WO2001000875A2

WO2001000875A2 - Novel methods and products for arrayed microsphere analysis

Info

Publication number: WO2001000875A2
Application number: PCT/US2000/017421
Authority: WO
Inventors: Walter Klimecki; George Maracas
Original assignee: Motorola, Inc.
Priority date: 1999-06-25
Filing date: 2000-06-23
Publication date: 2001-01-04
Also published as: WO2001000875A3; AU5764500A

Abstract

Among other things, the invention provides components, devices, and methods for arrayed microsphere analysis (AMA), and more particularly, for fluorescent arrayed microsphere analysis (FAMA).

Description

NOVEL METHODS AND PRODUCTS FOR ARRAYED MICROSPHERE

ANALYSIS

TECHNICAL FIELD OF THE INVENTION The present invention provides, among other things, components, devices, and methods for arrayed microsphere analysis (AMA), and more particularly, for fluorescent arrayed microsphere analysis (FAMA).

BACKGROUND OF THE INVENTION The identification and characterization of the constituent genes of the human genome involves two principal efforts. First, genes must be identified and sequenced. The gene sequence provides valuable clues as to the evolutionary derivation of newly identified genes, through sequence similarities with other known homologues. This type of sequence- homology information also provides clues as to possible functional relevance of novel genes. Second, and equally important sequence information, is that obtained by the analysis of gene expression. Gene expression analysis provides information regarding the functional relevance of a given gene product with respect to temporal, biochemical, and location- specific criteria. Thus, the knowledge of the levels of mRNA for a particular gene at particular times, under specific biochemical conditions, in a specific tissue, allows one to make predictions about the function of a gene for which functional significance is unknown; or to elucidate the fine points of biochemical pathway response and control for genes with well-characterized functions.

Presently, the techniques available to analyze mRNA levels are cumbersome. This has restricted the number of genes whose expression can be analyzed at a given time. One technique used commonly is RNA Northern Blot Analysis. In this technique RNA is isolated from a population of cells. The RNA is then size fractionated using gel electrophoresis. The fractionated RNA is then transferred to a membrane, where the RNA is immobilized. The membrane is then reacted with an excess of detection-labeled DNA ("probe") of known sequence, such that the probe specifically hybridizes with mRNA of the gene of interest. The greater the number of copies of mRNA for a particular gene, the greater the amount of hybridized, labeled probe on the membrane, yielding a larger signal.

While Northern Blot Analysis has proven an extremely effective method of RNA analysis, it often involves 72-96 hours of assay time to yield results, with analysis limited to 10-15 genes per experiment. In addition, since the signal from one probe molecule is well below the minimum detectable signal of the assay, the measurement of low levels of RNA is variable, and ranges from difficult to impossible. The difference between a negative signal and a minimally detectable signal is essentially an analog gradation which is dependent upon the background signal ("noise") of that particular experiment on that particular day. The signal is also highly dependent upon the efficiency of the reactions which label the probe. Other RNA analysis techniques, such as the RNAse Protection Assay, have the potential of improved sensitivity, but are equally as cumbersome, potentially even more complex, and do not address the problem of clear identification of low level signals.

Thus, in an effort to bypass the cumbersome nature of RNA analysis techniques, recent efforts have utilized the concept of "DNA Microarrays" to allow the analysis of RNA levels from extremely large numbers of genes in a single experiment (Schena et al., Science. 270, 467-70 (1995)). The principle of these array-based RNA analysis techniques involves attaching short oligonucleotide probes, with specificity to particular genes, to a solid substrate such as a glass slide or an acrylamide pad. Because of their small size, thousands of distinct oligonucleotides can be attached to a small area, in defined locations. RNA is isolated from two distinct populations of cells and kept separate. One population is considered a control. The RNA from this population is reverse-transcribed in the presence of fluorescence-labeled nucleotides of a single color (e.g., green). The other population of cells is the "test" population which is treated similarly, except that the fluorescence-labeled nucleotide is a distinct color (e.g., red). The RNA samples are then mixed 1 : 1, and the mixture is hybridized to the immobilized oligonucleotides. Fluorescence at each oligonucleotide location is measured and the ratio of the fluorescence (i.e., in this example, green fluorescence as compared to red fluorescence) at a location is a measurement of the ratio of control mRNA to test mRNA. The overall fluorescence intensity reflects the quantity of mRNA at that site. Indisputably, array analysis has greatly increased the number of genes which can be analyzed for expression in a single experiment. In spite of this, the array system still suffers from the problem of "analog" signal gradation, and consequently does not offer reliable, low level RNA analysis.

The present invention seeks to overcome these disadvantages attendant the prior art. The invention provides a novel method of RNA analysis which maintains the power of array analysis, while providing a discrete, "digital" signal of invariable intensity. This optimally is accomplished without significantly increasing the difficulty or labor input relative to currently used array-based systems of RNA analysis. These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION The present invention provides, among other things, components, devices, and methods for arrayed microsphere analysis (AMA), and more particularly, for fluorescent arrayed microsphere analysis (FAMA).

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts DNA (or cDNA) including one or more biotin-conjugated nucleotide(s) that is produced from a sample by isolating its DNA and labeling the DNA with at least one biotin-conjugated nucleotide, or that is produced from a sample by reverse transcription of sample RNA in the presence of at least one biotin-conjugated nucleotide. Symbols: B, biotin label. Figure 2 depicts the synthesis of probe DNA and attachment to microspheres.

Symbols: Circles labeled 1 -6, microspheres that optionally differ in color.

Figure 3 depicts the placement of the probe/mi crospheres of Figure 2 in separate microlocations of a multi-compartmentalized container (e.g., in separate wells of a microtiter plate). Symbols: Circles labeled 1-6, microspheres that optionally differ in color. Figure 4 depicts the hybridization of the probe/microspheres of Figure 3 in separate microlocations of a multi-compartmentalized container (e.g., in separate wells of a microtiter plate) with the biotinylated DNA (or cDNA) depicted in Figure 1 to obtain probe/microsphere/DNA (or cDNA) hybrids. Symbols: B, biotin label; Circles labeled 1 -6, microspheres that optionally differ in color. Figure 5 depicts the addition to the probe/microsphere/DNA (or cDNA) hybrids depicted in Figure 4 of streptavidin-conjugated magnetic particles to obtain streptavidin- conjugated magnetic particles/probe/microsphere/DNA (or cDNA) hybrids in the presence on non-bound probe/microsphere/DNA (or cDNA) hybrids. Symbols: B, biotin label; Circles labeled 1 -6, microspheres that optionally differ in color; Stars, streptavidin-conjugated magnetic particles.

Figure 6 depicts the streptavidin-conjugated magnetic particles/probe/microsphere/DNA or (cDNA) hybrids that are selectively captured and removed from non-bound probe/microsphere/DNA (or cDNA) hybrids by exposing the mixture depicted in Figure 5 to a magnetic field, followed by washing. Symbols: B, biotin label; Circles labeled 1 -6, microspheres that optionally differ in color; Stars, streptavidin- conjugated magnetic particles.

Figure 7 depicts recovery of the microspheres following removal (e.g., degradation) of bound DNA from the magnetic particles/probe/microsphere/DNA (or cDNA) hybrids of Figure 6 and washing. Symbols: Circles labeled 2. 3, 4, or 6, microspheres that optionally differ in color.

Figure 8 depicts a fluidic switching assembly having a multi-compartmentalized container (10) such as a microtiter plate, and a micropipettes or other tubing system (20) having the same footprint and the container and providing means of inputting reagants and components to the container, as well as a means for removing items from the container for routing (30), e.g., to the detection system.

Figure 9 depicts an integrated fluidic switching assembly having a multi- compartmentalized container ( 10) such as a microtiter plate or other miniature reaction chambers, and one or more channels (34 and 38) as a means of providing reagants and components to the container, as well as a means for removing items from the container for routing (30), e.g., to the detection system, and a separately housed magnetic particle positioning system (40) located on top of the micropipettes or other tubing system (20) having the same footprint and the container and which provide means of inputting reagents and components to the container, as well as a means for removing items from the container. Figure 10 depicts an integrated fluidic switching assembly having a multi- compartmentalized container ( 10) such as a microtiter plate or other miniature reaction chambers, and one or more channels (34 and 38) as a means of providing reagants and components to the container, as well as a means for removing items from the container for routing (30), e.g., to the detection system, and a separately housed magnetic particle positioning system (40) located on top of the micropipettes or other tubing system (20) having the same footprint and the container and which provide means of inputting reagents and components to the container, as well as a means for removing items from the container. This device differs from that depicted in Figure 9 in that the reaction chambers themselves are present inside of a housing, and in intimate association with the channels and micropipettes or other tubing system, thus allowing a "microchip" type assembly.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides, among other things, components, devices, and methods for arrayed microsphere analysis (AMA), and more particularly, for fluorescent arrayed microsphere analysis (FAMA). This invention offers a novel method of nucleic acid (e.g., cellular RNA or DNA) analysis which maintains the power of array analysis, while providing a discrete, "digital" signal of invariable intensity. This is accomplished without significantly increasing the difficulty or labor input relative to currently used array-based systems of DNA/RNA analysis.

