, in turn, the His-tagged biomolecule. The fluorogenic signal can then be detected either by direct visualization and/or by other means, such as photographically, e.g., film or charged coupled device (CCD) imaging.

Abstract: The present method provides methods and apparatus for separating proteins using a series of electrophoretic methods that utilize controlled fractionation and labeling techniques to resolve mixtures of proteins. The samples for each electrophoretic method other than the initial method, contain only a subset of proteins resolved in the preceding method. The methods can be used in a variety of different applications including, creating proteomic databases, comparative expression studies, diagnostics, structure activity relationships and metabolic engineering investigations.

Abstract: A method for separating and assaying lipoprotain to determine a degree of modification of a predetermined component in a specimen of lipoprotein, comprising the step of: determining a distance “a” from an application point of the standard sample to a fraction corresponding to the predetermined lipoprotein using an electrophoretic pattern obtained by electrophoresis of a standard sample; determining a distance “b” from the application point of the specimen to a fraction corresponding to the predetermined lipoprotein using electrophoretic pattern obtained by electrophoresis of a specimen; comparing the distance “a” and the distance “b” to determine a relative mobility “z(=b/a)” of the specimen to the standard sample, wherein the degree of modification of the predetermined component in the specimen being judged on the basis of the relative mobility “z”.

Abstract: An apparatus and a method for reading a gel electrophoresis pattern can read the gel electrophoresis pattern of a sample, such as a nucleic acid and a protein, with high sensitivity and without requiring the use of an expensive device structure such as a laser light source of unique type.

Abstract: A device, method and kit for the electrophoretic separation and purification of charged and neutral compounds in an analyte solution. The device comprises a chamber (1), at least one wall of which is composed of a chemical buffering system (4). A potential difference is applied across the buffering system, resulting in the charged and neutral compounds being differentially separated by extraction of the charged compounds into. the buffering system. The device also comprises means for collecting the separated compounds, preferably in ampholyte-free or buffer-free solution and optionally means for recycling the separated fractions.

Abstract: A method of assessing the age or expectation of life wherein the length of the telomeres is determined by a blot method and, optionally, by subsequently hybridizing the DNA gel-electrophoretically separated and bound to the membrane obtained through the blot method by means of a nucleic acid probe complementary to the telomeric sequence, and wherein the specimen DNA containing the telomeric DNA prior to the gel-electrophoretic separation is subjected to a full restriction digestion, characterized in that the separation of the digested DNA in the gel-electrophoresis underlying the blot method takes place at (a) <5 V/cm, and (b) for the duration of at least 6 hours.

Abstract: Methods and devices are provided for characterizing a duplex nucleic acid, e.g., a duplex DNA molecule. In the subject methods, a fluid conducting medium that includes a duplex nucleic acid molecule is contacted with a nanopore under the influence of an applied electric field and the resulting changes in current through the nanopore caused by the duplex nucleic acid molecule are monitored. The observed changes in current through the nanopore are then employed as a set of data values to characterize the duplex nucleic acid, where the set of data values may be employed in raw form or manipulated, e.g., into a current blockade profile. Also provided are nanopore devices for practicing the subject methods, where the subject nanopore devices are characterized by the presence of an algorithm which directs a processing means to employ monitored changes in current through a nanopore to characterize a duplex nucleic acid molecule responsible for the current changes.

Abstract: The proteins in a biological sample that is sought to be analyzed for its protein composition by am electrophoretic or chromatographic procedure are coupled to a dye in an unusually efficient manner by combining the sample with a solid dry composition containing the dye, a buffering agent, and in preferred embodiments, a denaturing agent as well. The solid and dry form of the composition prevents the dye from deteriorating or decomposing, and the combination of components in the composition allows the dye to couple to the proteins in a relatively uniform manner with no overstaining of the protein when the composition and the sample are heated together and held at an elevated temperature for a short period of time.

Abstract: Data traces from four channels of an automated electrophoresis detection apparatus are aligned by identifying peaks in each of the four data traces; optionally normalizing the data traces to achieve a uniform peak height; combining the four data traces in an initial alignment; and determining coefficients of shift and stretch for selected data points within each data trace. The coefficients are determined by optimizing a cost function which reflects the extent of overlap of peaks in the combined normalized data traces to which the coefficients have been applied. The cost function is optimized when the extent of overlap is at a minimum. The coefficients are then used to generate a warp function for each data trace. These warp functions are applied to their respective data traces to produce four warped data traces which are aligned to form an aligned data set. The aligned data set may be displayed on a video screen of a sequencing apparatus, or may be used as the data set for a base-calling process.

