WO2001044812A1 - Method for the simultaneous analysis and detection of multiple analytes by micro identification - Google Patents

Abstract

The present invention provides a micro system of identification of micro particles or beads. The identification tag is added to, or written onto, a uniformly manufactured particle or bead during the addition of a specific affinity ligand. The affinity ligand is reactive with a particular analyte or ligand of interest which might be present in a sample. Processes are provided for the preparation of the micro particle bead reagent sets as are various methods for their use including DNA sequencing, identification of single nucleotide polymorphisms, qualitation or quantitation of multiple analytes for various diagnostic purposes.

Description

METHOD FOR THE SIMULTANEOUS ANALYSIS AND DETECTION OF MULTIPLE ANALYTES BY MICRO IDENTIFICATION

BACKGROUND OF THE INVENTION

Recent advances in technology utilized in biomedical research have provided a vast amount of new information. In particular, new DNA and amino acid sequencing methods have provided information regarding genetic mutations which relate to various genetically based disease states. Consequently, efficient use of this information requires the ability to analyze multiple analytes in a single biological sample. Each sample must provide as much information as possible in order to reduce the number of samples which must be taken from a patient and to reduce the cost of conducting the various tests required.

Current technologies related to the analysis of multiple analytes in a single sample remain limited in the number of analytes which can be measured, or are unmanageable in terms of sample collection, sample classification, and the analysis of sample data. For example, in one assay micro-spheres of different sizes are used as supports and the identification of micro spheres associated with different analytes was based on distinguishing micro sphere size. (McHugh et al. in, Clinical Flow Cytometry, Bauer et al., eds., Williams and Williams, Baltimore, MD, pgs. 535-544). Also, certain prior assays merely replace a single enzyme linked immunoassay procedure with a flow cytometer-based assay. These methods were based on only the few characteristics of the particles under analysis and enabled simultaneous determination of very few analytes in the assay.

There are many ways micro-particles or -beads can be identified. Micro- beads can be colored in the manufacturing process, used to attach ligands, and analyzed for binding. (U.S. patent 5,736,330 and WO 97/14028). However, the colored beads must be manufactured, stored and handled separately prior to the attachment and assignment of a ligand. This can be particularly cost prohibitive and complicated when large numbers of different colored beads are needed. This would be the case where thousands, hundreds of thousands, or millions of beads are needed, as in the analysis of genetic mutations and for oligonucleotide sequencing.

One method used to overcome these limitations is the use of micro-arrays on a single solid support. The array is a linear or two dimensional array of discrete regions, each having a finite area, formed on the surface of a solid support. The method involves linking or immobilizing a particular known reagent at each of a number of selected array positions on the solid support. Each selected position contains a ligand for a particular analyte. The array is exposed to a sample to be tested and each selected position of the array is analyzed for an interaction between the ligand (probe) and analyte (target).

The present invention provides an assay with greater flexibility that can be adapted to more applications than the prior technology.

SUMMARY OF THE INVENTION The present invention provides a process for preparing a micro-particle reagent assemblage capable of simultaneously quantitating a plurality of analytes in a single sample. The method provides for the immobilizing of a first discrete, specific micro- identification tag and a first affinity ligand onto the surface of a set of micro-particles to form a micro-particle subset. The first affinity ligand is specific for a first analyte believed to be present in the sample to be tested. Additional affinity ligands are immobilized onto the surface of other micro-particles to form a plurality of micro-particle subsets which are combined to form a micro-particle bead reagent assemblage wherein each micro-particle subset and therefore the affinity ligand which has been immobilized on that micro-particle subset can be identified by the micro-identification tag immobilized onto the micro-particle subset. The individual particles within each set of micro -particles are identical and each bears a micro-identification tag that correspond 1 : 1 with an analyte-specific ligand also born by the micro-particle. Therefor, each set micro-particle set is uniquely identified for its capacity to bind a specific analyte.

In one particular embodiment of the present invention the micro-particle is a micro-sphere or a micro-cylinder. Several types of solid support materials are suitable for use as the micro-bead or micro-cylinder, including polyacrylate, polystyrene, polysaccharide, nitrocellulose, or a metallic colloid. Typically, the micro-particle will measure from about 1 to about 20 μm in diameter or axial dimension. The micro-tag can be incorporated within or immobilized to the surface of the particle and comprise molecules or sub-micron particles having a quantitative physical attribute that can be made to assume a number of discrete values. Typically, the micro-tag can comprise one or more sub-micro- particles, a fluorochrome, a chromophore, or a micro-tag. As an example, fluorescent molecules attached to the micro-particle can collectively give the micro-particle a discrete fluorescent profile. Examples of fluorescent molecules suitable for use in the present invention, include fluorescein, Nile Red, Texas Red, lissamine or phycoerythrin.

Immobilization of the micro-identification tag and the affinity ligand to a micro-particle to from a reagent bead set can be carried out either simultaneously or sequentially. In a preferred embodiment of the invention the chemistry to immobilize the micro-identification tag and affinity ligand is carried out simultaneously to result in a reagent bead set having a specific affinity ligand immobilized on a micro-bead which can be correlated with the identified micro-tag during sample analysis.

Affinity ligands suitable for use in the present invention include molecules that have high and specific affinities for a target analyte. Preferred ligands include, nucleic acids, oligonucleotides, polynucleotides, peptide nucleic acids, peptides, proteins, and fragments thereof, carbohydrates, fatty acids, other small molecules, i.e., enzyme substrates and products, and the like. Particularly preferred nucleic acids, oligonucleotides, and polynucleotides include RNA and DNA. Proteins of particular interest as affinity ligands can include antibodies, antigen binding fragments and derivatives thereof. Antibody derivatives are recombinant antigen binding molecules which comprise the antigen binding region of an antibody, i.e., a single chain antibody, chimeric antibody or a recombinant molecule which comprises a complementarity determining region.

The present invention further comprises a method for the simultaneous quantitation of multiple distinct analytes in a sample. The assay comprises the steps of admixing a sample suspected of containing one or more of the analytes with a detectable label and with a micro-particle reagent comprising a plurality of bead sets. Each bead set comprises a distinct micro-identification tag and a distinct affinity ligand immobilized on a micro-particle. The micro-particle bead reagent is incubated with the sample under conditions conducive to the formation of a complex between each distinct affinity ligand and its target analyte which can be present in the sample for a time period sufficient for complex formation. The presence of an analyte in the sample is determined by correlating the formation of an analyte/ligand complex and quantity of labeled analyte with the micro- identification tag associated with the analyte on the micro-particle. In a preferred embodiment, the micro-particles are micro-spheres comprising a detectable fluorescent micro-identification tag, the analytes are labeled with a soluble fluorescent molecule. After incubation, the microsperes with bound fluorescently labeled analyte are analyzed by a flow cytometer. The cytometer records the fluorescent identification of each microparticle and the amount of the corresponding bound analyte fluorescent label. Normalization of the cytometer data with standard curves from a calibrated microspere set provides a table of concentrations and coefficients of variation for each analyte.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 depicts an example absorbance arising from two spectral segments for fluorescent microsphere identification. Six intensities are consistently represented from spectral segment 2. Ten intensities are consistently represented from spectral segment 1. Fig. 2 depicts the fluorescent spectra of current and anticipated fluorophores.

( ~ ) : Excitation spectra. ( ~ ) : Emission spectra. ( I ): laser line for excitation. ( | | ): Spectral segment for detection.

