WO2002038812A2 - Method in quantitative analysis of gene and protein expression - Google Patents

Method in quantitative analysis of gene and protein expression Download PDF

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Publication number
WO2002038812A2
WO2002038812A2 PCT/US2001/047277 US0147277W WO0238812A2 WO 2002038812 A2 WO2002038812 A2 WO 2002038812A2 US 0147277 W US0147277 W US 0147277W WO 0238812 A2 WO0238812 A2 WO 0238812A2
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molecules
immobilized
target
labeled
molecule
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PCT/US2001/047277
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French (fr)
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WO2002038812A3 (en
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Richard V. Denton
James O. Bowlby, Jr.
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Denton Richard V
Bowlby James O Jr
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Application filed by Denton Richard V, Bowlby James O Jr filed Critical Denton Richard V
Priority to AU2002235170A priority Critical patent/AU2002235170A1/en
Publication of WO2002038812A2 publication Critical patent/WO2002038812A2/en
Publication of WO2002038812A3 publication Critical patent/WO2002038812A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the present invention relates to improved methods, compositions and arrays for detection and quantitation of biological or chemical molecules, whether natural or synthetic, including but not limited to areas such as diagnostics of diseases, infection, or other medical conditions, detection of environment hazards, SNP genotyping, search for specific binding partners, such as small molecule drug discovery, determination of gene and protein expression, etc., all relevant to the field of plants, human and animal health, bacteria, viruses, archebacteria, fungi, rickettsia, prions, mycoplasmas, and parasitic organisms.
  • Arrays such as DNA and protein arrays have gained much popularity in the recent years especially for the conduct of genomics, proteomics and diagnostics research. Arrays are used, for example, in comparing the gene expression between normal cells and tumor cells.
  • Nucleic acids from a normal cell sample may carry one detectable label, for example, a fluorescent label, and nucleic acids from a tumor cell sample may carry a different detectable fluorescent label.
  • the two samples are mixed and hybridized to a set of oligonucleotide probes on an array.
  • One hybridization signal from a given spot on the array may indicate hybridization mostly with nucleic acid from normal cells, another hybridization signal may indicate hybridization mostly with nucleic acids from tumor cells, and a mixed hybridization signal may indicate hybridization with both normal and tumor cells.
  • Assays of this type are qualitative at best and assay results represent only an approximate indicator of gene expression.
  • variation in spot sizes and, thus, the number of probes at each spot renders any comparisons between relative expression levels even more difficult to interpret.
  • U.S. patents 6,040,138 and 6,004,755 also attempt to improve the measurement quantitation in differential expression.
  • a relatively small number of normalization controls are introduced.
  • Normalization controls are oligonucleotide probes that are perfectly complementary to labeled reference oligonucleotides that are added to the nucleic acid sample.
  • the signals obtained from the normalization controls after hybridization provide a control for variations in hybridization conditions, label intensity, "reading" efficiency and other factors that may cause the hybridization signal to vary between arrays.
  • the signals (e.g., fluorescence intensity) read from all other probes in the array are calibrated by the signal (e.g., fluorescence intensity) from the control probes thereby normalizing the measurements.
  • our method does not rely on extrapolation from a relatively small set of normalization control probes, with potential attendant extrapolation errors, but rather provides quantitative measurements (i.e. absolute calibration) for each and every site or bead as desired.
  • U.S. patents that address the measurement of analyte concentrations include U.S. patent 5,516,635 (EP 0608370B1) and U.S. patent 5,837,551. These involve both a capture binding agent and a developing binding agent.
  • Related patents in this series include U.S. 5,599,720 (EP 0134215), US 5,171,695 (EP 0271974), U.S. 5,834,319 (EP
  • a method of determining target molecule concentration in an array-based assay where the assay includes the steps of:
  • the substrate is in the form of a planar surface such as a sheet, or in the form of a bead or a microsphere.
  • the method as described herein where the array contains immobilized molecules that are situated at different spots of the array, and the immobilized molecules at different spots are the same or different.
  • the target molecule is labeled with a second tracer moiety that is indirectly measurable.
  • the labeled immobilized molecules and the unlabeled immobilized molecules herein may be the same or different molecular species.
  • a method of determining target molecule concentration in an array-based assay further including the step of providing or determining a dissociation constant for the bound target molecules
  • the second tracer moiety contains an indirectly measurable moiety.