The AMA/FAMA method is summarized graphically in Figures 1-7, which are exemplary only, and in no way limiting of the invention. As can be seen from these figures, and as explained in greater detail below, Figure 1 depicts DNA (or cDNA) including one or more biotin-conjugated nucleotide(s) that is produced from a sample by isolating its DNA and labeling the DNA with at least one biotin-conjugated nucleotide, or that is produced from a sample by reverse transcription of sample RNA in the presence of at least one biotin- conjugated nucleotide(s). Figure 2 depicts the synthesis of probe DNA and attachment to microspheres. Figure 3 depicts the placement of the probe/microspheres of Figure 2 in separate microlocations of a multi-compartmentalized container (i.e., in separate wells of a microtiter plate), and Figure 4 depicts their hybridization with the biotinylated DNA (or cDNA) generated from the sample to obtain probe/microsphere/DNA (or cDNA) hybrids. As depicted in Figure 5, streptavidin-conjugated magnetic particles are added to the probe/microsphere/DNA (or cDNA) hybrids to obtain streptavidin-conjugated magnetic particles/probe/microsphere/DNA (or cDNA) hybrids in the presence on non-bound probe/microsphere/DNA (or cDNA) hybrids. Figure 6 depicts the streptavidin-conjugated magnetic particles/probe/microsphere/DNA (or cDNA) hybrids that are selectively captured and removed from non-bound probe/microsphere/DNA (or cDNA) hybrids by exposing the mixture depicted in Figure 5 to a magnetic field, followed by washing, and Figure 7 depicts recovery of the microspheres following optional removal (e.g., degradation) of bound DNA from the magnetic particles/probe/microsphere/DNA (or cDNA) hybrids.

Thus, in an especially preferred embodiment, the present invention provides, among other things, methods for analyzing the expression of a plurality of genes, e.g., present in a sample. Generally, the methods Arrayed Microsphere Analysis (AMA), and Fluorescent Arrayed Microsphere Analysis (FAMA), preferably comprise the following steps: (a) isolating from the sample the RNA produced by the genes;

(b) reverse transcribing the RNA into cDNA in the presence of one or more biotin- conjugated nucleotide(s) to obtain biotin-conjugated cDNA; (c) obtaining a plurality of single-stranded DNA probes that are capable of specific hybridization to said cDNA of interest, wherein the probes are modified to allow end- attachment to labeled microspheres;

(d) obtaining probe/microspheres by attaching to the modified single-stranded DNA probes microspheres of a certain type, with microsphere type being determined by the combination, to provide a unique identification in the method of the microsphere, of: (i) microsphere diameter, the microsphere being spherical; and (ii) microsphere color, wherein microsphere color is determined by the presence within the microsphere of one or more dyes, with each dye being of a different color, different intensity, or different color and different intensity;

(e) placing each probe/microsphere in a separate, specific microlocation of a multi- compartmentalized container and performing all subsequent steps in each the separate microlocation;

(f) hybridizing the biotin-conjugated cDNA to the probe/microspheres under conditions sufficient to effect specific hybridization and obtain probe/microsphere/ cDNA hybrids;

(g) washing the probe/microsphere/cDNA hybrids;

(h) obtaining streptavidin-conjugated magnetic particles and incubating the particles with the probe/microsphere/cDNA hybrids under conditions such that they bind to the biotin- conjugated cDNA present in the probe/microsphere/cDNA hybrids, and streptavidin- conjugated magnetic particles/ probe/microsphere/cDNA hybrids are obtained;

(i) using a magnet to capture the streptavidin-conjugated magnetic particles/ probe/microsphere/cDNA hybrids; and

(j) assessing the streptavidin-conjugated magnetic particles/ probe/ microsphere/cDNA hybrids to determine the number and type of microsphere captured per microlocation, and considering this information along with the type of probe employed per microlocation to analyze the expression of a plurality of genes present in the sample.

The key steps and considerations involved in AMA/FAMA are discussed below as follows: (1) sample (DNA or cDNA) preparation; (2) probe preparation; (3) microsphere (e.g., fluorescent microsphere, FM) preparation; (4) probe/microsphere attachment; (5) probe/microsphere placement to generate array; (6) probe/microsphere and DNA (or cDNA) hybridization; (7) selective capture of steptavidin-conjugated magnetic particle/probe/microsphere/DNA (or cDNA) hybrids; (8) potential for recovery of analyzed DNA (or cDNA); (9) assessment of captured microspheres; and (10) miniaturization of AMA/FAMA assay.

Sample Preparation According to the invention, the expression (or representation, as described below) is analyzed of a plurality of genes present in a sample. A "plurality of genes" means at least two genes, preferably from at least 2 to at least about 100,000 genes, and optimally, from at least 2 genes to as many genes as can feasibly be assessed in a particular array experiment. Feasibility assessment is based on cost, ease of manipulation, availability of instrumentation and software for analysis, sensitivity necessary or desired, etc. "Specific genes of interest" are those present in the sample whose expression (or representation) is being assessed in a particular experiment. Of course, a sample may contain many more genes than those whose expression (or representation) is being analyzed in an individual experiment, and separate experiments using the same sample can be employed to analyze expression (or representation) of different genes.

According to the invention, a "sample" desirably is any sample obtained from any natural source (i.e., isolated from nature). According to the invention, a sample comprises ribonucleic acid (i.e., RNA) when expression is being assessed, and comprises deoxyribonucleic acid (i.e., DNA) when representation is being assessed. In particular, preferably the sample comprises the RNA or DNA of a human or non-human animal.

However, the sample nucleic acid can be taken from any organism. For instance, the sample can be taken from mammals (such as humans, non-human primates, horses, dogs, cows, cats, pigs, or sheep), from viruses, from plants, from microorganisms, from other living things in the environment, etc. The biological sample (i.e., a sample containing DNA or RNA) can be isolated by any of a variety of means known to those skilled in the art. For instance, blood, tissue, or other bodily fluid samples can be taken from one or more individuals. Total RNA and/or messenger RNA (i.e., mRNA) desirably is isolated from a cell population to be analyzed. In general, mRNA should prove to be a superior sample for use in the method of the invention of analyzing expression as compared to total RNA. Any appropriate method such as is known in the art for isolation of total RNA or mRNA can be employed (see, e.g., Chirgwin et al., Biochemistry. 18. 5294 (1979), and others). Any commercially available kit for carrying out isolation of total RNA or mRNA similarly can be employed (e.g., Oligotex-dT resin, Qiagen, Valencia, California). Similarly, when representation of a particular DNA is being assessed, desirably any method for its isolation and/or any commercially available kits or reagents can be employed.

The RNA from the sample is reverse transcribed, using reverse transcriptase enzyme, typical reaction buffers and deoxynucleotide triphosphates which consist of at least one biotin-conjugated nucleotide; for example, dATP, dCTP, dGTP and biotin conjugated dTTP (or, biotin-conjugated dUTP substituted for biotin-dTTP). Any appropriate method such as is known in the art for RNA reverse transcription can be employed (see, e.g., Friedman et al., Nucleic Acids Res. 4(10), 3455-71 ( 1977)). Similarly, any commercially available kit for carrying out RNA reverse transcription similarly can be employed (e.g., StrataScript RT-PCR kit, Stratagene, La Jolla, California).

Following cDNA synthesis, optionally the cDNA is separated from the RNA. This separation preferably is carried out by digesting the RNA. Alternately, desirably the biotin- conjugated cDNA is separated from the RNA by means of the biotin incorporated in the biotin-conjugated cDNA. For instance, by purifying the biotin-conjugated cDNA by binding to streptavidin beads and/or streptavidin packed in a column, such as is known in the art. As indicated previously, in some applications, the nucleic acid of interest in the sample is DNA instead of RNA (i.e., cDNA transcribed from RNA, including from mRNA). For instance, RNA analysis is conducted where gene expression is being assayed. However, the presence and nature of the genomic DNA itself may be of interest in cases where gene copy number is being assessed, gene composition (i.e., presence of absence of mutations) is being assessed, etc. In these cases, biotinylated DNA suitable for analysis can be derived from the sample using methods commonly used by those familiar with the art. These methods include incorporation of biotin-derived nucleotide triphosphates onto the 3' end of the DNA using terminal deoxynucleotide transferase. Alternately, restriction endonucleases may be used to digest the double-stranded DNA in such a conformation that a 5' overhang is generated, which can then be a substrate for the incorporation of biotin-derived deoxynucleotide triphosphates by DNA polymerase, such as Klenow fragment, forming a blunt end of double-stranded DNA. Any other appropriate methods similarly can be employed. Thus, in an especially preferred embodiment, the present invention provides, among other things, methods for analyzing the representation of a plurality of genes, e.g., present in a sample. This method also is a preferred method of the invention for carrying out Arrayed Microsphere Analysis (AMA), and Fluorescent Arrayed Microsphere Analysis (FAMA), and preferably comprises the following steps: (a) isolating a sample to be tested for a DNA of interest;

(b) biotinylating the sample DNA to obtain biotin-conjugated DNA;

(c) obtaining a plurality of single-stranded DNA probes that are capable of specific hybridization to the DNA of interest, wherein the probes are modified to allow end- attachment to labeled microspheres;

(d) obtaining a plurality of microspheres of differing types, with microsphere type being determined by the combination to provide a unique identification of the microsphere of:

(i) microsphere diameter, the microsphere being spherical; and (ii) microsphere color;

(e) obtaining probe/microspheres by attaching the microspheres to the modified single-stranded DNA probes ;

(f) placing each probe/microsphere in a separate, specific microlocation of a multi- compartmentalized container so as to generate an array, and performing all subsequent steps in each of the separate microlocation;

(g) hybridizing the biotin-conjugated DNA to the probe/microspheres under conditions sufficient to effect specific hybridization and obtain probe/microsphere/DNA hybrids;

(h) washing the probe/microsphere/DNA hybrids; (i) obtaining streptavidin-conjugated magnetic particles and incubating the particles with the probe/microsphere/DNA hybrids under conditions such that they bind to the biotin- conjugated DNA present in the probe/microsphere/DNA hybrids, and streptavidin-conjugated magnetic particles/probe/microsphere/DNA hybrids are obtained;

(j) using a magnet to capture the streptavidin-conjugated magnetic particles/probe/microsphere/DNA hybrids; and