Figures 3 A through 3C depict three example processes of the present invention. Figure 3 A depicts bead set fabrication. Figure 3B depicts Analyte labeling with repart fluorophore and binding to microsphere set. Figure 3C depicts the analysis of the

Figures 4A and 4B provide example configurations for cytometry hardware amenable the analyze samples using an embodiment of the present invention. Figure 4A depicts a diagrammatic layout of the optics of a cytometry hardware configuration. The block diagram shows the layout of the 11 detectors on a modified FACSarPlus bench (Becton Dickinson). Figure 4B depicts a layout for a cytometry system for use in the present invention. The system is a hybrid consisting of a FACStarPlus bench, Cytomation electronics, and other electronics and computer systems. The Cytomation electronics is used to amplify (log or linear mode), evaluate, and digitize the signals from the PMTs.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention provides a Micro Identification System (MIDS) wherein a detectable identification tag can be added or written onto or into a particle or bead in the process of adding an affinity ligand specific for the analyte to be detected or quantitated. Labeling of the particle or bead in the process provides a correspondence between the detectable identification tag and the identity of the ligand. The number of reactions required for the addition of the identification tag and affinity ligand can vary between one and many millions. However, once the particle or bead set is made and the correspondence between the detectable identification tag and an affinity ligand is recorded, the bead sets can be mixed together to form a bead assemblage or reagent bead set for use in an assay. The individual bead sets therefore do not require separate storage and handling prior to mixing and use as an assemblage.

Generally, the bead assemblage set is used by coincubating the bead assemblage with a fluid unknown under conditions conducive to analyte binding to specific bead-bound receptor (ligand). To determine and record the amount of analyte interacting with a bead, each bead is individually analyzed for the amplitude of the label signal associated with the receptor and for the specific identification tag of the bead. Computational analysis, i.e., by an accounting software, provides a correspondence between the identification tag of the bead and the intensity of the analyte label.

Micro-particles can be fabricated in sizes ranging from 50 nonometers top 100 micrometers. One particular example of a micro-particle for use in the present invention comprises micro-beads, which are typically sized between about 5 μm and about 15 μm, preferably between about 5 μm and about 10 μm. Commonly micro-particles or micro- beads comprise polyacrylate, polystyrene, polyacrylamide, polysaccharide, metallic colloids, and the like. The surface of the micro-particle or -bead can be derivatized with a functional group for conjugating or covalently binding the affinity ligand or micro-identification tag to the surface of the particle. Commonly used functional groups include, but are not limited to, hydroxyl, thiol, aldehyde, or carboxylic acid groups. Typical coupling reagents for attaching an affinity ligand to a micro-particle or micro-bead include, but are not limited to, carbodiimides, glutaraldehyde, and the like.

In one particular embodiment of the present invention, the micro-particle or - bead can be written and read much the same way a computer compact disc. In this embodiment, a bead set would be assigned a detectable identification tag which is written onto the beads as they are being dispensed into a reaction vessel to add a specific ligand. Once a number of bead sets have been made they can be mixed together to form a bead assemblage. The bead assemblage can be contacted with a sample to be analyzed and labeled receptor. After an incubation period sufficient for association of labeled ligand with a specific bead tag, analysis would be performed to register amplitude of associated label and identification tag of the particle. The identification tag can be a digital signal of Os and Is or a more complex four or six bit code. The particle can be a micro-cylinder with locations to write the code. The cylinder would pass through the dispensing machine such that the positioning of the cylinder would allow for the writing of the identification tag. Likewise, the final analysis of the particle would comprise reading the identification tag in a similar manner.

In a further embodiment, a micro-particle or -bead can be identified by attaching a tag to the bead. For example, a detectable micro-fabricated tag can be chemically linked to the bead prior to, or after the linking of the affinity ligand to the bead. The analysis would comprise, the reading of the micro-identification tag, such as for example, by flow analysis, which would orient the tag to be read.

In a modification to the micro-fabricated tag, small particles or beads can be sequentially added to the micro-particle to encode the identification tag. The beads used to fabricate the identification tag would be smaller than the micro-particle. As an example, if the micro-particle is 5 μm the beads used to form the micro-fabricated identification tag would be equal to, or less than, 1 μm in size. In a particular example, the fabricated micro- tag beads can be white or black in color added sequentially in a digital like pattern to form a detectable code on the bead. In another example, the code pattern can be sequentially added beads of, for example, four colors to mimic four bit information. The micro-fabricated identification tag can be added to the ends of the ligands on the beads or can be attached directly to the bead via their own anchoring tether. As in the prior embodiment, flow orientation of the bead through a detector would allow for the reading of the micro- identification code during analysis. In a particular embodiment of the present invention, the micro-particles are micro-spheres in which descrite fluorescent tags provide identification as well as quantitation of specific anlaytes. The fluorescent microsphere as envisioned in this embodiment provides a unique combination of fluorescent intensities in distinct segments of the visible and infrared spectrum. The number of identifications achievable is the combinatorial product of the number of intensities that can be resolved within each segment. An example emerging from two spectral segments having ten and six available inensities (respectively) appears in Figure 1.

Where the number of intensities is a constant among all spectral segments, the number of achievable identifications is equal to the number of intensities raised to the power of the number of spectral segments. Table 1 provides an example of the number of achievable identifications in a matrix of two dozen test cases. In practice, the number of intensities is variable among spectral segments, but the mean of the number of intensities are represented in Table 1. The resulting number of identifications range from tens to millions, and is equal to the number of analytes that can be analyzed simultaneously by the methods of the present invention. The large number of identifications possible make the methods described herein applicable in a broad range of contexts including analysis of genetic, clinical, microbial and environmental samples.

TABLE 1

Matrix of Numbers of Achievable Fluorescence Identification

Number of Number of Number of Unique Spectral Segments Intensities/Segment Fluorescent Identifications

6 3 729

6 4 4096

6 5 15625

6 6 46656

7 3 2187

7 4 16384

7 5 78125

7 6 279936

8 3 6561

8 4 65536

8 5 390625

8 6 1679616

9 3 19683

9 4 262144

9 5 1953125

9 6 10077696

10 3 59049

10 4 1048576

10 5 9765625

10 6 60466176

The identification of fluorescent labeled microspheres emerges from a mixture of fluorophores covalently attached to the microsphere surface. The individual fluorophores are either small organic molecules, proteins, or excimer-loaded particles. Small molecules or proteins currently used in fluorescent assay methods include, for example, fluorescein, phycoerythrin (PE), Cy5PE, Cy7PE, Texas Red, allophycocyanin (APC), Cy7APC, Cascade Blue, Cascade Yellow, and the like. (See Table 2 and Figure 2). Additional flurophores useful in the methods of the present invention will likely be discovered and developed to include fluorophores (and excimer-loaded particles) with currently unrepresented Stokes-shift profiles. Examples of Stokes profiles include absorbances in the far red and emission in the infrared, and absorbance in the violet and emission in orange or red (See, Figure 2). Therefore, the number of flurophores available for use in the present invention is ultimately about twelve. All but one of the available fluorophores can be used for microsphere identification; the one remaining must be a dedicated "report fluorophore" - - to report the level of analyte present in a sample unknown, as described more fully below.

TABLE 2

Laser Assignments and Molecular Profiles of Known Fluorophores

Assigned Laser Fluorophore Fluorophore Molecular

Fluorophore and Wavelength Molecular Profile Weight (daltons)

Cascade Blue Krypton 407/413 small organic molecule 666 (cadaverine derivative) Cascade yellow Krypton 407/413 small organic molecule 563 (succinimidyl ester derivative)

Fluorescein and analogs Argon 488/514 small organic molecule 389 (thiocyanate derivative) phycoerythrin Argon 488/514 protein 240000

PECy5 Argon 488/514 protein/tanden dye conjugate 242000

PECy7 Argon 488/514 protein tanden dye conjugate 242000

Texas Red He/Ne 633 small organic molecule 691 (cadaverine derivative)

APC He/Ne 633 protein 104000

Cy7APC He/Ne 633 protein/tanden dye conjugate 106000

Any analyte that can be labeled with a fluorophore (the report fluorophore) and bound specifically to a microsphere-bound ligand (the microsphere ligand) can be included in multiplex analysis by the method of the present invention. An analyte can be labeled either directly (e.g., through transcription with labeled nucleotides in the case of nucleic acids), or indirectly through binding to a labeled ligand (the "fluorophore ligand") (Figure 3), examples of which include labeled antibodies and oligonucleotides). The labeled analyte binds to its specific microsphere ligand, and provides report fluorescence to the microsphere. In all cases, analyte labeling must not interfere with analyte binding to the microsphere ligand. In the case of indirect analyte labeling, the report fluorophore and the microsphere ligands must be noncompetitive with one another for binding to the analyte.