  • the method of the present invention optionally includes the step of determining fractional occupancy of the immobilized molecules by the bound target molecules.
  • a method as described herein where the steps of labeling probe or target molecules and/or measuring probe and target signals are repeated sequentially for each target molecule concentration to be determined, and the target molecule concentrations obtained thereby are compared.
  • a method as described herein where target molecules of different dilutions are employed in a series of concentration determinations to arrive at a dissociation constant for the target molecules.
  • a. method of determining expression of a target molecule in a sample including the steps of: (a) providing a first composition having an array of molecules immobilized on a substrate, where the array contains a plurality of spots, at least one spot having more than one immobilized molecule, where the immobilized molecules are attached to the substrate, and at least one of the immobilized molecules is labeled with a first tracer moiety to form a labeled immobilized molecule that yields a probe signal; (b) providing a second composition having a plurality of target molecules, where at least one target molecule is directly labeled with a second tracer moiety that yields a target signal;
  • target molecules are genes, gene fragments or molecules resulting from expression or reverse transcription of genes or gene fragments; and where concentration of target molecules is related to expression thereof.
  • a method of comparing expression of a first target molecule with expression of a second target molecule including the steps of: (a) providing a first composition having an array of molecules immobilized on a substrate, where the array contains a plurality of spots, at least one spot having more than one immobilized molecule, where the immobilized molecules are attached to the
  • first and second target molecules are genes, gene fragments or molecules resulting from expression or reverse transcription of genes or gene fragments; and where concentration of target molecules is related to expression thereof.
  • the immobilized molecules and target molecules are each oligonucleotides or nucleic acids. Further, optionally, such methods are applicable to the determination of gene or protein expressions. Still optionally, the immobilized molecules situated at different spots of the array may be the same or different, such as in their specificities. Additionally, still optionally, the immobilized molecules that are different from spot to spot are all labeled with the same tracer moiety.
  • the plurality of target molecules in the second composition may be the same or different.
  • the tracer moieties for labeling the immobilized molecules and target molecules are also the same or different.
  • a method as described herein for an array-based assay where the array is supported by a substrate and the substrate has a planar surface, or is a bead or a microsphere.
  • the second composition contains target molecules from more than one sample population, where the tracer moiety for labeling the target molecules from one sample population are different from the tracer moiety for labeling target molecules from another sample population, and concenfration of the target molecules from the one sample population is compared with the concentration of target molecules from the other sample population.
  • a method of comparing gene or protein expression of two or more target molecules from two or more target sample populations where the target molecules from the different target population contains the same molecular species, where at least the target molecules from one of the sample populations are directly or indirectly labeled with a second tracer moiety.
  • At least one type of target molecules are labeled with a tracer moiety that carries a directly measurable or indirectly measurable moiety.
  • the second tracer moiety carries an indirectly measurable moiety
  • tlie target molecules are modified to link, by covalent bonding, to the indirectly measurable moiety.
  • kits that contains: (a) a composition having an array of molecules immobilized on a substrate, where the array contains a plurality of spots, at least one spot having more than one immobilized molecules, where the immobilized molecules are attached to the substrate; and (b) information on at least one dissociation constant for target molecules of one specificity bound to the immobilized molecules.
  • kits as described herein where the kit optionally includes a first tracer moiety for labeling or spiking the immobilized molecules, and/or instructions for at least a second tracer moiety for labeling the target molecules. Moreover, in another embodiment, the kit includes a plurality of different tracer moieties for labeling target molecules of different specificities. In a further embodiment, the kit as described herein includes instructions for determining dissociation constants for target molecules of different specificities bound to immobilized molecules, and/or instructions for determining gene or polypeptide expression.
  • kits containing an array as described herein where the array has for a substrate a planar surface, such as, for example, a sheet, a slide, a silicon wafer, or the substrate may be in the form of beads or microspheres, with one bead or microsphere representing one spot, for example.
  • kits having: (a) a composition that contains an array of molecules immobilized on a substrate, where the array includes a plurality of spots, at least one spot having more than one immobilized molecule, where the immobilized molecules are attached to the substrate; (b) information on at least one dissociation constant for one kind of target molecules bound to the immobilized molecules; (c) a first tracer moiety for labeling or spiking the immobilized molecules; (d) instructions for at least a second tracer moiety for labeling target molecules; (e) instructions for determination of dissociation constant for any target molecules; and (f) instructions for determination of target concentration or gene or protein expression.