(k) assessing the streptavidin-conjugated magnetic particles/probe/microsphere/DNA hybrids to determine the number and type of microsphere captured per microlocation, and considering this information along with the type of probe employed per microlocation to analyze the representation of the DNA present in the sample. Incorporation of a biotinylated nucleotide into the sample DNA or cDNA reverse transcribed from sample RNA allows subsequent conjugation with streptavidin (as well as providing a possible means of purifying nascent cDNA). While it is preferred according to the invention that biotinylation be employed as a means of providing later conjugation, any appropriate means that can be employed in the methods and devices according to the mvention can be used instead of biotmylation If biotmylation is changed as the means of labeling, then, as later descπbed, the binding partner for biotin (which is attached to a magnetized particle, 1 e , preferably streptavidin) also must be changed Binding partners analogous to (1 e , having the same function as) biotin/streptavidm are well known in the art In general, and as later descπbed, it is desirable that only a single sample be analyzed in one microlocation of a multi-compartmentalized container However, numerous genes (1 e , expression and/or representation) can be assessed m that microlocation, as will become apparent from the descπption of the invention provided herein

Pi obe Pi epω ation

Desirably a "probe" according to the invention is a single stranded oligonucleotide or polynucleotide, or any nucleic acid that can function as a probe as described herein Single- stranded DNA probes, with sequences complimentary to the DNA (or cDNA) sequences from specific genes of interest (I e , "target genes") are obtained These probes thus are capable of specific hybπdization to the cDNA of interest Preferably the probe ranges from about 1 to about 5000 bases in length desirably from about 1 to about 500 bases in length, especially from about 1 to about 300 bases in length, and particularly from about 1 to about 10 bases in length

For relatively short probe sequences (l e . <40-50 bases), synthesis desirably is accomplished on a DNA synthesizer (e g , on a Cruachem PS 250 DNA Synthesizer, or other automated DNA synthesizer) using standard chemistπes (e g , phosphoramidite chemistry) as discussed, for instance, in Beucage et al , Tetrahedron. 48, 2223-231 1 ( 1992), U S Patents 4,415,732, 4,458,066, 4,725,677, 4,973,679. and 4,980,960 Typically, synthetic oligonucleotides desirably are further puπfied, e g , by HPLC followed by a 20% polyacrylamide gel/7 M urea For longer probes, specific single stranded DNA preferably is synthesized using the Polymerase Cham Reaction (PCR), followed by an isolation technique to obtain puπfied populations of the desired single-stranded probe Alternately, the oligonucleotides or polynucleotides of any size can be purchased as commercially prepared Regardless of the manner in which probe is prepared, preferably, duπng probe synthesis, provisions are made for one end of the probe to be anchored to the labeled microsphere (e g , the fluorescent microsphere. FM) This desirably is accomplished by using an amine-conjugated base at one end of the probe, although other means of modification that provide for end-attachment also can be employed 5' amine modification is a standard modification by most oligonucleotide synthesis facilities Preferably, the DNA probes are present in an array (i.e., as described further below). Since the probes are synthesized or otherwise obtained, they are of a known sequence, and are selected to be complementary to the DNA targets present in a sample being assessed. In an array according to the invention, desirably the sequence and/or position of every probe on the array is known, such that the probe array can be employed in sequencing and diagnostic applications, in forensics analyses, determinations of paternity, veterinary applications (e.g., thoroughbred testing), and the like.

In particular, preferably in the method of the invention, a different probe is employed for each microlocation of the array. Optionally, single probe is employed for hybridization at each array microlocation. As further described below, it also is desirable in some embodiments that more than one probe is hybridized at each array microlocation. Along these lines, desirably from 3 to 10 different probes are hybridized at each array microlocation. However, more generally, more than one probe optionally is hybridized at each microlocation, with the only limit on the number of probes that can be so hybridized being the ability to obtain and successfully detect such hybridization.

Should it be necessary or desirable according to a particular application, the probe itself also can include a detectable label. It is not necessary, however, according to the invention that each probe be labeled. In fact, it is important that any label that may be used for the probe does not interfere with the capture and/or detection of the microspheres according to the invention. Thus, it is desirable that the probe not be labeled with a fluorescent or luminescent label, and that the probe not include biotinylated nucleotides. With those provisos considered, any of the conventionally used methods of labeling (e.g., radioisotopes, enzymic, redox or other electrochemical labels, or other labels such as ligands, antigens, and the like) can be used according to the present invention to optionally label the probe and as set out, for instance, in: U.S. Patents 4,563,417, 4,581,333 and 4,582,789; EP Applications 1 19448 and 144914; and Prober et al., Science. 238. 336-340 (1987). Means of probe labeling, and other means of labeling to detect hybridization and other bioconjugation events are well known in the art.

Microsphere Preparation

Once the probes as described above have been obtained, the present invention calls for obtaining probe/microspheres by attaching to the modified single-stranded DNA probes microspheres of a certain type. According to the invention, "microsphere type" desirably is determined by the combination, to provide a unique identification in the method of the invention, of (1) microsphere diameter, the microsphere being spheπcal, and (n) microsphere color

The "diameter" of the microsphere preferably ranges from about 0 001 microns to about 100 microns, and most desirably ranges from about 0 02 microns to about 20 microns The microsphere can be of any mateπal that would function in the methods of the invention Preferred mateπals for the microspheres include, but are not limited to, mateπals such as latex and polystyrene The microspheres can be synthesized or purchased from commercial suppliers, e g , from Molecular Probes, Eugene, Oregon, as well as other vendors

The "color" of the microsphere color is determined by the presence within the microsphere of one or more dyes, with each dye being of a different color, different intensity, or different color and different intensity Namely, the dye preferably is either a fluorescent or luminescent dye. such as are known in the art. e g , AMCA. fluorescein. Rhodamme 6G, The Alexa Dye Seπes from Molecular Probes (Eugene, Oregon) Cy3, tetramethylrhodamine, ssamme rhodamme B, Texas Red, organic luminescent dyes, as well as others Each dye desirably is of a color that can be distinguished from other color dyes using conventional instrumentation, such as flow cytometers and fluorescent particle counters made, for instance, by Beckman-Coulter, Fullerton, California Alternately, each dye desirably can be of the same color, but of an intensity of color that can be distinguished from other intensity of color dyes using conventional instrumentation In some embodiments, it may be desirable to use a single dye per microsphere to identify the microsphere Preferably, more than one dye (e g , fluorescent and/or chemiluminescent, same or different colors, same or different intensities of color) can be used per microsphere to provide a unique identification of the microsphere The most important consideration for the microspheres is that the dyes in each microsphere are present alone or m combinations or at intensity levels which can be detected with minimal overlap by the assay detection system (1 e each dye or dye combination can be easily, discretely identified by the analysis instrumentation)

Thus, a plurality of microspheres can be used in the invention wherein the different types of microspheres each have (1) the same size but differ in color, (2) the same color but differ in size, (3) differing size and diffeπng color, (4) the same size and color and differ m intensity of color, (5) the same size but differ in color and intensity of color, (6) the same color but differ in size and intensity of color, (7) diffeπng size and diffeπng color and diffeπng intensity of color, or (8) any other combination of size/ color/intensity that uniquely identifies the microsphere Since luminescent and fluorescent dyes are detected by different means, the combination in a microsphere of luminescent and fluorescent dyes further can be employed to uniquely identify the microsphere A particularly preferred microsphere according to the invention, how ever, is a fluorescent microsphere (l e , FM) Furthermore, the combination within a microsphere of more than one color and/or intensity of color dye adds additional options for unique identification of the microsphere As further descπbed below, it is important to the invention that each microsphere be capable of being uniquely identified However, placement in the array, and attachment to a particular probe, further provide another level of complexity This additional level of complexity means that it is possible that a microsphere of a defined type (I e , size, color, intensity of color) can be used more than once in an array, for instance, by attachment to differing probes known to be present in different parts of the array

Pi obe/Mici osphei e Attachment

Probe attachment to microspheres can be accomplished by any one of several possible techniques For instance, one such technique employs microspheres with carboxyl groups on their surface These microspheres desirably have their surface-carboxyl groups activated with a carbodnmide They then optimally are reacted with the am e-con ugated probes, forming a covalent, amide bond (e g , as descπbed in Molecular Probes Product Information Sheet, "Working with FluoSpheres® Fluorescent Microspheres", Section 7, "Covalent Coupling of Proteins to Carboxylate-Modified Microspheres", ©1997, as well as by other means)

The attachment of the probes to the microspheres desirably is at a ratio of probe number per microsphere so as to allow the most accurate and feasible correlation between microsphere number enumerated at the conclusion of the assay and the number of nucleic acid molecules which are the target of that particular probe in the sample to be assayed The number of probes to be attached per microsphere is expeπmentally determined, and preferably is vaπed to accommodate individual expression levels For example, for a very low abundance mRNA or DNA (l e , a low level expression or low copy number gene), a low probe/microsphere ratio would produce a system in which microsphere number would more closely reflect actual number of nucleic acid copies At the extreme end. one probe attached per one microsphere would produce a 1 1 correspondence between microsphere number and nucleic acid copy number Alternatively, in order to avoid "saturating" the number of available probe-micro spheres by high-expression level genes, a higher probe/microsphere ratio would be desirable Of course, the number of probes attached per microsphere is a factor that must be assessed in the end stage analysis of gene expression In cases where it is anticipated that the gene has a high level of expression, it may be desirable to dilute the sample, or run the assay on various dilutions of sample, and still employ a one probe per microsphere. Alternately, it may be desirable to run an assay in duplicate, or triplicate, and compare results, for instance, with the first assay containing all probe/microsphere combinations at a ratio of one probe per microsphere, the second assay containing all probe/microsphere combinations at a ratio of ten probes per microsphere, and the third assay containing all probe/microsphere combinations at a ratio of one hundred probes per microsphere. These are all obvious and routine manipulations that are well within the skill and understanding of those conducting the assays.