Because small fluorophores are lease likely to influence the physio-chemical behavior of analytes and ligands, preferred report fluorophores are those with molecular weights of less than 1000 daltons (See, for examples, Table 2).

The present invention can comprise three distinct processes. The first process comprises microsphere set fabrication wherein microspheres are concurrently assigned

(through surface coupling) both a fluorescent identifier and a ligand. The correspondence between each ligand and each fluorescence identifier is one to one. A microsphere "set" comprises a plurality of identical microspheres whose fluorescence identifier and ligand are distinct from those of any other microsphere "set." Further, the microsphere set is "calibrated" by correlation to a reference standard of known analyte concentration. For example, a binding assay can be used to determine the concentration of analyte comprising a microsphere set by comparison to a standard curve determined using a number of samples with known concentrations of the analyte.

Following fabrication and calibration of a plurality of microsphere sets, a microsphere "assemblage" is formed by combining any number of microsphere sets. In one particular embodiment of the present invention it is only necessary to calibrate approximately 10% of the microsphere sets within a microsphere assemblage, so that raw data provided by the uncalibrated sets in the assemblage can be normalized by reference to the calibration curves of the calibrated microsphere sets.

The second process comprises two steps: (i) analyte labeling in the sample fluid unknown, and (ii) contacting the sample with the microsphere assemblage under conditions conducive to binding of the analytes to ligand immobilized on the microspheres for a sufficient time period. When the analyte is indirectly labeled, the two steps can be carried out concurrently. When the analyte is directly labeled, labeling of the analyte precedes the contacting step.

The third process comprises analyzing the microsphere assemblage (now comprising bound fluoropore-labeld analytes), to determine the level of analyte (report fluorescence). In one particular embodiment of the present invention analysis can be accomplished by, for example, flow cytometry and the like. Levels of a report fluorescence are first assigned to each fluorescent identification and provide estimates of frequency of each analyte in the entire analyte population. Figure 3C depicts an example of report fluorescence where the fluorescence identification emerges solely from two spectral segments. These frequencies are then converted to concentrations of analytes of interest through normalization with the calibrated curves of the calibrated micro-sphere sets within the reagent bead assemblage. A table of absolute concentrations of each analyte of interest results. In another example, the methods of the present invention can be used for

DNA sequencing, single nucleotide polymorphism identification, and other diagnostic uses.

These methods can comprise the synthesis or attachment of a specific nucleic acid, polynucleotide, or oligonucleotide to a bead set. When the oligonucleotide is synthesized on the surface of the bead, a subset of the growing polynucleotide chains can be extended with, for example, a series of small particles or beads. The particles can be, for example, of different colors arranged so as to impart the sequence of the oligonucleotide attached to the bead. Only a limited number of the oligonucleotides on a bead are labeled by extension because it is likely the interaction between the ligand and an analyte could be inhibited should all of the oligonucleotides comprise a label. Use of four different colored beads for oligonucleotide extension can provide a label comprising the same number of beads as the number of nucleotides in the oligonucleotide probe attached to the bead.

Detection of the micro-identification tag can be by an optical device. Typically, the optical device can be a laser capable of detecting both the micro-identification tag and the label used to detect the ligand of interest. The MIDS technology, as described herein, has many applications. For example, in one embodiment of the present invention, the ligand attached to the beads can be a nucleic acid, e.g., RNA, mRNA, cDNA, or genomic DNA, and the like. The bead reagent sets of the present invention prepared with immobilized nucleic acid sequences can be used for large scale hybridization assays in numerous genetic applications, including genetic and physical mapping of a genome, monitoring gene expression, DNA sequencing, genetic diagnosis, genotyping of organisms, and the like.

For gene mapping, a gene or cloned DNA fragment can be hybridized to a pooled bead reagent set, and the identity of the DNA elements applied to the bead reagent set is unambiguously established by the identification of the bead subsets of the pooled bead reagent set which are detected. One application of the present invention for creating a genetic map is described by Nelson et al. (Nature Genetics 4:11-18 (1993)). In constructing a physical map of the genome, reagent bead assemblage sets of immobilized cloned DNA fragments are hybridized with other cloned DNA fragments to establish whether the cloned fragments in the probe mixture overlap and are therefore contiguous to the immobilized clones of the bead assemblage.

The reagent bead assemblage containing immobilized DNA fragments can also be used for genetic diagnosis. To illustrate, a bead assemblage comprising multiple forms of a mutated gene or genes can be probed with a labeled mixture of a patient's DNA. The patient's labeled DNA will preferentially interact with only one of the immobilized versions of the gene. Detection of this interaction and correlation with the specific gene mutation can lead to a medical diagnosis.

In a particular example of genetic diagnosis using MIDS, a ligand, which is an oligonucleotide complementary to normal and to an abnormal genotype, is attached to bead sets. Nucleic acid material is obtained from patients, labeled, and hybridized to the pooled bead reagent. Hybridization to oligonucleotides of normal genotypes infers normal phenotypes and hybridization to oligonucleotides of abnormal genotypes infers abnormal phenotypes. In a more complex embodiment of genetic diagnosis using MIDS, a complete

DNA sequence of a gene or gene set implicated in a normal or disease state is determined. This allows for not only the assessment of the presence or absence of known disease causing mutations, but also the accumulation of information about additional allelic differences which may be correlated with a specific disease state. Implementation of a fully developed MIDS technology can provide for the determination of the complete expressed genomic sequence of an individual in an over night analysis. Such a complete set of genetic information for an individual would allow the delineation of all known disease states and disease propensities of an individual.

As a particular example, in the genetic disease Porphyria, a number of the genes responsible for the condition are known, as are many of the mutations in the nucleotide sequences of the genes. Micro-bead sets with each oligonucleotide sequence of all of the heme pathway enzymes each having a particular micro-identification tag, covering the gene sequences in a nested manner, can be made. Bead sets will be fabricated for oligonucleotides representing all known porphyrin mutants and wild type sequences. The bead sets are mixed together to form the reagent bead set or microsphere assemblage. The genomic DNA from a patient sample would be isolated and the DNA of the heme pathway enzymes amplified by PCR. A fluorescent nucleotide can be added to the PCR reactions to provide a labeled amplified PCR product representing the DNA of the patient. The fluorescent labeled PCR product would be hybridized to the reagent bead set, washed, and analyzed by a fluorescent assay for the amount of fluorescent labeled DNA and also detection of the micro-identification tag. An analysis system would correlate an identified micro-identification tag with the oligonucleotide sequence which was immobilized on the bead surface, and therefrom the genetic basis for the disease determined. Microsphere assemblages of immobilized DNA fragments can also be used in