Abstract: A photovoltaic device is employed to facilitate interactions between immobilized and dissolved biological materials. Such interactions including nucleotide hybridization, peptide-peptide interaction and peptide/nucleotide binding. Through the force of the photovoltaic effect, which is caused by illuminating the photovoltaic device with a light source, the efficiency of interaction between immobilized biomedical materials on the surface of the device and free biological materials is remarkably enhanced. Such photovoltaic device may be a microfabricated device having an array of microlocations capable of being used as a biological screening tool with high throughput applications.

Abstract: An alignment plate is provided with a plurality of holes for guiding a pipette tip toward a sample plate of a MALDI mass spectrometer. Each of the holes is provided with a conical upper contour in order to guide the pipette tip toward a specific location on the sample plate. Two companion alignment plates are used in order to overlay two separate arrays of samples on the sample plate. For instance, a first of two alignment plates is formed with a 10×10 array of holes so that a 10×10 array of samples is deposited by the pipette tip onto the sample plate. The second of the two alignment plates is formed with a 9×9 array of holes so that a 9×9 array of samples is deposited on the sample plate at locations offset from the 10×10 array of samples already on the sample plate. The number of samples loaded on the sample plate is large and the space on the sample plate is more fully utilized.

Abstract: Methods and reagents for fluorescent analysis of serum proteins separated by electrophoresis. This assay allows for the immediate quantitation of resolved proteins in biological materials. The electrophoresed sample is treated with a fixative composition and stained using an ANS-based stain solution. No pre-stain washing or post-stain washing and drying is required. The fluorescent serum protein assay allows for automation of serum protein analyses.

Abstract: Marker compounds suitable for gel electrophoresis are disclosed. The compounds are natural, non-natural compounds or a mixture thereof, but not a protein. The compounds comprise at least one monomer unit, at least one functional group unit and optionally at least one core unit. Also contemplated is a method for positioning the marker compounds or a set of the compounds according to the invention and a method for the detection and/or quantification of s sample molecule in a two-dimensional gel.

Abstract: The invention provides a method for producing antibodies that selectively recognize short, modified amino acid motifs substantially independent of the surrounding amino acid context in which the motif occurs. A novel class of motif-specific, context-independent antibodies is also provided. The invention encompasses modified motifs consisting of single modified amino acids, for example phosphotyrosine or acetylated lysine, as well other modified motifs of multiple amino acids, such as kinase consensus substrate motifs and protein-protein binding motifs relevant to cell signal transduction. Also provided are methods of profiling large and diverse protein populations on a genome-wide basis by utilizing the antibodies of the invention, and methods for the positive identification of cellular phosphoproteins using one or more motif-specific, context-independent antibodies of the invention coupled with protein database searching.

Abstract: A method is disclosed for the detection of a His-tagged target biomolecule while said molecule remains in the gel in which it has been separated from other constituents by electrophoresis. The method involves, while the separated His-tagged biomolecule remains in the gel, forming a chelate between the His-tagged molecule, a lanthanide metal ion which exhibits fluorescence, and a sensitizer which enhances the fluorescence of the ion. The gel is then exposed to a u.v. light source to thereby generate a fluorogenic signal indicating the presence of the chelate and, in turn, the His-tagged biomolecule. The fluorogenic signal can then be detected either by direct visualization and/or by other means, such as photographically, e.g., film or charged coupled device (CCD) imaging.

Abstract: Method and composition for detecting one or more selected polynucleotide regions in a target polynucleotide. In the method, a mixture of sequence-specific probes are reacted with the target polynucleotide under hybridization conditions, and the hybridized probes are treated to selectively modify those probes which are bound to the target polynucleotide in a base-specific manner. The resulting labeled probes include a polymer chain which imparts to each different-sequence probe, a distinctive ratio of charge/translational frictional drag, and a detectable label. The labeled probes are fractionated by electrophoresis in a non-sieving matrix, and the presence of one or more selected sequences in the target polynucleotide are detected according to the observed electrophoretic migration rates of the labeled probes in a non-sieving medium.

Abstract: An electrophoresis method, comprising simultaneously electrophoresing a test sample containing a plurality of molecules labeled with a plurality of luminescent reagents and a marker sample containing a plurality of molecules with known molecular weights labeled with the same plurality of luminescent reagents.

Abstract: A method for the indirect extraction of DNA greater than 300 kb in size from non-cultivatable organisms contained in an environmental sample is disclosed. The method comprises isolating the organisms from the sample and embedding the isolated organisms in a block of agarose where the organisms are subsequently lysed and the DNA subjected to a first alternating field electrophoresis to extract DNA fragments which are large in size and separate them from the cell debris. The first electrophoretic migration is followed by an enzymatic restriction step and additional electrophoretic migrations. The invention also encompasses DNA obtained by the method.