Probe/Microsphere Placement to Generate Array

The method of the invention desirably is carried out by placing each probe/microsphere in a separate, specific microlocation of a multi-compartmentalized container so as to generate an array, and performing all subsequent steps in each of the separate array microlocations. Thus, according to the invention, each probe/microsphere is placed in a specific microlocation of a multi-compartmentalized container. A "multi- compartmentalized container" is a multiwell plate (e.g., a microtiter plate having separate wells) or other suitable container as described below. Preferably each microlocation of the multi-compartmentalized container is separately addressable, e.g., to facilitate probe/microsphere placement.

A "microtiter plate" is a plate comprising many microlocations which is commercially available. Generally, a microtiter plate comes in a variety of sizes and configurations, e.g., 96-wells, 384-wells, etc. Any appropriately sized microtiter plate can be employed in the invention. Furthermore, multi-compartmentalized containers having microlocations even smaller than those present on commercially available microtiter plates also can be used. For instance, the reactions can be carried out in separation microlocations of a microchip, e.g., as where the microchip's surface is etched to create separate indentations, or where holes are created within,with each indentation or hole serving as a microlocation where an independent probe hybridization reaction can take place, or in microwells cast in plates, elastomers or metals, etc.

The material of the multi-compartmentalized container is made optionally is any solid substrate that can be employed in the invention, e.g., film, glass, Si, modified silicon, ceramic, plastic, resins, or any type of appropriate polymer such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, or polydimethylsiloxane (PDMS). Preferred substrates according to the invention are glass and plastic. The solid substrate can be any shape or size, and can exist as a separate entity or as an integral part of any apparatus (e.g., cuvette, plate, vessel, device, and the like). It further is assumed that the material of the substrate is such that it does not interfere with the hybridization reaction, and can withstand any necessary manipulations, e.g., heating, centrifugation, etc., that are needed in order to effect hybridization.

By "array" (or "matrix") is meant an arrangement of microlocations (i.e., "locations") on the substrate. The microlocations can be arranged in two dimensional arrays, three dimensional arrays, or other matrix formats. The number of microlocations can range from one microlocation to a plurality of microlocations (e.g., from two to hundreds of thousands). Desirably an array is comprised of microlocations in rows and columns. Microlocations can be of any shape, and preferably, are either round, square, or rectangular. The thickness and dimensions of the multi-compartmentalized container, and/or the arrays produced according to the invention can vary dependent upon the particular needs of the user.

Thus, according to the invention, specific probes, attached to specifically colored and sized microspheres are then placed into an arraying container such as a multi-well plate (e.g., having 96-, 384-, or 1536- wells). As discussed above, while it is possible that more than one different probe/microsphere could have the same microsphere size and color, desirable each probe within a single microlocations (e.g., well) will be attached to a microsphere of a unique combination of color and size. Thus, for example, one well could contain probes for the BRCA1 gene and the BRCA2 gene and the c-Myc gene. There could be thousands of BRCA-1 -specific probe/microspheres in the array, thousands of BRCA-2-specific probe/microspheres in the array, etc. Importantly, it may be desirable for a certain experiment that all the BRCA-1 -specific probe/microspheres in the array will be of the same color-size-intensity combination, and that color-size-intensity combination is not repeated within that well. Consequently, the combination of color, size, intensity and well location (i.e., microlocation, or exact position on the multi-compartmentalized container) of microspheres uniquely identify those microspheres as the probes for a specific gene. It is this novel utilization of color, size, intensity and well location which confers the array capability to AMA/FAMA. The theoretical underpinnings of the invention further are described in Example 1.

The absolute number of probe/microspheres which can be placed in a single microlocation desirably is experimentally optimized in the development of the assay for particularized uses. This number will depend upon the volume of the microlocation (i.e., the volume of the well), the number of probe/microsphere replicates per gene, probe length, as well as a variety of other factors (i.e., factors inherent to each particularized use).

By comparison, as a less preferred embodiment, but still another way to do the assays according to the invention, the position of one microsphere in the assay relative to any other could be randomized. This would be accomplished by having unique identifiers for microsphere type (e.g., size, color, and/or intensity of color), as previously described, and only using a particular type of microsphere to attach to a specific type of probe (e.g., using a particular microsphere only once in an assay). In this embodiment, hybridization could be carried out in a container that is not compartmentalized (e.g., in a fluid-filled sac). The number of recovered particular microspheres (e.g., size, color, and/or intensity) in this case would tell the number of hybridizations per particular probe. Of course, this embodiment allows a substantially lesser number of nucleic acid species to be assessed.

The present invention accordingly further provides an improvement of an array-based hybridization method for nucleic acids, the improvement comprising the use of fluorescent or luminescent microspheres for attachment to single stranded probes to obtain probe/microspheres, the probes directed to (i) genes of interest present in the sample, or (ii) cDNA reverse-transcribed from RNA produced by specific genes of interest present in the sample, wherein the combination of color, and size of probe/microspheres uniquely identify those probe/microspheres as specifically hybridizing to:

(a) the specific gene itself, and the level of signal detected corresponds to the level of representation of the gene in the sample; or

(b) cDNA reverse-transcribed from RNA produced by a specific gene, and the level of signal detected corresponds to the level of expression of the gene. Furthermore, the invention also desirably provides an improvement of an array-based hybridization method for nucleic acids, the improvement comprising the use of fluorescent or luminescent microspheres for attachment to single stranded probes to obtain probe/microspheres, the probes directed to (i) genes of interest present in the sample, or (ii) cDNA reverse-transcribed from RNA produced by specific genes of interest present in the sample, the hybridization method is carried out by placing each probe/microsphere in a separate, specific microlocation of a multi-compartmentalized container and performing all subsequent steps in each separate microlocation, wherein the combination of color, size, and position of microlocation of the probe/microspheres uniquely identify those probe/microspheres as specifically hybridizing to:

(a) the specific gene itself, and the level of signal detected corresponds to the level of representation of the gene in the sample; or (b) cDNA reverse-transcribed from RNA produced by a specific gene, and the level of signal detected corresponds to the level of expression of the gene.

Probe/Microsphere and DNA (or cDNA) Hybridization

According the invention, preferably the biotin-conjugated DNA (or cDNA) produced from the sample is hybridized to the probe/microspheres under conditions sufficient to effect specific hybridization and obtain probe/microsphere/DNA (or cDNA) hybrids. This preferably is done by dividing up sample DNA (or cDNA) into aliquots for distribution into each microlocation (e.g., each well of a microtiter plate). Probe/microspheres desirably are mixed with the biotin-conjugated DNA (or cDNA) and hybridized under standard conditions using standard buffers within each well (as further described below). Washing of the probe/microsphere/DNA (or cDNA) hybrids is then carried out to wash non-hybridized DNA (or cDNA) will be washed away from hybridized DNA (or cDNA) using standard stringency hybridization and washing conditions. Desirably, all the probe/microspheres are collected following the wash step(s) using centrifugation to "pellet" the probe/microspheres. At this point some of the pelleted probe/microspheres will be hybridized to DNA (or cDNA), while some of the recovered probe microspheres will not. Selective capture of those pelleted probe/microspheres which are hybridized to DNA (or cDNA) is done in the next step of the process.

Generally, according to the invention, hybridization is carried out by standard means. Reaction conditions for hybridization include provision of appropriate salts and buffers with each enzyme incubation. The reaction conditions for hybridization desirably are maintained such that the probe/microspheres stably and specifically hybridize to the target biotin- conjugated DNA (or cDNA). The hybridization reaction mixture preferably is maintained under hybridizing conditions for a time period sufficient for the probe/microspheres to hybridize to complementary nucleic acid sequences present in the biotinylated DNA (or cDNA) sample to form probe/microsphere/DNA (or cDNA) hybrids. Such "hybridizing conditions" (i.e., "hybridization conditions") includes subjecting a hybridization reaction mixture to time, temperature, and pH conditions needed to allow the probe/microspheres to hybridize with the complementary nucleic acid sequence of the sample DNA (or cDNA). Such time, temperature, and pH conditions are well known in the art, and depend, for instance, on the length and composition (e.g., guanidine and cytosine content) of the probe to be hybridized, the degree of complementarity between the probe and the sample DNA (or cDNA), the stringency of the hybridization employed, and the presence of salts or additional reagents in the hybridization mixture such as may affect the kinetics of the hybridization. As defined herein, "stably" (or stable) hybridizing means that the hybridization desirably has a T_m greater than the temperature under which the hybridization is to be carried out (i.e., typically from about 20°C to about 40°C). "Specific" hybridization means that the length and/or sequence complexity of the probes employed for hybridization is/are sufficient to prevent any non-desirable spurious hybridization such as might occur between sequences that are only partially complementary.

Typical hybridization conditions include the use of solutions buffered to pH values ranging from about 4 to about 9, hybridization temperatures ranging from about 18°C to about 70°C, and for time periods ranging from about 0.5 seconds to 24 hours. Hybridization preferably is carried out for from about 15 to about 30 minutes at room temperature in a solution that comprises about 1.5 M NaCl and 10 mM EDTA, although other hybridization conditions also desirably can be employed.

Typically, preferably hybridization is carried out in a hybridization mixture that contains up to about 50% deionized formamide and about 10% dextran sulfate in 2X SSC (with 20X SSC containing 175.32 g sodium chloride and 88.23 g sodium citrate, brought to 1 liter with distilled water), or an equivalent reaction mixture in which hybridization can be carried out. Optimally, prior to hybridization, the probe/microspheres are heated to approximately 95°C for from about 1 to about 10 minutes in order to eliminate any secondary structure formation in the strands and/or any binding between strands. The heated mixture preferably is allowed to cool to about room temperature prior to use in hybridization.