DNA probe diagnostics. For example, the identity of a pathogenic microorganism can be established unambiguously by hybridizing a sample of the unknown pathogen's DNA to a reagent bead assemblage comprising bead sets containing many types of known pathogenic DNA. A similar technique can also be used for unambiguous genotyping any organism. The bead reagent assemblage can be used to determine the concentrations of all the analytes, i.e., proteins, or the like, known to be normally present in a patient specimen. This assay can provide information similar to that of an ELISA analysis, but rather than information for one or a few proteins it will allow for multiplex analysis of tens to millions of biological molecules. In yet another embodiment of the present invention, the MIDS technology can be used for DNA sequence analysis. In this embodiment, the ligand added to a bead set is an oligonucleotide representing each of the sequence permutations for a given length of oligonucleotide normally found in a sample to be tested. Each bead set will have a unique oligonucleotide and an associated distinct micro-identification tag. When all the bead sets are completed, they are mixed together to form the bead reagent assemblage. To utilize the MIDS bead reagent assemblage for DNA sequence analysis, nucleic acid to be sequenced is labeled. The labeled nucleic acid is hybridized to the bead reagent. Individual beads are analyzed for amplitude of hybridization signal and for bead identification. For specific methods, see for example, Bains et al., J. Theor. Biol. 135:303-307 (1988), Drmanac, et al., Genomics 4:114-128 (1989), Drmanac et al., Science 260:1649-1652 (1993) and Southern et al., Genomics 13:1008-1017 (1992); each incorporated herein by reference in their entirety.) Computational analysis comprises, determining a correlation between positive hybridization and sequence of an oligonucleotide associated with a distinct micro- identification tag. Starting from one such oligonucleotide sequence the computer searches for four other oligonucleotides with a sequence shift of one nucleotide and containing the four different possibilities for the nucleotide position extending one nucleotide from the first oligonucleotide. Assessment is performed to determine which of these four oligonucleotides shows positive hybridization. The one that is positive provides the sequence of the position plus one from the position of the original oligonucleotide. This computational analysis is repeated until the entire sequence of the nucleic acid under analysis is completed.

In another embodiment of the present invention the MIDS technology is applied to chemical libraries. The high through-put screening of a chemical library is the first step in the identification of a lead compound for small molecule therapeutics, veterinary medicines, and food crop effectors. In this case an organic molecule is synthesized on or attached to a micro-particle or -bead set and designated with a specific distinct micro- identification tag. After preparing bead sets comprising a distinct micro-identification tag for each compounds of a library, the bead sets are mixed to form a bead reagent assemblage. The bead reagent assemblage is co incubated with a labeled target biological molecule.

Individual beads are analyzed, the beads with associated labeled target biological molecule are scored, and the identity of the compound on the bead is determined by cross referencing the bead micro-identification tag stored in the data base with the compound composition. In yet another embodiment, MIDS can be used to identify any object of manufacture or production as a means of tracking the origin and identity of the object. Each production of the object run can be assigned a specific unique micro-identification tag containing a specific identifier for the producer and the production run. The micro- identification tag would be unobservable to visual inspection, but easily detected by an MIDS assay system. This method would be particularly useful for objects of manufacture or production where diversion, relabeling, pirating, theft, authenticity, illegal disposal, or environmental contamination are a concern. The expense per user or per production run would have to be low but the potential applications are vast.

In a specific example of this embodiment of the present embodiment, MIDs can be used in the fuel industry, wherein each producer can be assigned a distinct micro- identification tag. Each production run of product by a producer would contain the assigned unique identification tag. Theft or diversion of fuel could be easily determined by testing a sample of fuel for the micro-identification tag. Also, the identity of a manufacturer fuel which has been spilled in an environmental accident can be made so that the proper party responsible for clean-up can be assured. In another example, a part used to manufacture a final product, such as an automobile part, can be provided a unique micro-identification tag indicating not only the producer of the particular part, but also the manufacturer of the automobile, and to specify the particular vehicle. The tracing and validation of, not only the automobile, but also each part of the completed automobile would be feasible using MIDS. The following examples are offered by way of illustration, not by way of limitation.

Example 1

In the following example a concerted process utilizing carboxylase-modified microspheres and concurrent assignment of a fluorescent identifier and a microsphere ligand for the detection of a fluorophore labeled analyte is described.

I. Set fabrication

Carboxylate coated microspheres (6 μm) were covalently modified with a cocktail of identification fluorophores (except FITC) and microsphere ligands through carbodiimide conjugation chemistry. Briefly, 2.0 x 10⁷ microspheres (100 μl; Polysciences) were first washed twice with 0.1 M Na CO₃ utilizing filtration with a 0.45 μm pore size centrifugal device (Pall-Gelman), then washed twice with reaction buffer (0.1 M MES (morpholinoethane-sulfonate); 0.15 M NaCl, pH 4.7). Microsphere carboxylates were converted to N-hydroxysuccinimidyl esters by incubating the beads for 15 min at room temperature (with mixing) in one ml of reaction buffer with 2 mM (l-ethyl-3-(3- dimethylamino-propyl)-carbodiirnide hydrochloride (EDC)) and 5 mM sulfo-NHS (N- hydroxysulfo-succinimide). Both soluble reactants were introduced as solids immediately before the reaction.

The EDC was quenched by the addition of β-mercaptoethanol to a final concentration of 20 mM. The reaction buffer was then replaced and the microspheres were concentrated by filtration and re-suspension in 100 μl of reaction buffer. Beads (4 x 10⁵ for each microsphere set) were suspended in 100 μl reaction buffer with a specific distinct cocktail of identification fluorophores and a specific distinct ligand, and incubated at room temperature for two hours with mixing. The concentration in the cocktail of both the identification fluorophores and the microsphere ligand were pre-established for each microsphere set so as to maximize both identification resolvability and dynamic report range. To normalize report fluorescence among microsphere sets, a ligand to a reference analyte was included in the cocktail for all microsphere sets to permit comparing microsphere ligand availability among the sets. The microspheres were then washed twice with storage buffer (1 X PBS; 1% bovine serum albumin; 0.05% sodium azide; 0.1% Tween 20) and suspended in 500 μl storage buffer. The cytometric characteristics of each fabricated set were verified before combination into reagent bead assemblages.

If non-protein fluorophores and ligands were used, an aliphatic carbon spacer was added by means of a reactive amine-, hydrazine- or hydrazide- derivatives to maximize availability of the reactive moiety for carbodiimide-mediated conjugation.

II. Labeling of analytes in the unknown and binding to the microsphere assemblage Two separate applications of the MIDS assay are described herein. One application is to a solution of nucleic acid analytes, and in the other is to a solution of protein analytes.

A. Nucleic acid analyte unknown:

Direct labeling of the nucleic acid unknown was first achieved by transcription (e.g., PCR, "recessed 3' fill in" and "random priming") or by modification (e.g., enzymatic 5' end modification or chemical cross-linkage) to incorporate fluoroscein isothiocyanate (FITC) into unknown nucleic acid strands which bind specifically to the microsphere-ligands. In the case of indirect labeling, fluorophore ligands were prepared as FITC-conjugated oligonucleotide 20-mers complementary to nucleic acids in the unknown. The nucleic acid unknown (directly labeled, or if indirectly labeled, with FITC-conjugated oligonucleotides as follows), FITC-conjugated oligonucleotides (for indirect labeling, each at a final concentration of 0.1 mM) and 10 μg fragmented salmon sperm DNA were combined in 30 μl of hybridization buffer and denatured at 100°C for ten minutes. (Hybridization buffer constitutes 2.25 M tetramethyl ammonium chloride; 0.1% (v/v) Tween 20; 40 mM Tris-Cl, pH 8.0; 2 mM EDTA). After cooling to 37°C, 30 μl of microsphere assemblage (pre-sonicated for three minutes) containing 1 x 10 microspheres of each microsphere set were combined with the nucleic acid cocktail and incubated at 37°C for 30 minutes with continuous mixing. Four hundred μl of 37°C hybridization buffer was added, sonication was repeated and the suspension immediately analyzed by cytometry.