Abstract: Methods, compositions and kits are disclosed for determining one or more target polypeptides in a sample where the target polypeptides have undergone phosphorylation. A mixture comprising the sample and a first reagent comprising a cleavage-inducing moiety and an IMAC resin for a binding site on a target polypeptide is subjected to conditions under which binding of respective binding moieties occurs. The binding site is the result of phosphorylation activity involving the target polypeptide. The method may be employed to determine the target polypeptide itself. In another embodiment the presence and/or amount of the target polypeptide is related to the presence and/or amount and/or activity of an agent such as an enzyme involved in phosphorylation of the target polypeptide.

Abstract: The optical imaging of two-dimensional solute zone arrays in electrophoresis gels is corrected for nonuniformities in the optical system such as those arising from the light source or from light dispersion underneath the gel. The correction is achieved by the use of a reference plate that responds to a light source uniformly along its length and width by being either uniformly light absorptive or uniformly light transmissive, or by emitting light upon excitation. Thus, any nonuniformities or deviations in the image of the reference plate arise only from nonuniformities or deviations within the optical system. Analogous corrections are made in other two-dimensional assay images, such as microarrays and microtiter plates.

Abstract: The present teachings relate, among other things, to polynucleotide sequencing, fragment analysis and sample/lane tracking, and to polynucleotide sequencers and analyzers that employ optical detection techniques. Embodiments of the present teachings are described which include, for example, the addition of a calibration standard to a sequencing reaction. Information such as peak spacing and peak shape can he extracted from the standard.

Abstract: The invention relates to a method and a device for the isolation and purification of nucleic acids. According to the invention, after decomposition of a sample the nucleic acids present in said sample are isolated and purified.

Abstract: Methods, compositions and kits are disclosed for determining one or more target polypeptides in a sample where the target polypeptides have undergone a post-translational modification. A mixture comprising the sample and a first reagent comprising a cleavage-inducing moiety and a first binding agent for a binding site on a target polypeptide is subjected to conditions under which binding of respective binding moieties occurs. The binding site is the result of post-translational modification activity involving the target polypeptide. The method may be employed to determine the target polypeptide itself. In another embodiment the presence and/or amount of the target polypeptide is related to the presence and/or amount and/or activity of an agent such as an enzyme involved in the post-translational modification of the target polypeptide.

Abstract: The claims provide for detecting prions in a sample by (a) subjecting a sample to membrane-based electrophoresis to separate and/or concentrate at least some prions present in the sample; and (b) detecting and or identifying the presence of the separated and/or concentrated prions.

Abstract: A universal gel and methods of use in detecting the presence, or absence, of one or more target molecules in a test sample comprising the formation of a universal gel hybridization complex, wherein an adapter molecule is hybridized to a target molecule and universal capture probe is disclosed.

Abstract: A method for identifying a microorganism, wherein the method comprises the steps of: 1) preparing one or more kind(s) of double-stranded DNA fragments by the random PCR using at least a part of a genome of an organism of interest, 2) applying the double-stranded DNA fragments which were prepared in step 1 to temperature gradient gel electrophoresis (TGGE) or denaturant gradient gel electrophoresis (DGGE), 3) extracting identification dots of each DNA fragment from the electrophoresis pattern which was obtained in step 2, 4) determining PaSS and/or genome semi-distance from the identification dots which were obtained in step 3, and 5) analyzing the PaSS and/or genome semi-distance which were/was obtained in step 4, wherein pseudo-absolute position of identification dots is determined by the locational relation to standard DNA in the presence of standard DNA as the starting dots in TGGE or DGGE.

Abstract: Electroosmotic flow controllers and methods of fluid flow control are described. The invention uses an electroosmotically generated flow component in conjunction with a pressure driven flow component to modulate fluid flow. The devices and methods of the invention may include salt bridges for making electrical connection between a power supply and a channel filled with a porous dielectric material and a fluid. Embodiments including flow controllers and flow splitters are described as is their use in a variety of fluid handling applications.

Abstract: The preferred embodiments described herein provide an apparatus and method for intersecting a light beam and a focal point of a detector at an object of interest. In one preferred embodiment, an apparatus is provided comprising a first support element and a first member carrying a light source and a detector. The first member is pivotable between first and second positions, and when the first member is in the first position, a light beam generated by the light source and a focal point of the detector intersect at an object of interest carried by the first support element. When the first member is in the second position, the light beam and the focal point of the detector intersect at an object of interest carried by a second support element disposed on the first support element.