Desirably the sample DNA (or cDNA) is prehybridized in the hybridization mixture at about 50°C for about 5 minutes. The probe/microspheres are then added, and incubation is allowed to proceed, preferably at a temperature that ranges from about room temperature to about 37°C (depending on probe characteristics), desirably for at least from about 10 seconds to overnight, preferably for about 2 hours, and even more preferably for at least about 10 seconds to about 10 minutes, to effect the hybridization of the probe/microsphere to the hybridizing strand of the DNA (or cDNA) sample. Optionally, the hybridization temperature can be increased or decreased from room temperature. Preferably, washing of the probe/microsphere/DNA (or cDNA) hybπds is earned out The cooled mixture preferably is washed to remove unhybπdized DNA from the mixture, desirably which is accomplished by successive incubations in wash solution (e g , as is well known in the art), wherein fresh wash solution is employed for each wash Alternately the wash preferably is accomplished by continuous flow of fresh wash solution over the probe/microsphere/DNA (or cDNA) hybπds Typical characteπstics of washing include a temperature of approximately 65 °C, a low salt concentration, and the presence of a detergent such as sodium dodecyl sulfate For instance, post-hybπdization washes of about 5 minutes each can be done as follows preferably 4X SSC for up to two washes, optimally followed by 2X SSC for up to two washes, desirably followed by a 0 1 X SSC wash, and optionally followed by a 0 05X SSC wash Wash conditions used m the invention can vary depending on many factors that have been well descπbed in the art, e g , probe length, probe composition, etc Furthermore, it may be desirable to carry out the wash at room temperature, and using a higher salt concentration (or even the same buffer as is employed for hybπdization)

Selective Captui e of Probe/Mtci osphei e/DNA (or cDNA) Hybrids

According to the invention streptavidin-conjugated magnetic particles are employed to selectively capture the probe/miciosphere/DNA (or cDNA) hybπds Such streptavidm- conjugated magnetic particles can be purchased from any appropπate commercial vendor (or synthesized), and desirably, are obtained from Dynal, Lake Success, New York Preferably, the streptavidin-conjugated magnetic particles are incubated with the probe/microsphere/DNA (or cDNA) hybπds under conditions such that they bind to the biotin-conjugated DNA (or cDNA) present in the probe/microsphere/DNA (or cDNA) hybπds. and streptavidin-conjugated magnetic particles/probe/microsphere/DNA (or cDNA) hybπds are obtained Preferably this incubation is earned out with an excess of streptavidin- conjugated magnetic particles using standard conditions Binding can be earned out by any appropπate means, and particularly, can be earned out by the means descπbed by the commercial vendor of the streptavidin-conjugated magnetic particles (e g , in the Dynal product literature, the binding buffer is approximately 1M NaCl)

Following standard wash steps (e g , with buffer), preferably a magnet is employed to capture the streptavidin-conjugated magnetic particles/probe/microsphere/DNA (or cDNA) hybπds The magnet optionally can be a permanent-type magnet, or can be an electromagnetic field The magnetic field applied to the mixture consolidates a "pellet" comprised of streptavidin-conjugated magnetic particles/probe/microsphere/DNA (or cDNA) hybrids. Optionally, the non-bound magnetic particles are washed away from this pellet (e.g., with buffer). Subsequently, the pellet consolidated by the magnetic field is isolated.

Assessment of Captured Microspheres

In the next step of the method, the captured microspheres are assessed. Namely, using methods analogous to flow cytometric fluorescent particle detection, preferably each well is aspirated of its streptavidin-conjugated magnetic particles/probe/microsphere/DNA (or cDNA) hybrid, and these hybrids desirably are passed through a fluorescence analysis system capable of simultaneously measuring the dye color and intensity via fluorescence emission (e.g., at one or more excitation wavelengths) or luminescence, and the microsphere diameter via a measurement such as electrical conductance or low angle forward light scatter. Preferably, the number and type of microsphere captured per microlocation is determined, and this information is assessed along with information regarding the type of probe employed per microlocation, to analyze the expression of a plurality of genes present in the sample.

It may be desirable to purify the microspheres from other attached components prior to microsphere analysis. This can be done by a variety of means. For instance, optionally the streptavidin-conjugated magnetic particles/probe/microsphere/DNA (or cDNA) hybrid pellet is exposed to denaturing conditions under a magnetic field in order to remove hybridized DNA (or cDNA) conjugated to the magnetic particles from the streptavidin-conjugated magnetic particles/probe/microsphere/ DNA (or cDNA) hybrids. The probe/microspheres (i.e., the fraction not attracted to the electric field) are then collected by centrifugation. Optionally, the probe can be degraded, e.g., with DNAse. The resultant microsphere optionally is washed, and subjected to further analysis. Thus, the invention desirably provides a means of assessing the streptavidin- conjugated magnetic particles/probe/microsphere/DNA (or cDNA) hybrids to determine the number and type of microsphere captured per microlocation, and considering this information along with the type of probe employed per microlocation to analyze the expression of a plurality of genes present in the sample.

Potential for Recovery of Analyzed DNA (or cDNA)

In some instances, post-analysis recovery of the sample DNA (or cDNA) is desirable. For example, in homology-based hybridization experiments, in which the exact sequence of the DNA (or cDNA) is unknown at every base, recovered sample could be further analyzed for exact sequence data, either by direct sequencing or by using the recovered sample as a template for further amplification followed by sequencing. In another example, the assay could be used to screen for rare events (e.g., mutation events), such as DNA or mRNA from rare tumor cells in a predominately normal tissue sample. In these types of applications, it could be valuable to evaluate the DNA (or cDNA) bound to the microsphere as a way to verify that the positive signal, in fact, came from the mRNA of concern. In cases in which the outcome of the assay would direct future therapy of a patient, such verification would minimize the chance of false positive signals.

Two considerations are key to this sample recovery. First, the hybridized DNA (or cDNA) must be retained on the microsphere throughout analysis. Second, a technique must be used to allow sample recovery. Fluorescence Activated Cell Sorting (FACS) is a common technology that is available in most hospitals and universities, with equipment for carrying out FACS being sold by Becton-Coulter and other commercial vendors. FACS uses the principle of flow cytometry, and adds electrically induced deflection of certain droplets to collect them. FACS using the principle of flow cytometry and the electrostatic deflection of selected particles into a container desirably is one technique which could be employed according to the invention to accomplish selective recovery of sample DNA.

The biotinylated DNA (or cDNA) can be recovered from the process as described above. Namely, as the streptavidin-conjugated magnetic particles/probe/microsphere/DNA (or cDNA) hybrid pellet is exposed to denaturing conditions under a magnetic field, biotinylated DNA (or cDNA) will be recovered bound to the streptavidin-conjugated magnetic particles and subjected to further analysis, such as PCR or sequencing. Optionally, the DNA (or cDNA) can then be recovered from the streptavidin-conjugated magnetic particles, for instance, by treating with hot formamide (e.g., at a temperature of from about 70-95°C, optimally about 90°C).

Thus, the present invention optionally provides a method wherein the DNA (or cDNA) is recovered from the streptavidin-conjugated magnetic particles/probe/microsphere/DNA (or cDNA) hybrids. This allows further manipulation and assessment of the DNA (or cDNA).

Miniaturization ofAMA/FAMA assay

An AMA/FAMA assay that detects and analyzes many targets in parallel requires handling of a large number of fluids and microspheres. While these requirements can be satisfied using conventional microtiter plate robotic fluid handling systems, scaling the process up to detect over ten times more samples can become unwieldy. Two approaches described below optimally can be employed to increase throughput of this assay. Like numbering is employed in Figures 8 and 9 to depict like components in the devices, (a) Parallel fluidic switching assembly A preferred parallel fluidic switching assembly is shown in Figure 8. As can be seen from this figure, a multi-compartmentalized container (10), preferably a conventional microtiter plate is used to perform the assay. In the simplest case, desirably conventional dispensing robotics are used to supply the microlocations (e.g., microtiter wells) with hybridization and washing reagents and probe microspheres. The fluidic switching assembly desirably consists of a series of micropipettes or other tubing arrangement (20) to supply the microlocations and which have the same footprint (i.e., configuration, so they components mesh) as that of the appropriate microtiter plate. A magnetic assembly (e.g., electromagnets located in the supplying micropipettes or tubing anangement, or an assembly equivalent to electromagnets) optimally is programmed to magnetically attract labeled microspheres from specific wells and draw them up into the appropriate pipette or tube. Movement of the microspheres in a transport liquid (e.g., buffer) would draw the microspheres into the switching assembly and then provide routing (30) into the appropriate detection system. Using this preferred embodiment, optimally each microlocation (i.e., each well of the microtiter plate) is interrogated and analyzed under computer control without the need for robotic handling of the finished assay products.

Figure 8 is depicted as having a single port (30) for providing input and output to the multi-compartmentalized container. However, multiple ports can be configured in the device, for instance, multiple ports for input and/or output, and with the ports for inputting and aspirating off reagents and components (e.g., buffers for hybridization, probe/microspheres, magnetized particles, etc.) being separated. It further is possible that the micropipettes or other tubing arrangement itself provides a means for inputting and aspirating off reagents and components. Further not depicted in Figure 8 is a configuration wherein a magnetic particle positioning system is housed separately from the series of micropipettes or other tubing arrangement, and is employed to correctly position magnetic particles (i.e., either before or after hybridization to probe/microspheres). In this scenario, essentially, the electromagnets are positioned not within the micropipettes or tubing arrangement itself, but are sequestered in a separate area, e.g., positioned above the micropipettes or other tubing arrangement (20), as depicted by (40) in Figure 9, and as further described below. Alternately, the electromagnets can be position below the multi-compartmentalized container, i.e., either alone, or, in addition to electromagnets positioned above the micropipettes or other tubing arrangement.