B. Protein analyte unknowns:

FITC-conjugated analyte-specific antibodies and the microsphere assemblage were first combined so that each immobilized ligand was present at about 10 μg/ml, and 1 x 10³ microspheres of each microsphere set were present in a volume of 50 μl in binding buffer (Ix PBS; 1% (w/v) BSA; 0.1% Tween 20). The suspension was sonicated for 3 minutes and 50 μl of unknown protein solution in physiological saline was added and incubated at room temperature with continuous mixing for 30 minutes. Binding buffer

(400μl) was added, sonicated and cytometry was immediately performed.

III. Cytometric analysis

The cytometer used for analysis of the microsphere assemblage combined a modified FACstarPlus optical bench (Becton Dickinson) with MoFlo (Cytomation) electronics and custom interfaces both between these two components and with the user workstation (Figures 4 A and 4B). The software was a combination of FloJo (Tree Star, Inc.) and a custom developed package designed to manage the accounting of reported identification fluorescence. Laser excitation in three parallel non-colinear beams was provided by an argon ion laser, part of whose output was directed to a dye head tuned to 595 nm, and by a krypton laser.

Before assay analysis, the cytometer was first optimized for signal stability and for dynamic range using singly-stained microspheres. Next the cytometer was compensated by an analog algorithm for same-laser spectral overlaps, and finally run again with singly-stained microspheres to generate a digital compensation matrix for use after data collection.

Assay suspensions were analyzed in their entirety, and data was collected un- gated. Collected data was digitally compensated before gating, first by forward versus side scatter to exclude microsphere aggregates, and then to exclude all events that lie outside of predetermined fluorescent identification regions. A report fluorescence, with coefficient of variation, was assigned to each fluorescent identification by the software; each assignment was based upon at least one hundred events. Report fluorescence signals were then converted to absolute concentrations by normalization with calibration curves of standard microsphere sets. Final data analysis was provided as a table of relative frequencies, absolute concentrations, and coefficients of variation for each analyte measured.

Example 2 The present example provides a description of various methods which can be used to immobilize nucleic acids to polystyrene micro-beads.

The covalent attachment of nucleic acids to polystyrene beads can be achieved with a number of methods and several possible chemistries. There are two central considerations in the development of beads modified by the addition of nucleic acids. First, the linkage between the nucleic acid and the bead can be designed such that the beads and the nucleic acids are modified with a fluorophore simultaneously, consecutively, or in small alternating batches. Secondly, the chemistry of the linkage between the nucleic acid and the bead, and the chemistry of the linkage between the fluorophore and the bead can be identical, modified, or entirely different.

Time of reaction to form the linkage, when defined in the context of the physical manipulations required of the beads, can be a major factor to select the type of linkage desired. Inceptive beads obtained from a manufacturer represent the starting material for all linkage chemistries. The beads can be activated to specifically react with a distinct chemically active group. Thus, activated beads that can form a permanent linkage with any molecule containing, for example, a reactive amine, can be used to attach fluorophores or nucleic acids containing amine groups. The time of reaction to form the linkage is important in defining the topography or landscape of the final bead product. Linking four distinct fluorophores to activated beads in the absence of any other molecule resulted in a bead topography best described as a coating of randomly attached fluorophores. If both fluorophores and nucleic acids were attached simultaneously, then the topography of coated fluorophores were studded with attached nucleic acids.

Therefore, to obtain a random distribution of fluorophores and nucleic acids coating the surface of the beads, the linkages can be performed simultaneously using the same chemistry, or sequentially using different chemistries, each for an activated group randomly generated on the surface of the bead. The preferred method of attachment is to simultaneously attach both the fluorophores and the nucleic acids to the beads.

The second consideration in the attachment of nucleic acids to polystyrene beads is the linkage chemistry itself. One method which provides an easily manageable, strong linkage comprises avidin-biotin conjugation, wherein avidin containing moieties were attached to the beads and biotin was attached to the nucleic acid. Subsequent incubation of the beads and nucleic acids produced a bead-nucleic acid linkage. Biotin derivatized nucleic acids can be prepared during the synthesis of oligonucleic acids, or biotin can be added to a nucleic acid by various methods as described herein below. Alternatively, other linkage chemistries are available that generally provide permanent covalent attachments. For example, one of the first methods developed for immobilizing DNA to a solid support comprises the covalent attachment of DNA to a cyanogen bromide (CNBr) activated support and subsequent incubation with the nucleic acid. However, this method is prone to provide a linkage which lacks long term stability of the covalent linkage to the support (an inherent instability issue of the cyanate ester/amine linkage) and furthermore results in random linkage sites on the nucleic acid. Thus, a single nucleic acid can attach at more than one site, potentially interfering with subsequent manipulations of the immobilized nucleic acid, such as, for example, hybridization. An alternative method comprises linking the nucleic acid to a solid support by immobilization of a periodate prepared aldehyde of the nucleic acid to a hydrazide modified support. While the chemistry is different than the CNBr approach, the method can still provide linkage through inconsistent positions within the nucleic acid. A third linkage chemistry comprises carbodiimide conjugation which was briefly described in Example 1. Carbodiimide conjugation provides a covalent bond between the fluorophore and the bead as well as permanent covalent linkage between a nucleic acid and the beads. It is preferred that modified nucleotides be included in the nucleic acid at defined positions to ensure reproducible, defined attachment of a nucleic acid to a bead.

Two forms of nucleic acid can be immobilized on the solid support. Single stranded oligonucleotides represent one form. Typically, single stranded oligonucleotides are synthesized and can be modified at defined nucleotide positions. The modifications available include, but are not limited to, biotinylated nucleotides, as well as nucleotides containing other chemically reactive groups (allowing, for example carbodiimide conjugation). A second form of nucleic acid available to be immobilized on a solid support comprises double stranded linearized segments of DNA obtained by fragmentation of DNA isolated from a cell. For example, fragments of double stranded DNA can include restriction digestion fragments of a plasmid. If an appropriate restriction enzyme is chosen for the digestion, double stranded nucleic acids can be prepared that contain a 5' overhang. The overhangs can be filled in with nucleotides containing modifications, such as biotin or other chemically reactive groups. The limitation of this approach, however, is that restriction enzymes intended to cut only known sequences will likely cut within the unknown sequence of the insert sequences as well. For example, if an unknown gene sequence were to be linked to beads, and a restriction enzyme approach were taken, there is no way to guarantee that the unknown sequence will not be cut as well. Such an event would render the attached nucleic acid unusable in hybridization or other measurement techniques.

To overcome this limitation, a preferred approach is to generate a single stranded nucleic acid modified by, for example, biotin (or another chemically reactive group) in a two step approach starting from a double stranded template. It is also preferred that the modification be inserted at a known position and that the nucleotide fragment have both vector and insert sequence. In this way, the vector sequence will provide a consistent method to quantitate the amount of DNA linked to the bead, as well as provide a standard to compare between different sets of beads linked at different times and with different nucleotide insert sequences. As a particular example, a double stranded template can be isolated or prepared, a DNA PCR fragment can be generated encompassing both the T7 and Sp6 promoter regions as well as insert DNA sequence. The fragment then serves as a substrate for PCR with a biotinylated primer. Only the resulting single stranded DNA containing the biotinylated (or chemically modified) nucleotide will be attached to the beads. Also, because the primer can be chosen to encompass vector sequence, primer extension can be used to measure the length of the attached DNAs to the beads. Furthermore, oligonucleotides or other nucleic acids can be used in hybridizations to the single stranded linked nucleic acids. Sequencing of the linked nucleic acids can also be achieved.

Example 3

Hybridization of labeled nucleic acids to single stranded oligonucleotides immobilized on micro-beads for use in expression analysis requires conditions that are sufficiently stringent to optimize specificity, but are sufficiently flexible to allow formation of stable hybrids at an acceptable rate. Typically, hybridization solutions include one of the following salts: SSC, tetraethylammonium chloride (TEACL) or tetramethylammonium chloride (TMACL). The T_M for hybridization of oligonucleotide sequences less than 20 nucleotides in length is strongly influenced by the GC content of the oligonucleotide and it's complementary sequence when the hybridization solution includes SSC salts. The contribution of base composition to duplex stability in the hybridization of short oligonucleotides to complementary sequences is minimized in solutions including either TEACL or TMACL salts (Jacobs et. al., Nuc. Acids Res. 16:4637-4650 (1988)).