Abstract: The present invention relates to an electrophoretic method to effect differential net migration, the extent of said migration being dependent on molecular size, of electrically charged macromolecules through a gel support in a single dimension, which method comprises subjecting electrically charged macromolecules applied to a gel support to an electric field oriented along a single axis within the gel for a time period sufficient to effect migration in the direction of the oriented field and form a separation pattern in order of the respective molecular weights of the macromolecules in a distance of about 0.5 to about 20 millimeters, said gel support comprising about 3 to about 40 percent acrylamide, said electric field being applied in an amount of about 5 to about 100 volts per millimeter of gel support along the axis length, and said gel support having a width perpendicular to said axis of about 0.1 to 1.5 millimeters.

Abstract: The present invention provides an integrated, fully automated, high-throughput system for two-dimensional electrophoresis comprised of gel-making machines, gel processing machines, gel compositions and geometries, gel handling systems, sample preparation systems, software and methods. The system is capable of continuous operation at high-throughput to allow construction of large quantitative data sets.

Abstract: This invention provides devices and methods for the simultaneous separation and purification of molecular samples by electrophoresis. In a preferred embodiment, the separation and purification of molecular samples is provided in a two-dimensional array. In a further embodiment of the invention, gas removal passageways are provided to remove gas produced by electrodes that are immersed in aqueous solution.

Abstract: An apparatus for detecting fluorophore labelled substances in an electrophoretic separation medium comprises illuminating means (1) for illuminating the fluorophore labelled substances (8), detecting means (7) for detecting fluorescence emitted by said fluorophore labelled substances upon illumination, and illumination varying means (9) for varying the illumination of the fluorophore labelled substances (8) to vary the intensity of the fluorescence.

Abstract: The present invention provides a detection cell that comprises a body having a first end that defines a first opening, a second end that defines a second opening, and an internal surface that defines an interior cavity that fluidly connects the first opening and the second opening; wherein the interior cavity is capable of holding a support matrix for receiving a chemical species through the first opening, passing the chemical species through the interior cavity, and discharging the chemical species through the second opening; and wherein at least two portions of the body are capable of providing a first and a second guiding region each having an index of refraction less than that of the support matrix. A system utilizing the detection cell of the present invention is also provided. In another embodiment, the present invention provides a method for guiding light through a support matrix through which a chemical species is passing.

Abstract: The invention relates to methods, compositions, and systems for calibrating a fluorescent polynucleotide separation apparatus. One aspect of the invention is multiple color calibration standards and their use. A multiple color calibration standard is a mixture of at least two polynucleotides of different length, wherein each of the polynucleotides is labeled with a spectrally distinct fluorescent dye. Another aspect of the invention is to produce total emission temporal profiles of multiple color calibration standards for use in calibrating fluorescent polynucleotide separation apparatus. The peaks corresponding to the fluorescently labeled polynucleotides in the total emission temporal profile may be detected using a peak detector that is driven by changes in the slopes of the total emission temporal profile.

Abstract: A bio-separation system using an efficient, compact, portable, interchangeable, reusable, recyclable, multi-channel cartridge, has integrated pre-aligned optics and an integrated reagent reservoir. The cartridge supports, for example, multiple capillaries for CE separation. An integrated reservoir containing a separation support medium (e.g., a gel buffer) is common to all capillaries. The chemistry of the medium and the characteristics of the capillaries (e.g., capillary size, coating and length) are defined for each cartridge. Different cartridges can be easily interchanged in the bio-separation system to suit the particular sample based separation. The reservoir is coupled to an air pressure pump that pressurizes the gel reservoir to purge and fill the capillaries with buffer as the separation support medium.

Abstract: Disclosed are methods and reagents for detecting nucleotide mismatches (for example, due to sequence variances) in a nucleic acid sample involving the use of a nucleic acid probe derived from a hemizygous cell. Methods for determining the haplotype of a nucleic acid sample are also disclosed. Also disclosed are methods for producing the probe and kits containing the probe.

Abstract: A denaturation fingerprinting method (dnF) involves subjecting a nucleic acid segment of interest to bidirectional cycle sequencing using oppositely oriented primers and incorporating two different dideoxynucleotides (ddNTPs) in the sequencing reaction. The resulting fragments are separated by denaturing electrophoresis. In one embodiment, designated dnF2R, reactions and electrophoretic separation using the two ddNTPs are conducted separately. In an alternative embodiment, designated dnF1R, one of the ddNTPs has a mobility altering modification such that electorphoretic separation occurs when both ddNTPs are employed in the same reaction. The methods are useful for detecting genetic mutations.