  • kits as described herein where the immobilized molecules and target molecules are nucleic acids or oligonucleotides.
  • the kit herein contains immobilized molecules that are polypeptides or proteins.
  • kits that contains: (a) a composition having an array of molecules immobilized on a substrate, where the array includes a plurality of spots, at least one spot having more than one immobilized molecule, where the immobilized molecules are attached to the substrate, and at least one immobilized molecule is labeled with a first tracer moiety to form a labeled immobilized molecule; (b) information on at least one dissociation constant for one kind of target molecules bound to the immobilized molecules; (c) instructions for at least a second tracer moiety for labeling target molecules; (d) instructions for determination of dissociation constant for any target molecules; and (e) instructions for determination of target concentration or gene expression.
  • kits containing: (a) a composition having an array of molecules immobilized on a substrate, where the array includes a plurality of spots, at least one spot containing more than one immobilized molecules, where the immobilized molecules are attached to the substrate, and at least one immobilized molecule is labeled with a first tracer moiety; and (b) information on at least one dissociation constant for target molecules of one specificity bound to the immobilized molecules.
  • the kit as described herein, further including instructions for determination of expression of a target molecule, where the target molecule is a gene or gene fragment, or result of expression or reverse transcription of a gene or gene fragment.
  • kits that contains: (a) a composition having an array of molecules immobilized on a substrate, where the array contains a plurality of spots, at least one spot having more than one immobilized molecules, where the immobilized molecules are attached to the substrate, and at least one immobilized molecule is labeled with a first tracer moiety; and (b) instructions on determining dissociation constant for target molecules bound to the immobilized molecules.
  • the immobilized molecules are any suitable probe for capturing a target molecule that is conventional in the art.
  • probes include: nucleotides, polynucleotides, DNA, cDNA, RNA, mRNA, cRNA, peptide nucleic acids, oligonucleotides, polypeptides, antibodies, enzymes, hormones, cytokines, antigens, other proteins, peptides displayed on phages and other peptides, carbohydrates, polymers containing alpha-, beta-, and omega-amino acids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, mixed polymers, small molecule drugs, fragments, analogs and optical isomers thereof, or combinations thereof that form chimeric molecules.
  • composition having an array of molecules immobilized on a substrate, where the array contains a plurality of spots, at least one spot having:
  • the composition described herein where the proportion is any proportion greater than about 0.16%.
  • proportions suitable herein may be one selected from the group of ranges of: about 0.16% to about 1%, about 1% to about 2%, about 2% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 50%, about 50% to about 70%, or about 70% to about 100%.
  • composition as described herein where the proportion of the first amount to the second amount can be the same or different for different spots of the array.
  • the array is supported by a substrate that conventional in the art.
  • the substrate may be planar in surface, such as on a glass slide or a flat sheet of nitrocellulose, or the substrate may be in the form of a bead or a particle such as a microsphere.
  • the substrate may be glass or silicon or a synthetic material. Examples of substrates include solid-phase synthesis supports, fibers, capillary tubes, silicon wafers, slides, membranes, filters or other sheets.
  • composition as described herein where the immobilized molecules may be attached to the substrate by covalent or non-covalent bonding, either directly or indirectly through a linker.
  • the first tracer moiety may be attached to the immobilized molecules also by covalent or non-covalent bonding, either directly or indirectly.
  • a composition as described herein where the first tracer moiety is removable from the immobilized molecules after attachment. This facilitates reading of the target signal without interference from the probe signal.
  • the first tracer moiety remains attached to the immobilized molecule, and the probe signal can be read in the presence or absence of the target signal.
  • the target signal can be determined by subtraction.
  • the first or second tracer moiety may be the same or different and are each directly measurable.
  • the first or second tracer moiety may be indirectly measurable.
  • the tracer moiety is optionally directly or indirectly measurable and can be any label that is conventional in the art.
  • the first and second tracer moiety may be a radioactive isotope, an enzyme including one catalyzing light emission such as luciferase , a luminescent label, or a bead or microsphere containing one or more of such.
  • luminescent labels include quantum dots, fluorescent labels, energy transfer dyes, chemiluminescent labels such as phosphorescent dyes, bioluminescent labels such as phycobilisomes, colorimetric labels, and combinations thereof.
  • the tracer moiety may be indirectly measurable using any indirectly measurable molecules conventional in the art, such as conventional binding pairs, one of which can carry a directly measurable moiety.