(b) Integrated fluidic switching assembly As depicted in Figure 9, in this preferred embodiment, desirably the multi- compartmentalized container (10) is a microtiter plate, or a plate containing an anay of microlocations (i.e., "reaction chambers") having dimensions less that 5 mm x 5mm (i.e., smaller than the typical microtiter plater). The reaction chambers preferably are etched or molded into glass or plastic or other appropriate material and, when attached to the fluidic switching assembly, form an environment that is sealed from the ambient room conditions and thus reduces the incidence of contamination by unknown and unwanted environmental agents.

Preferably the fluidic switching assembly consists of one or more etched or molded channels (34 and 38) in glass or plastic that serve to move the fluids, microspheres and magnetic particles and other reagents (optimally using hydrostatic pressure), and serve as an inlet into the reaction chambers. Similarly, desirably the fluidic switching assembly consists of one or more etched or molded channels (30) in glass or plastic that serve to move the fluids, microspheres and magnetic particles and other reagent (optimally using hydrostatic pressure), and serve as an outlet from the reaction chambers, e.g., to a detection device. It is possible for a channel to serve as inlet and outlet, i.e., with appropriate partitioning.

These channels desirably connect to a series of micropipettes or other tubing system (20) having the same footprint and the multi-compartmentalized container, and which provide means of inputting reagents and components, etc. to the container, as well as a means for removing these items from the container. Within the channels and/or series of micropipettes or other tubing system (i.e., 20 both in the present device depicted in Figure 9, as well as that depicted in Figure 8), a series of micromachined valves desirably are employed to control the quantity and path of fluids/components through the system (i.e., from the reagent inputs into the appropriate reaction chamber, out of the appropriate reaction chamber and to the detection system). These opening and closing of these valves preferably is controlled by hydrostatic pressure, puffs of air, and/or are electrically activated.

A magnetic particle positioning system (40) located above the reaction vessel plate (or optimally is integrated in series of micropipettes and tubing (20), as depicted in Figure 8) preferably is used to stir, attract and repel the magnetized particles. While the magnetic particle positioning system is positioned above the reaction vessel plate (10) and above the series of micropipettes or other tubing system (20) in Figure 9, this magnetic particle positioning system optionally can be positioned below the series of micropipettes or other tubing system (20). and below and directly attached to the reaction vessel plate ( 10). Furthermore, optionally there can be more than one magnetic particle positioning system, for instance, one positioned above and directly attached to the series of micropipettes or other tubing system (20). and one positioned below and directly attached to the reaction vessel plate (10).

This magnetic positioning system (i.e., as well as that depicted in Figure 8) desirably consists of a multitude of computer controlled electromagnets, or permanent magnets controlled by electromagnets, which control the trajectory of the magnetic particles through the system. Thus, desirably, a combination of magnetic positioning and/or fluid pressure are used to move the magnetic particles into the detection system for analysis. The electromagnets themselves desirably are any item comprised of metal wire wound around a ferrous core (or other magnetizable core) with enough repetitions of winding such that the item is capable of functioning as an electromagnet. These electromagnets and/or permanent magnets can be prefabricated and deposited into a device, or constructed as part of the device, e.g., using photolithography techniques. Preferably according to the invention, the number of electromagnets is equivalent to the number of microlocations (e.g., reaction chambers) of the multi-compartmentalized container, and directly controls input to and aspiration from each of the microlocations (e.g., reaction chambers) of the multi-compartmentalized container.

Accordingly, the positioning of the magnets desirably minors that of the anay. Alternately, a magnet can control more than one microlocation. Under this scenario, the positioning of the magnets desirably can mirror that of the anay, or. can be substantially different from that of the anay. When permanent magnets are employed, these magnets can be controlled, for instance, by energizing or de-energizing the magnet, i.e., as with an electric charge.

Electromagnets can be controlled, for instance, by switching the field of the applied cunent. Preferably the electromagnets are controlled by an appropriate power source, e.g., a DC power source capable of providing from about 0 to about 50 milliAmps per electromagnet coil. Desirably, each of the electromagnets is independently controlled, e.g., making use of a multiplexer to control signal inputs/outputs, and computer programming, such as are well known in the art.

In addition to the device configurations shown in Figures 8 and 9, the device can have the configuration depicted in Figure 10. Namely, in this device, the reaction chambers themselves are present inside of a housing, and in intimate association with the channels and micropipettes or other tubing system, thus allowing a "microchip" type assembly. For instance, the reaction chambers actually can be the wells of a microtiter plate, or etched onto the surface of a glass slide, or other appropriate material. Of course, the type, number, and location of the input/output channels, and the number and the location of the magnetic particle positioning system with respect to the chambers can vary, all as previously described. Accordingly, as described above, the present invention desirably provides a fluidic switching assembly (i.e., parallel or integrated) that combines pressure and magnetic elements to move liquids and particles from a microtiter plate into a detection system. In particular, desirably the method of the invention is carried out using a multi- compartmentalized container that is integrated onto a fluidic switching system that contains computer controlled magnetic particle position control. Sequential energizing of adjacent electromagnets can be used to move magnetic particles among different well and through the fluidic system.

While the foregoing discusses two approaches that optimally can be employed to increase throughput of this assay, other approaches and instrumentation such as would be convenient for a particular application of the method of the invention optionally can be employed instead of these (e.g., modification of the apparatus and method described in PCT International Application WO 98/38490, herein incorporated in its entirety by reference).

Alternative means of Effecting Microsphere/Magnetic Particle Binding and Capture

An important part of the present invention is the manner in which binding between the sample nucleic acid and the magnetic particle is effected. The foregoing description is directed toward use of streptavidin and biotin as means of binding. However, as previously indicated, there are alternative means of ensuring such binding. All that is required is that there be provided a "first binding partner" and a "second binding partner" which together constitute a binding pair. Of course, further according to the invention, the first and second binding partner are attached to their respective molecules (i.e., either a nucleic acid, or a magnetic particle) in some fashion, e.g., for instance, by a covalent interaction (e.g., chemical linkage and/or fusion), or by a noncovalent interaction. In particular, preferably a first binding partner is attached to a nucleic acid of interest, and a second binding partner is attached to the magnetic particle.

Namely a "binding pair" is comprised of (1 ) a first binding partner that is capable of specifically binding to a second binding partner, and (2) a second binding partner that is capable of specifically binding to a first binding partner. The binding pair thus optimally binds so as to form a complex. A "complex" of the first and second binding partner is any interaction, e.g., covalent or noncovalent, between the first and second binding partner, and, preferably, is a noncovalent interaction. Preferably according to the invention, this complex is one that can be dissociated - not spontaneously, but only with application of the appropriate conditions.

The complex is brought about by any means of contacting the first binding partner with the second binding partner. Such "contacting" can be done by any means known to those skilled in the art, and described herein, by which the apparent touching or mutual tangency of the first and second binding partner can be effected. In a prefened embodiment of the invention one of the binding partners preferably is an antibody (e.g., a polyclonal, monoclonal, bispecific, and/or single-chain antibody). Such an antibody includes, but is not limited to, immunoglobulin molecules and immunologically active portions of immunoglobulin molecules such as portions containing a paratope (i.e., an antigen binding site), such that the antibody comprises, for example, either intact immunoglobulin molecules or portions thereof, such as those known in the art as Fab, Fab', F(ab') and F(v). The antibody can be, for example, a monoclonal antibody, a polyclonal antibody, a single-chain antibody (e.g., directly fused to an oligonucleotide, for instance), and a bispecific antibody (e.g., that in and of itself can be a bispecific molecule having one paratope directed to an epitope of a first binding partner, and another paratope directed to an epitope of a second binding partner).

Prefened antibodies according to the invention are, of course, those that are directed against the other binding partner that constitutes the binding pair. The antibody can be produced by any suitable technique, e.g., conventional techniques for preparing monoclonal, polyclonal, single-chain, and bispecific antibodies, as well as more cunent recombinant DNA techniques that are familiar to those skilled in the art. In particular, bispecific antibodies can be made by a variety of means, e.g., chemical techniques (see, e.g., Kranz et al., Proc. Natl. Acad. Sci.. 78. 5807 (1981)), for instance, disulfide cleavage reformation of whole IgG or, preferably, F(ab')₂ fragments; fusions of more than one clone to form polyomas that produce immunoglobulins having more than one specificity (see, e.g., Segal et al., In Cunent Protocols in Immunology. Coligan et al. (eds.), vol. 1, 2.13.1 -2.13.16 (John Wiley & Sons, Inc. (1995))); or by genetic engineering.