One embodiment of the present invention provides a method for the synthesis of a product that includes short (20mer) oligonucleotides of specific sequence attached to micro-beads. It is estimated that up to 500,000 different sequences can be represented in this manner. A reagent bead assemblage comprising thousands of different genes can be incubated with an analyte and the interaction between the bead reagent and analyte correlated to the expression levels of a specific gene. Efficient hybridization of the analyte to the beads is ensured and potential problems caused by nucleic acid secondary structure are reduced, by incubating randomly fragmented nucleic acid analyte with the reagent bead assemblage under the following preferred conditions: about 94°C for 35 minutes in 40 mM Tris acetate (pH 8.1), 100 mM potassium acetate and 30 mM magnesium acetate. After a sufficient time of incubation, samples can be washed, precipitated and resuspended in quaternary alkylammonium hybridization solution (IX: 2.4 M tetraethylammonium chloride (TEACL), 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS and 100 μg/ml denatured, fragmented salmon sperm DNA) and heated at 95°C for 5 minutes. After heating the analyte/hybridization solution, prewarmed (33°C) bead mixture can be added. It is particularly preferred that for a probe of 20 nucleotides in length the hybridization reaction mix be incubated at about 33°C for approximately 1 hour with mixing. After hybridization, the analyte/probe hybrid can be washed at about 40°C for 15 minutes in 6 X SSC to remove weakly hybridized analyte.

The use of oligonucleotides attached to beads has also been indicated for application in sequencing. As described in detail above, the use of alkylammonium salts in hybridization solutions allows for duplex stability based upon the length of an oligonucleotide and not the base composition. Thus, a mixture of oligonucleotides of the same length, but different sequence composition, can be hybridized under the same conditions without compromising the stability of select oligomers. Short oligonucleotides with a single base mismatch to the complementary sequence have a thermal stability profile that is distinct from a perfectly matched hybrid such that the perfect and mismatched species can be differentiated by their melting temperatures.

Alkylammonium salts have not been demonstrated as being useful for hybridization of oligonucleotides shorter than 11 nucleotides in length. Conditions for hybridization with oligonucleotides of less than 11 nucleotides will therefore have to be optimized for use in MIDS. One possibility for hybridization of oligonucleotides as short as 8 nucleotides in length has been described by Drmanac et al., DNA Cell Biol. 9:527-534 (1990)). In this method hybridization occurs at low temperatures and high detergent concentration. For use in the present invention, sequencing reactions that include octamers with a single end base variance, samples can be incubated at 15°C for 3 hours in hybridization solution consisting of 0.5 M Na₂HPO₄ (pH 7.2) and 7% sodium lauroyl sarcosine. After hybridization the analyte/octamer hybrid can be washed in 6 X SSC at about 7°C for approximately 90 minutes. Hybridization efficiency is dependent upon probe concentration, therefore, the length of time for hybrid formation as well as hybridization temperature may need to be optimized empirically. Methods for optimization are well known to an artisan skilled in the nucleic acid art. Example 4 The present example provides methods for the stabilization of hybridization between oligonucleotides immobilized on micro-beads and nucleic acids. The hybridization strength of a short unmodified (7 or 8-mer) DNA oligonucleotides (oligo) on cRNA is not strong enough to remain bound during, for example, fluorescent activated cell sorter (FACS) analysis. To increase the binding strength of the immobilized oligo to the cRNA several options are available:

One available option is to extend the oligonucleotide from the 3 ' end along the hybridized cRNA utilizing a polymerase with a mixture of 4 nucleotides. The product cDNA/cRNA hybrid will be of sufficient length to remain stable and immobilized on the micro-bead surface throughout the required manipulations for analysis. A similar approach has been tested with oligonucleotides attached to a microchip hybridized to fluorescent labeled oligonucleotides (Fotin et al., Nucleic Acids Research 26:1515-21 (1998), incorporated herein by reference). Reverse transcriptase has the highest activity copying RNA to cDNA and would be preferred, but most DNA polymerases can provide similar activity with varying degrees of efficiency. It may be necessary to screen the available polymerases using optimal temperatures and hybridization buffer conditions optimum for the binding of oligonucleotides in MIDS. One particular advantage of hybrid stabilization by polymerization is that fluorescent modified deoxynucleotide triphosphates (dNTPs) can be incorporated into the growing DNA strand. The incorporated detectable label can be used as a primary signal for analysis, i.e., FACS, and the like, or to intensify the signal from the fluorescent modified cRNA being sequenced. Incorporation of a fluorescent label can also be used to amplify and or to shift the wavelength of emission through interactions with a second different fluorophore on the cRNA by scintillation proximity events.

Another advantage of hybrid stabilization by polymerization is that temperatures significantly higher than the T required for the length of the original oligonucleotide can be used for annealing. Standard hybridization strategies rely on a stable interaction such that a polymerase can extend from a very transient annealing positioning event. Therefore, once extension has begun the stable annealing temperatures rise as the oligonucleotide lengths increases.

One potential disadvantage of hybrid stabilization by polymerization is that annealing buffer conditions would be limited to conditions that are compatible with polymerase activity. High salts of various forms are often used to stabilize short sequences.

Some salts (TMCL and TEACL) have also been used to eliminate the differences in binding strength between A-T and G-C base interactions, as discussed above. Polymerases are strongly inhibited under these conditions. Buffer conditions that are compatible with both of annealing of an oligonucleotide and polymerase extension may require optimization.

Optimization can include adjustment of the magnesium ion or manganese ion concentrations in the reaction buffer. It is well known that an increase in the Magnesium ion (or Mn⁺) concentration in a polymerization buffer can induce hyperactivity of the polymerase and lead to an increase the T_M of the oligonucleotide. This would allow for a higher reaction temperature and faster polymerization. It should also be noted that an occasional miss- incorporation of nucleotides would not decrease the T_M of the product sufficiently to cause inconsistencies in data analysis during FACS.

In another embodiment, stabilization of an oligonucleotide/cRN A hybrid can be accomplished by the attachment of a short random oligonucleotide to the previously immobilized oligonucleotide by a DNA ligase. Addition of the random sequence extends the region hybridized and therefore stabilizes the duplex. The incorporation of a short random 4-mer oligonucleotide with the low complexity of 256 different sequences will allow, for example, a T4 bacteriophage or E. coli DNA ligase, and the like, to create a long and thermodynamically stable cRNA/cDNA hybrid strong enough to withstand further analysis. Longer length random oligonucleotides can also be used, but slower reaction kinetics due to the higher complexity of the mixture result. It is preferred to use an E. coli DNA ligase because it lacks RNA-specific activity. Also, poly-inosine or other "universal base" oligonucleotides can be substituted. These oligonucleotides have the advantage of being less base sensitive (with potentially faster extension rates). The faster extension rates would have to be compared to the interference in the formation of specific hybrid duplexes incurred by the formation of concatamers with "universal base" oligonucleotides hybridizing to each other. It should be noted that stabilization of hybridization using polymerization by ligation has the advantage of being useful with both cRNA and PCR generated DNA for sequencing templates. There is potential that the ligase may have reduced activity with an oligonucleotide of four bases. One approach to obtaining an enzyme with higher activity is to select for a desired rate of activity by evolving the ligase in vitro. Briefly, the process comprises producing pools of mutated T4 or E. coli DNA ligase clones in an IPTG inducible E. coli expression system. Recombinant protein would be purified with, for example, a cobalt or nickel resin and placed into wells of a multiwell plate. Enzyme activity can be measured by assaying a ³²P gamma labeled single sequence 4-mer oligonucleotide ligated onto a 30 mer deoxynucleotide oligonucleotide hybridized to a single species cRNA tethered to beads by the 5' end. Unbound oligonucleotide would be removed by filtering the reaction mixture through the bottom of a filter plate or by aspiration. Multiple washes would ensure low background radiation. Wells containing a desired rate of ligase activity would be identified by increased immobilization of P label. Detection of ligation events would be very sensitive due to high catalytic efficiency of ligase and the sensitivity of the detection method. The method provides for a rapid screening method for very large pool sizes. DNA isolated from clones corresponding to wells having the desired enzyme activity can be split into smaller pools and screened. The desired clones would then undergo further rounds of selection to localize clones having the desired increase in ligase activity.