Abstract: The present invention relates to an electrophoretic method to effect differential net migration, the extent of said migration being dependent on molecular size, of electrically charged macromolecules through a gel support in a single dimension, which method comprises subjecting electrically charged macromolecules applied to a gel support to an electric field oriented along a single axis within the gel for a time period sufficient to effect migration in the direction of the oriented field and form a separation pattern in order of the respective molecular weights of the macromolecules in a distance of about 0.5 to about 20 millimeters, said gel support comprising about 3 to about 40 percent acrylamide, said electric field being applied in an amount of about 5 to about 100 volts per millimeter of gel support along the axis length, and said gel support having a width perpendicular to said axis of about 0.1 to 1.5 millimeters.

Abstract: The present invention provides an integrated, fully automated, high-throughput system for two-dimensional electrophoresis comprised of gel-making machines, gel processing machines, gel compositions and geometries, gel handling systems, sample preparation systems, software and methods. The system is capable of continuous operation at high-throughput to allow construction of large quantitative data sets.

Abstract: The present invention provides an integrated, fully automated, high-throughput system for two-dimensional electrophoresis comprised of gel-making machines, gel processing machines, gel compositions and geometries, gel handling systems, sample preparation systems, software and methods. The system is capable of continuous operation at high-throughput to allow construction of large quantitative data sets.

Abstract: The present invention discloses a method of separating or distinguishing carbohydrate substances. More particularly the method includes using fluorescent labeling reagents which have a positive charge when bound to a carbohydrate, involves separating the labeled carbohydrate substances, such as by performing electrophoresis to cause differential migration of different labeled carbohydrate substances, or by isoelectric focusing in a pH gradient. The fluorescent labeling reagents are preferably cyanine dyes.

Abstract: Experiments were performed to test for a set of SSCP conditions that would detect virtually all mutations in a nucleic acid being analyzed. The effects of buffer, gel matrix, temperature, and additive were all tested. Dideoxy fingerprinting was used as a tool to generate a large statistical sample (about 1,500) of mutation-containing single-stranded segments in order to evaluate adequately the sensitivity under a given condition. Mutations in exons H and B/C of the factor IX gene were utilized. SSCP sensitivity, as conveniently measured by the average SSCP efficiency, varied with conditions. Correlation coefficients (R) identified pairs of conditions that were either close to independent or complementary. Five conditions were selected with sufficient redundancy to detect all the mutations in the set tested. The sensitivity of multi-conditional SSCP (SSCP5) was determined by blinded analyses on samples containing mutations in all the eight exon regions in the factor IX gene.

Abstract: A molecular recognition layer 21 is formed on a sensor face 6a of a chemical CCD 1 having such a structure that a plurality of potential wells 6 constituted to change a depth corresponding to a chemical quantity are arranged two-dimensionally, electric charges are injected into the potential wells 6, and the chemical quantity is converted into an electric charge corresponding to the sizes of the potential wells. Thus, it is provided a novel and useful molecular recognition type chemical CCD capable of measuring an ultramicro chemical substance with an ultrahigh sensitivity at a molecular level.

Abstract: The present invention provides an integrated, fully automated, high-throughput system for two-dimensional electrophoresis comprised of gel-making machines, gel processing machines, gel compositions and geometries, gel handling systems, sample preparation systems, software and methods. The system is capable of continuous operation at high-throughput to allow construction of large quantitative data sets.

Abstract: A proteome analyzer includes a separation cassette having a first dimension separation compartment for separation of protein samples by isoelectric focusing and a second dimension separation compartment for separation of protein samples by SDS-polymer network electrophoresis. The first dimension compartment is a reservoir in which a porous material having capillary channels is disposed. The protein samples are disposed in the capillary channels and, in the presence of a pH gradient, are focused spatially by isolectric focusing upon application of an electric field. The second dimension compartment consists of two glass or plastic plates separated by a separation medium. The separation medium is an ultra-thin layer of a low concentration linear polymer supported by an inert matrix. The spatially focused protein samples are contacted with the separation medium in the presence of an electric field to initiate second dimension separation.

Abstract: An auto allele caller executed in a computer system for identifying alleles from a trace is described. The auto allele caller applies a typical shape of an allele for a marker to the trace to identify potential allele calls that match to the typical shape of the allele at the marker and assigns a quality factor to the allele calls.

Abstract: The present invention provides an integrated, fully automated, high-throughput system for two-dimensional electrophoresis comprised of gel-making machines, gel processing machines, gel compositions and geometries, gel handling systems, sample preparation systems, software and methods. The system is capable of continuous operation at high-throughput to allow construction of large quantitative data sets.