In particular an especially prefened antibody according to the invention is that directed against the agent digoxigenin. Preferably, digoxigenin is attached to the sample nucleic acid, and the anti-digoxigenin antibody is attached to the magnetic particles. Of course, it further is possible according to the mvention to carry out capture of the sample nucleic acid without use of magnetic particles. This can be done, for instance, by binding the second binding partner not to a magnetic particle, but instead, to a coated surface (e.g., slab, bead, or other) Use of such a surface coated with the second binding partner optimally provides for capture on the coated surface of the first binding entity associated with the nucleic acid. Such capture on a surface still allows for the nucleic acid to be assessed either while still attached, or, following its dissociation from the coated surface. As an example, in the case where the coated surface is a well, e.g., a well of a microtiter plate, the further analysis of the nucleic acid (e.g., PCR) can be done directly in the well. Other vanations and manipulations would be apparent to one skilled in the art Alternately, as with release from the magnetic particles as previously descπbed, release can be effected with use of the appropπate denaturant, such as hot formamide

Thus, the present invention further provides a method for analyzing the expression of a plurality of genes present in a sample, wherem the method compπses the steps. (a) isolating from the sample the RNA produced by the genes;

(b) reverse transcnbing the RNA into cDNA;

(c) attaching a first binding partner to the cDNA to obtain a first binding partner- conjugated cDNA,

(d) obtaining a plurality of single-stranded DNA probes that are capable of specific hybπdization to the cDNA of interest, wherein the probes are modified to allow end- attachment to labeled microspheres:

(e) obtaining a plurality of microspheres of diffeπng types, with microsphere type being determined by the combination to provide a unique identification of the microsphere of (l) microsphere diameter, the microsphere being spheπcal. and

(ii) microsphere color;

(f) obtaining probe/microspheres by attaching the microspheres to the modified single- stranded DNA probes;

(g) placing each probe/microsphere in a separate, specific microlocation of a multi- compartmentalized container so as to generate an anay, and performing all subsequent steps in each the separate microlocation;

(g) hybπdizing the first binding partner-conjugated cDNA to the probe/microspheres under conditions sufficient to effect specific hybπdization and obtain probe/microsphere/cDNA hybπds. (h) washing the probe/microsphere/cDNA hybrids;

(i) obtaining magnetic particles that are coated with a second binding partner and incubating the coated particles with the probe/microsphere/cDNA hybrids under conditions such that they bind to the first binding partner-conjugated cDNA present in the probe/microsphere/cDNA hybrids, and second binding partner-coated magnetic particles/probe/microsphere/cDNA hybrids are obtained;

(j) using a magnet to capture the second binding partner-coated magnetic particles/probe/microsphere/cDNA hybrids; and

(k) assessing the second binding partner-coated magnetic particles/probe/microsphere/cDNA hybrids to determine the number and type of microsphere captured per microlocation, and considering this information along with the type of probe employed per microlocation to analyze the expression of a plurality of genes present in the sample.

Also, this method desirably can be employed for nucleic acid hybridization, in this instance, preferably comprising the steps of:

(a) isolating a sample to be tested for a DNA of interest;

(b) attaching a first binding partner to the DNA to obtain a first binding partner- conjugated DNA;

(d) obtaining a plurality of microspheres of differing types, with microsphere type being determined by the combination to provide a unique identification of the microsphere of: (i) microsphere diameter, the microsphere being spherical; and

(ii) microsphere color;

(f) placing each probe/microsphere in a separate, specific microlocation of a multi- compartmentalized container so as to generate an anay, and performing all subsequent steps in each of the separate microlocation;

(g) hybridizing the first binding partner-conjugated DNA to the probe/microspheres under conditions sufficient to effect specific hybridization and obtain probe/microsphere/DNA hybrids; (h) washing the probe/microsphere/DNA hybπds,

(1) magnetic particles that are coated with a second binding partner and incubating the particles with the probe/microsphere/DNA hybπds under conditions such that they bind to the first binding partner-conjugated DNA present in the probe/microsphere/DNA hybπds. and second binding partner-coated magnetic particles/probe/microsphere/DNA hybπds are obtained.

(j) using a magnet to capture the second binding partner-coated magnetic particles/probe/microsphere/DNA hybπds; and

(k) assessing the second binding partner-coated magnetic particles/probe/microsphere/DNA hybπds to determine the number and type of microsphere captured per microlocation, and consideπng this information along with the type of probe employed per microlocation to analyze the representation of the DNA present m the sample

Preferably these methods are earned out where the first binding partner is digoxigenin, and the second binding partner is an anti-digoxigenm antibody However, other prefened first and second binding partners similarly can be employed

The following example further illustrates the present invention but. of course, should not be construed as in any way limiting its scope.

Example f Theoretical Underpinnings of the Invention

This Example descnbes some of the theoretical underpinnings of the invention Using distinct fluorescent dyes (and/or luminescent dyes) either alone or in combinations of two dyes per microsphere. the number of available anay elements (number of genes which theoretically could be probed in one expenment) can be descπbed by the following equation.

T_a = (L)(S)[0 5(X) + 0 5(X²)]

where:

T_a = Total Anay Elements L = Number of Microlocations (e.g., number of wells)

S = Number of microsphere sizes X = Number of Individual Fluorescent Colors used alone or in dual combination For X, the color can be due to use of distinctly different dyes (i.e., different color dyes), or, use of the same dye at distinctly different intensity levels (which analytical instrumentation can easily distinguish). Using the above equation, as an example, with a 384-well plate, 15 available colors, and 3 microsphere sizes, the Total Anay Elements =138,240. Of course other equations also could be used to describe the anays according to the invention.

A gene with a relatively high level of expression leads to more "copies" of biotinylated DNA (or cDN A) and thus a greater number of specifically-identifiable captured microspheres.

Software can be employed to automatically associate microlocation position, fluorescence/luminescence, and size with particular probe and output number of microspheres (measurement of level of expression) for that particular probe (expression for that particular gene). For instance:

PROBE GENE NUMBER MICROSPHERES DETECTED

AG418 BRCA1 8 9 7 6

BCV54 MDR1 4 3 1

PVT39 UVrexl 2

Example 2: Advantages of the Invention and Illustrative Uses

This Example describes some of the advantages of the invention, and some illustrative uses.

In the present invention, the concunent use of microlocation position in a multi-well plate, several distinct fluorescent "colors", and several distinct microsphere sizes, are combined to produce anays with extremely large numbers of anay elements. The use of magnetic capture to selectively capture the hybridized microspheres is advantageously employed. The number of spheres of a given microlocation position, fluorescent color, and size are analyzed to reflect the abundance of the nucleic acid species for which that element was designed to probe. These elements as well as potentially others, act in combination to produce the present unexpected and novel invention.

The improvements over the prior art that are accomplished by this invention include, but are not limited to, the following. First and foremost, hybridization is carried out in solution, i.e., as opposed to in the solid phase, which generally is less useful for analyzing rare sequences. AMA/FAMA provides a novel system with which to analyze the expression and/or representation of extremely large numbers of genes in a single experiment. Thus, the level of mRNA for a specific gene in the sample is reflected in quantal, digital events, with genes of low abundance mRNA yielding a signal of equal strength and intensity as that of high abundance genes. Low abundance mRNAs simply generate fewer event numbers compared to high abundance mRNAs. Furthermore, the measured sample can be recovered following analysis for further experimentation. Still further improvements would be apparent to those skilled in the arts.

AMA/FAMA provides a methodology enabling analysis of genetic information in an anay-type system, in which even genes with low level of expression produce strong, discrete signals. Typical analyses could use the flow cytometric equipment which is already commonplace in most hospitals and research laboratories. AMA (e.g., FAMA) also provides numerous other advantages.

Potential applications for the invention include, but are not limited to, basic research involving gene expression and clinical analysis of the expression of clinically relevant genes, for example, the expression of genes conferring resistance to anti-cancer drugs in a human tumor. Other applications are such as would be apparent or obvious to one skilled in the art. There are no restrictions or limitations as to the use of the methodology described herein. Basically, the invention can be employed in any experiment that requires hybridization-based analysis of gene expression (i.e., where RNA is the nucleic acid species of interest in a sample), or hybridization-based analysis of gene representation (i.e., where DNA, such as genomic DNA, is the nucleic acid species of interest in a sample).

Accordingly, the present invention provides not only a novel means of hybridization, but also, an improvement over other hybridization methods. In such an improvement of an anay- based hybridization method for analyzing gene expression, the improvement preferably comprises the use of fluorescent or luminescent microspheres for attachment to single stranded probes directed to genes of interest present in the sample to obtain probe/microspheres, the hybridization method carried out by placing each probe/microsphere in a separate, specific microlocation position of a multi-compartmentalized container and performing all subsequent steps in each separate microlocation, wherein the combination of color, size, and microlocation position of probe/microspheres uniquely identify those probe/microspheres as specifically hybridizing to RNA for a specific gene, and the level of signal detected conesponds to the level of expression of the gene.

All of the references cited herein are hereby incorporated in their entireties by reference. While this invention has been described with an emphasis upon a prefened embodiment, it will be obvious to those of ordinary skill in the art that variations in the prefened composition and method may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for analyzing the expression of a plurality of genes present in a sample comprising the steps of: (a) isolating from said sample the RNA produced by said genes;

(b) reverse transcribing said RNA into cDNA in the presence of one or more biotin- conjugated nucleotide(s) to obtain biotin-conjugated cDNA;

(c) obtaining a plurality of single-stranded DNA probes that are capable of specific hybridization to said cDNA of interest, wherein said probes are modified to allow end- attachment to labeled microspheres;

(d) obtaining a plurality of microspheres of differing types, with microsphere type being determined by the combination to provide a unique identification of said microsphere of:

(e) obtaining probe/microspheres by attaching said microspheres to said modified single-stranded DNA probes;

(f) placing each probe/microsphere in a separate, specific microlocation of a multi- compartmentalized container so as to generate an anay, and performing all subsequent steps in each said separate microlocation;

(g) hybridizing said biotin-conjugated cDNA to said probe/microspheres under conditions sufficient to effect specific hybridization and obtain probe/microsphere/cDNA hybrids;

(h) washing said probe/microsphere/cDNA hybrids; (i) obtaining streptavidin-conjugated magnetic particles and incubating said particles with said probe/microsphere/cDNA hybrids under conditions such that they bind to said biotin-conjugated cDNA present in said probe/microsphere/cDNA hybrids, and streptavidin- conjugated magnetic particles/probe/microsphere/cDNA hybrids are obtained;

(j) using a magnet to capture said streptavidin-conjugated magnetic particles/probe/microsphere/cDNA hybrids; and

(k) assessing said streptavidin-conjugated magnetic particles/probe/microsphere/cDNA hybrids to determine the number and type of microsphere captured per microlocation, and considering this information along with the type of probe employed per microlocation to analyze the expression of a plurality of genes present in the sample.

2. The method of claim 1. wherein microsphere color is determined by the presence within the microsphere of one or more dyes, with each dye being of a different color, different intensity, or different color and different intensity.

3. The method of claim 1. wherein said dye is a fluorescent or luminescent dye.

4. The method of claim 1. which further comprises a step following step (b) wherein said cDNA is separated from said RNA.