DNA ligase can also be mutated and bound to bacteriophage P3 or P4 protein by recombinant DNA methods known to the skilled artisan. For example, a photo- activatable crosslinking agent can be introduced as a modification on the short oligonucleotide to trap the phage with enhanced activity on the bead. Beads can be isolated and the phage removed by DNase treatment prior to amplification in an E. coli host.

C. Stabilization of Hybridization Product Using a Minor Groove Binding Element Minor groove binding elements have been used as agents to increase the binding of oligonucleotides to nucleic acid strands. The dihydropyrroloindole tripeptide minor groove binder CDPI3 conjugated to a dTTP 8-mer increased the T_M of a hybrid with poly (dA) by 43 to 44 °C and to poly (rA) by half that (Lukhtanov et al., Bioconjug. Chem. 6:418-426 (1995)). When attached to the end of oligonucleotides, CDPI3 has been shown to enhance the stability of the hybridization of both GC rich and AT rich DNA sequences.

(Katyavin, Nuc. Acids Res. 25:3718-3723 (1997)) The binding of the minor groove binding element was higher with AT rich sequences than with GC rich sequences. If this were to increase hybridization strength of A-T interactions to that of G-C interactions it would be an ideal anchor for the oligonucleotide in bead based sequencing applications. Another group bound CDPI3 has also been bound to the 5' end of the 10-mer TGATTATCTG-3' and found that it which increased the T_M by 30 °C (Kumer et al., Nuc. Acids Res. 26:831-838 (1998)) In general, minor groove binding elements have some sequence specificity. Ideally, this element should bind equally to all sequences or with slightly higher affinity to A-T rich sequences. Other elements have been added to the ends of oligonucleotides to increase hybridization stability. In particular, amino acids, i.e., aromatic and positively charged amino acids, especially Histidine, have been added and appear to confer the highest increase in T_M- The binding of shorter oligonucleotide probes can be regulated by the use of a varying a set of peptides, bringing all 8 mer sequences to approximately the same T_M*

D. Use of modified nucleotides to Stabilize Hybridization Products

A potential factor which may reduce the efficiency of the methods of the present invention for oligonucleotide sequencing strategies is provided by the difference in the strength of the binding between base pairs. The G-C nucleotide pairing is much stronger, with 3 hydrogen bonds, than the A-T pairing, with only 2. One way to equilibrate the difference between the binding energy of the nucleotide base pairings comprises the utilization of modified nucleotides to increase the strength of the A-T pairing or to decrease that of the G-C interaction. The use of a "G-clamp" heterocycle dCTP analogue has been demonstrated to significantly increase the binding affinity of an oligonucleotide to RNA by causing the formation of a fourth hydrogen bond (Flanagan et al., Proc. Natl. Acac. Sci USA 96:3513-3518 (1999), incorporated herein by reference). A similar modification on the other pyrimidine, dTTP, may enhance A-T interactions in a like fashion, creating an "A-clamp." Similarly, the addition of a tail on the 3' or 5' end of the oligonucleotide probe composed of a "universal base" would confer greater temperature stability during the annealing step. The increased temperature stability would increase the yield and specificity of the reaction. For example, 5-nitroindole tails of 3 or 4 residues have been shown to improve the performance of an 8-mer oligonucleotide in cycle sequencing (Ball et al., Nuc. Acids Res. 26:5225-5227(1998), incorporated herein by reference) 5-Nitroindole has also been added to the 3' and 5' ends of tethered oligonucleotides to adjust binding affinity and cancel out variations due to differing base compositions (Fotin et al., Nuc. Acids Res. 26:1515-1521 (1998)) These methods would be expected to provide similar increases in temperature stability in the methods of the present invention when used for nucleotide sequencing. Other "universal bases" can also be used, but may not be as effective. For example, deoxyinosine has historically been used as a universal base. However, similar to certain other "universal base" analogues, it is not totally non-discriminating in forming base pairings. Deoxyinosine has much higher binding to dCTP than to dGTP with others universal bases falling in between. 3-Nitroindole was the least discriminating of several tried in a study (Bergstrom et al., Nuc. Acids Res. 25:1935-1942 (1997)), but has a low binding affinity. 5-Nitroindole had much higher binding strength but varies more between the bases.

It is likely that a combination of the above described methods will provide optimal methods for using the MIDS assay system for various uses.

Example 5 The following example provides a description of the use of the methods of the present invention for DNA sequence analysis. Data for analysis with use of MIDS for DNA sequencing results from hybridization of oligonucleotides and a target DNA and an inference of DNA sequence from the known sequence of the oligonucleotides immobilized on the identified solid surface. The length of the oligonucleotide immobilized establishes the complexity of the oligonucleotide and is related to the size of the target DNA to be sequenced. In the following example, oligonucleotides of 7 nucleotides in length are used. A random library of oligonucleotides having 7 nucleotides provides 16,384 unique oligonucleotides. The library was synthesized in a 384 well format or representing a total of 43 plates. The library provides all the possible sequence permutations of a 7 nucleotide oligonucleotide. Because a 7 nucleotide oligonucleotide with randomly chosen sequence has the probability of hybridizing once to a nucleic acid of 16,384 nucleotides, the complexity of DNA sequence analysis would initially be simplified by analyzing DNA fragments of less than 16,000 nucleotides. After each bead set is given a unique identification tag, as described in the above example, the bead sets would be mixed together to produce the bead reagent assemblage.

In this example the DNA sequence of a cDNA clone can be determined. The cDNA clones can be selected from a cDNA library or from a normalized cDNA library. Briefly, a population of bacteria containing cDNA clones would be plated on agar plates at a density sufficient to produce isolated colonies. Bacteria from isolated colonies were transferred to culture media in the 384 well plates. The plates would then be incubated over night for growth. Inocula from these plates would be used to grow cultures over night for plasmid DNA preparations using, for example, a Promega paramagnetic bead preparations. Plasmid DNA would then be linearized by digestion with a restriction endonuclease

(preferably having am 8 base pair recognition sequence ) at the 5' end of the cDNA insert. Fluorescent labeled cRNA would be synthesized from the RNA polymerase promoter at the 3' side of the cDNA insert, using an RNA polymerase, ribonucleotides, and a fluorescent labeled ribonucleotide. cRNA from each cDNA clone would be hybridized to the bead reagent assemblage. Maintaining a low temperature, the oligonucleotide-cRNA complex would be diluted into an appropriate polymerase buffer solution and stabilized by extension with a polymerase as described in Example 4 above. The bead reagent assemblage would then be washed to remove any unassociated cRNA. The stringency of the wash would be determined empirically.

The beads are analyzed by a specially configured flow cytometer. As each bead passes through the flow cytometer, the amplitude of the hybridization signal as determined from the fluorescent labeled cRNA hybridized to the oligonucleotide immobilized on the bead, and the distinct bead identification is recorded. A look-up table previously established from information production of the bead sets is used to establish correspondence between oligonucleotide and bead identification. The identification data determined from flow cytometry, is compared to the look-up table to determine the identity of the oligonucleotide. Then positive or negative hybridization is recorded for the determined oligonucleotide. The sequence of the positively hybridizing oligonucleotides is used to build over lapping nucleotide sequence by computational analysis revealing the nucleotide sequence of the cDNA target nucleic acid.

Example 6 Genetic Diagnosis of Porphyria using MIDS Technology

In the following example, the use of the micro-identification system of the present invention for the genetic diagnosis of porphyria is described. Porphyria results from a deficiency in any one or more of the eight enzymes that are in the biosynthetic pathway for the production of heme. Heme is an important molecule that is a co-factor of hemoglobin required for carrying oxygen and carbon dioxide. It is also a component of cytochromes that are indispensable for energy metabolism of cells. All cells of biological systems have and require heme for viability. Homozygous mutation in any of the eight heme biosynthetic enzymes is lethal. People suffering from porphyria are therefore heterozygous for a mutation in one of the heme biosynthetic enzymes. Porphyria is manifested in many ways, but the most common symptom presented is severe stomach pain. Other typical symptoms include mental deficiencies and or skin lesions. The incidence of porphyria in the general population is about 1 in 10,000 to 1 in 5,000. Typically, the slight difference in the amount of heme biosynthetic enzymes synthesized from one rather than two genes is not sufficient to manifest disease symptoms. Onset of porphyria symptoms is typically seen in the late teens or early twenties. Acute incidence of porphyria can be fatal, but usually results in symptoms which are unpleasant, disturbing, poorly understood by medial doctors. Because the disease is poorly understood by the medical community and the patient it is highly under diagnosed. Although specific treatments of porphyria are being developed, current methods of treatment are nonspecific.

Treatment usually involves hospitalization to relieve the pain and for the administration of various medications not specific to treat the disease condition. Acute attacks appear to be precipitated by environmental agents and hormonal changes. Hormonal changes in the menstrual cycle of women can be very troublesome. Other precipitating agents include pharmaceutical drugs and recreational drugs, i.e., alcohol and tobacco. Currently, porphyria is best managed by avoiding precipitating causes of acute incidence. Precipitating agents usually are quite personal and are only determined by trial and error or are unavoidable, such as the normal hormonal changes in women. The direct or indirect neurological effects and chronic difficulties associated with the disease often results in these patients being labeled as hypochondriacs. The lack of definitive symptomolgy relating to porphyria has lead to many cases going undiagnosed until the disease becomes a chronic condition which negatively influences the quality of life for a patient.

Genetic diagnosis of porphyria is particularly demanding because of the many mutations that cause the disease. Porphobilinogen deaminase (PBGD), one of the heme biosynthetic enzymes, has been mapped to contain over a hundred known mutations which can lead to causing porphyria. Employment of commonly used genetic diagnostic procedures for the diagnosis of porphyria would not be practical to screen for each of the known mutation in just this one enzyme of the heme pathway. Conditions having such a complex genetic basis are readily diagnosed by use of the MIDS diagnostic system described by the present application.

There are two basic approaches to using MIDS for the genetic diagnosis of porphyria. The first comprises utilization of hybridization of oligonucleotides of normal and mutant sequence to the specific regions of DNA where the mutations are known to exist. The second approach is to use MIDS to sequence the DNA of the gene under diagnosis.

Hybridization to known mutated sequences is a less complex approach which can provide a definitive diagnosis for all mutations known to cause porphyria. Gene sequencing is more complex but can detect porphyria where a causative mutation is known, but also can detect those mutations that are yet to be discovered. While specific examples have been provided, the above description is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents.

All publications and patent documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent document were so individually denoted.

Claims

WHAT IS CLAIMED IS:

1. A process for preparing a micro particle reagent set capable of detecting a plurality of analytes in a single sample comprising: a) immobilizing a first specific micro-identification tag and a first affinity ligand, specific for a given first analyte in the sample, to form a micro-particle subset; b) repeating step (a) to form a plurality of micro-particle subsets; and c) mixing the plurality of micro-particle subsets to produce a micro- particle reagent assemblage, wherein each micro-particle subset and, therefore, the affinity ligand, which has been immobilized to the micro-particle, is identifiable by the immobilized micro-identification tag on the micro particle subset

2. The process of claim 1 , wherein the micro particle is a micro-bead or a micro-cylinder.

3. The process of claim 2, wherein the micro-bead comprises polyacrylate, polystyrene, polysaccharide, nitrocellulose, or a metallic colloid.

4. The process of claim 1, wherein the micro-identification tag comprises a colored micro-particle, a fluorophore, a chromophore, or a fabricated micro-tag.

5. The process of claim 4, wherein the fluorochrome associated with the micro-particle subset comprises a fluorophore cocktail comprising a mixture of fluorophores having substantially different emission spectra.

6. The process of claim 4, wherein the fluorophore comprises fluorescein, Nile Red, Texas Red, lissamine, phycoerythrin, or a combination thereof.

7. The process of claim 1 , wherein the micro-identification tag and the affinity ligand are immobilized onto the micro particle sequentially or simultaneously.

8. The process of claim 7, wherein the affinity ligand is immobilized onto the micro-particle subsequent to the micro-identification tag.

9. The process of claim 7, wherein the micro-identification tag is applied to the micro-particle subsequent to the affinity ligand.

10. The process of claim 1, wherein the affinity ligand is a nucleic acid, an oligonucleotide, a polynucleotide, a peptide nucleic acid, a peptide, a protein, a protein fragment, a carbohydrate, or a fatty acid.

11. The process of claim 10, wherein the nucleic acid is RNA or DNA.

12. The process of claim 9, wherein the protein is an antibody or an antigen binding fragment or derivative thereof.

13. The process of claim 12, wherein the antibody or antigen binding fragment or derivative thereof comprises a polyclonal antibody, a monoclonal antibody, a Fab, a F(ab')₂, a Fv, a chimeric antibody, a complementarity determining region grafted recombinant antigen binding protein, or a single chain antibody.

14. A method for the simultaneous determination of multiple distinct analytes, comprising: a) admixing a sample suspected of containing one or more distinct analytes with a micro-particle reagent assemblage of claim 1 ; b) incubating the sample with the reagent bead assemblage under conditions conducive to complex formation between the analyte and the affinity ligand immobilized on the micro-particle subsets for a time period sufficient for complex formation; and c) determining the presence of each distinct analyte by correlating the formation of an analyte/affinity ligand complex with the micro-identification tag of a micro-particle subset.

15. The method of claim 14, wherein the micro-particle is a micro-bead or a microcylinder.

16. The method of claim 15, wherein the micro-bead comprises polyacrylate, polystyrene, polysaccharide, nitrocellulose, or a metallic colloid.

17. The method of claim 14, wherein the micro-identification tag comprises a colored micro-particle, a fluorophore, a chromophore, or a fabricated micro-tag.

18. The method of claim 17, wherein the fluorophore associated with the micro-particle subset comprises a fluorophore cocktail comprising a mixture of fluorophores having substantially different emission spectra.

19. The method of claim 17, wherein the fluorophore comprises fluorescein, Nile Red, Texas Red, lissamine, phycoerythrin, or a combination thereof.

20. The method of claim 14, wherein the affinity ligand is a nucleic acid, an oligonucleotide, a polynucleotide, a peptide nucleic acid, a peptide, a protein, a protein fragment, a carbohydrate, or a fatty acid.

21. The method of claim 20, wherein the nucleic acid is RNA or DNA.

22. The method of claim 20, wherein the protein is an antibody or an antigen binding fragment or derivative thereof.

23. The method of claim 22, wherein the antibody or antigen binding fragment or derivative thereof comprises a polyclonal antibody, a monoclonal antibody, a Fab, a F(ab')₂, a Fv, a chimeric antibody, a complementarity determining region grafted recombinant antigen binding protein, or a single chain antibody.