5. The method claim 4, wherein said separation is carried out by: (a) digesting said RNA; or (b) separating said biotin-conjugated cDNA from said RNA by means of biotin incorporated in said biotin-conjugated cDNA.

6. The method of claim 1 , which further comprises a step following step (j) wherein: (i) said cDNA present in said hybrid is degraded, and the resulting microsphere is washed; or

(ii) said cDNA is recovered from said streptavidin-conjugated magnetic particles/probe/microsphere/cDNA hybrids.

7. The method of claim 1 , wherein one probe is hybridized at each anay microlocation.

8. The method of claim 1. wherein more than one probe is hybridized at each anay microlocation.

9. The method of claim 8, wherein a different probe is employed for each anay microlocation.

10. The method of claim 8, wherein from 3 to 10 different probes are hybridized at each anay microlocation.

1 1. A method for nucleic acid hybridization comprising the steps of: (a) isolating a sample to be tested for a DNA of interest;

(b) biotinylating said sample DNA to obtain biotin-conjugated DNA;

(c) obtaining a plurality of single-stranded DNA probes that are capable of specific hybridization to said DNA of interest, wherein said probes are modified to allow end- attachment to labeled microspheres; (d) obtaining a plurality of microspheres of differing types, with microsphere type being determined by the combination to provide a unique identification of said microsphere of:

(i) microsphere diameter, the microsphere being spherical; and (ii) microsphere color; (e) obtaining probe/microspheres by attaching said microspheres to said modified single-stranded DNA probes;

(f) placing each probe/microsphere in a separate, specific microlocation of a multi- compartmentalized container so as to generate an anay, and performing all subsequent steps in each of said separate microlocation; (g) hybridizing said biotin-conjugated DNA to said probe/microspheres under conditions sufficient to effect specific hybridization and obtain probe/microsphere/DNA hybrids;

(h) washing said probe/microsphere/DNA hybrids;

(i) obtaining streptavidin-conjugated magnetic particles and incubating said particles with said probe/microsphere/DNA hybrids under conditions such that they bind to said biotin-conjugated DNA present in said probe/microsphere/DNA hybrids, and streptavidin- conjugated magnetic particles/probe/microsphere/DNA hybrids are obtained;

(j) using a magnet to capture said streptavidin-conjugated magnetic particles/probe/microsphere/DNA hybrids; and (k) assessing said streptavidin-conjugated magnetic particles/probe/microsphere/DNA hybrids to determine the number and type of microsphere captured per microlocation, and considering this information along with the type of probe employed per microlocation to analyze the representation of said DNA present in the sample.

12. The method of claim 1 1, wherein microsphere color is determined by the presence within the microsphere of one or more dyes, with each dye being of a different color, different intensity, or different color and different intensity.

13. The method of claim 1 1 , wherein said dye is a fluorescent or luminescent dye.

14. The method of claim 1 1 , which further comprises a step following step (j) wherein:

(i) said DNA present in said hybrid is degraded, and the resulting microsphere is washed; or

(ii) said DNA is recovered from said streptavidin-conjugated magnetic particles/probe/microsphere/DNA hybrids.

15. The method of claim 1 1 , wherein one probe is hybridized at each anay microlocation.

16. The method of claim 1 1 , wherein more than one probe is hybridized at each anay microlocation.

17. The method of claim 16, wherein a different probe is employed for each anay microlocation.

18. The method of claim 16, wherein from 3 to 10 different probes are hybridized at each anay microlocation.

19. In an improvement of an anay-based hybridization method for nucleic acids, said improvement comprising the use of fluorescent or luminescent microspheres for attachment to single stranded probes to obtain probe/microspheres, said probes directed to (i) genes of interest present in said sample, or (ii) cDNA reverse-transcribed from RNA produced by specific genes of interest present in said sample, wherein the combination of color, and size of probe/microspheres uniquely identify those probe/microspheres as specifically hybridizing to:

(a) the specific gene itself, and the level of signal detected conesponds to the level of representation of said gene in said sample; or (b) cDNA reverse-transcribed from RNA produced by a specific gene, and the level of signal detected conesponds to the level of expression of said gene.

20. In an improvement of an anay-based hybridization method for nucleic acids, said improvement comprising the use of fluorescent or luminescent microspheres for attachment to single stranded probes to obtain probe/microspheres, said probes directed to (i) genes of interest present in said sample, or (ii) cDNA reverse-transcribed from RNA produced by specific genes of interest present in said sample, said hybridization method is carried out by placing each probe/microsphere in a separate, specific microlocation of a multi-compartmentalized container and performing all subsequent steps in each separate microlocation. wherein the combination of color, size, and position of microlocation of said probe/microspheres uniquely identify those probe/microspheres as specifically hybridizing to:

(a) the specific gene itself, and the level of signal detected conesponds to the level of representation of said gene in said sample; or

(b) cDNA reverse-transcribed from RNA produced by a specific gene, and the level of signal detected conesponds to the level of expression of said gene.

21. A fluidic switching assembly that uses electromagnets or permanent magnets to control movement of materials into and out of a multi-compartmentalized container.

22. The assembly of claim 21 , wherein said electromagnets or permanent magnets are computer controlled.

23. The assembly of claim 21, which is connected to a detection device.

24. The assembly of claim 21 , having the structure depicted in Figure 8.

25. The assembly of claim 24, further comprising one or more ports for input and/or output.

26. The assembly of claim 24, further comprising a magnetic particle positioning system positioned below the multi-compartmentalized container.

27. The assembly of claim 21 , having the structure depicted in Figure 9.

28. The assembly of claim 27, further comprising one or more ports for input and/or output.

29. The assembly of claim 27, further comprising a magnetic particle positioning system positioned below the multi-compartmentalized container.

30. The assembly of claim 21 , having the structure depicted in Figure 9.

31. The assembly of claim 30, further comprising one or more ports for input and/or output.

32. The assembly of claim 30, further comprising a magnetic particle positioning system positioned below the multi-compartmentalized container.

33. The method of claim 1 , which further comprises use of a fluidic switching assembly that uses electromagnets or permanent magnets to control movement of materials into and out of said multi-compartmentalized container.

34. The method of claim 1 1, which further comprises use of a fluidic switching assembly that uses electromagnets or permanent magnets to control movement of materials into and out of said multi-compartmentalized container.

35. A method for analyzing the expression of a plurality of genes present in a sample comprising the steps of:

(a) isolating from said sample the RNA produced by said genes;

(b) reverse transcribing said RNA into cDNA;

(c) attaching a first binding partner to said cDNA to obtain a first binding partner- conjugated cDNA;

(d) obtaining a plurality of single-stranded DNA probes that are capable of specific hybridization to said cDNA of interest, wherein said probes are modified to allow end- attachment to labeled microspheres; (e) obtaining a plurality of microspheres of differing types, with microsphere type being determined by the combination to provide a unique identification of said microsphere of:

(f) obtaining probe/microspheres by attaching said microspheres to said modified single-stranded DNA probes;

(g) placing each probe/microsphere in a separate, specific microlocation of a multi- compartmentalized container so as to generate an anay, and performing all subsequent steps in each said separate microlocation;

(g) hybridizing said first binding partner-conjugated cDNA to said probe/microspheres under conditions sufficient to effect specific hybridization and obtain probe/microsphere/cDNA hybrids;

(h) washing said probe/microsphere/cDNA hybrids; (i) obtaining magnetic particles that are coated with a second binding partner and incubating said coated particles with said probe/microsphere/cDNA hybrids under conditions such that they bind to said first binding partner-conjugated cDNA present in said probe/microsphere/cDNA hybrids, and second binding partner-coated magnetic particles/probe/microsphere/cDNA hybrids are obtained; (j) using a magnet to capture said second binding partner-coated magnetic particles/probe/microsphere/cDNA hybrids; and

(k) assessing said second binding partner-coated magnetic particles/probe/microsphere/cDNA hybrids to determine the number and type of microsphere captured per microlocation, and considering this information along with the type of probe employed per microlocation to analyze the expression of a plurality of genes present in the sample.

36. The method of claim 35, wherein said first binding partner is digoxigenin, and said second binding partner is an anti-digoxigenin antibody.

37. A method for nucleic acid hybridization comprising the steps of:

(a) isolating a sample to be tested for a DNA of interest;

(b) attaching a first binding partner to said DNA to obtain a first binding partner- conjugated DNA; (c) obtaining a plurality of single-stranded DNA probes that are capable of specific hybridization to said DNA of interest, wherein said probes are modified to allow end- attachment to labeled microspheres;

(e) obtaining probe/microspheres by attaching said microspheres to said modified single-stranded DNA probes ;

(f) placing each probe/microsphere in a separate, specific microlocation of a multi- compartmentalized container so as to generate an anay, and performing all subsequent steps in each of said separate microlocation;

(g) hybridizing said first binding partner-conjugated DNA to said probe/microspheres under conditions sufficient to effect specific hybridization and obtain probe/microsphere/DNA hybrids;

(h) washing said probe/microsphere/DNA hybrids;

(i) magnetic particles that are coated with a second binding partner and incubating said particles with said probe/microsphere/DNA hybrids under conditions such that they bind to said first binding partner-conjugated DNA present in said probe/microsphere/DNA hybrids, and second binding partner-coated magnetic particles/probe/microsphere/DNA hybrids are obtained;

(j) using a magnet to capture said second binding partner-coated magnetic particles/probe/microsphere/DNA hybrids; and (k) assessing said second binding partner-coated magnetic parti cles/probe/microsphere/DNA hybrids to determine the number and type of microsphere captured per microlocation, and considering this information along with the type of probe employed per microlocation to analyze the representation of said DNA present in the sample.

38. The method of claim 37, wherein said first binding partner is digoxigenin, and said second binding partner is an anti-digoxigenin antibody.