  • an indirectly measurable molecule may be one of a pair of binding partners, including but not limited to: an antibody/antigen pair, a biotin/avidin or strepavidin pair, a digoxigenin/anti-digoxigenin pair, a carbohydrate/lectin pair, a pair of complementary oligonucleotides or nucleic acids that hybridize to each other, a receptor/ligand pair, or a synthetic pair that is chemically synthesized to bind to each other with specificity.
  • Nucleic acid derivatives that are capable of hybridizing to a complementary molecule, such as peptide nucleic acids may also be used as indirectly measurable moieties.
  • binding pairs are used where the molecule to be labeled is chemically modified to comprise one member of the binding pair, the other member of the binding pair being linked or otherwise associated with a directly measurable moiety.
  • composition as described herein where the first tracer moiety is removable from the immobilized molecules and the removal is effected enzymatically, chemically, by light activation or other energy activation, or by a change in temperature.
  • the immobilized molecule is any molecule that is suitable for capturing a target molecule.
  • suitable immobilized molecules include, but is not limited to nucleotides, polynucleotides, DNA, cDNA, RNA, mRNA, cRNA, peptide nucleic acids, oligonucleotides, polypeptides, antibodies, enzymes, hormones, cytokines, other antigens or proteins, peptides displayed on phages or other peptides, carbohydrates, polymers containing alpha-, beta-, and omega-amino acids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, mixed polymers, fragments, analogs or optical isomers thereof, or combinations thereof that form chimeric probes.
  • compositions as described herein where the immobilized molecules situated at different spots of the array are the same or different in the specificities in recognizing or capturing different target molecules.
  • the target molecules situated at one spot may be the same or different in their specificities in binding to the immobilized molecules.
  • a method of determining target concentration in a sample in an array-based assay having the steps of:
  • a method of determining target concentration having the steps of:
  • target concentration determining target concentration.
  • target molecules are oligonucleotides or nucleic acids.
  • the target molecules are any target or analytes conventional in the art.
  • the target molecules may be nucleotides, polynucleotides, DNA, cDNA, RNA, mRNA, cRNA, peptide nucleic acids, oligonucleotides, polypeptides, antibodies, enzymes, hormones, cytokines, other antigens and proteins ' , peptides displayed on phages and other peptides, carbohydrates, polymers containing alpha-, beta-, and omega-amino acids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, mixed polymers, fragments, analogs and optical isomers thereof, and combinations thereof that form chimeric probes.
  • the target molecules may have originated from human or other animal host sources and may be derived from humans or other animals or from infectious or parasitic organisms, such as bacteria, viruses, fungi, prions, etc.
  • a method as described herein where the method is applied to the determination of concentration of a number of target molecules at one spot, where the target molecules may have the same or different binding specificities to different immobilized molecules.
  • a composition as described herein where the first tracer moiety is directly or indirectly attached to the immobilized molecule. Where the first tracer moiety is indirectly attached to the immobilized molecules, such attachment is through an intermediate molecule.
  • the method herein provides an intermediate molecule that is commonly shared among all the immobilized molecules for all the spots of the array.
  • the first tracer moiety is attached to an intermediate molecule, before or after a target molecule has contacted the immobilized molecules, and the probe signal is measured in the presence or absence of the target molecule.
  • FIG. 1 is a diagrammatic representation of an array of the present invention showing an expanded view of one spot on an array, as an example, showing target molecules (two represented here) that have been labeled with a tracer moiety (squares) binding to immobilized probes that have been labeled (4 immobilized probes out of 6 are shown labeled) with another tracer moiety (circles).
  • FIG. 2 is a diagrammatic representation of a process for measuring target signal and probe signal, as an example.
  • FIG. 2A is a- diagrammatic representation of an expanded view of one spot on an array, as an example, showing binding of target molecules that have been each labeled with a tracer moiety (squares) to immobilized molecules that have not been labeled.
  • FIG. 2B is a diagrammatic representation of an expanded view of one spot on an a ⁇ -ay, as an example, showing the addition of a plurality of tracer moieties (circles) to the array for the labeling of the immobilized molecules.
  • FIG. 2C is a diagrammatic representation of an expanded view of one spot on an array, as an example, showing the bound target molecules each labeled with a tracer moiety (squares) binding to immobilized molecules, some of which have been labeled with another tracer moiety (circles).
  • FIG. 3 is a diagrammatic representation of a process for measuring target signal and probe signal, as an example.
  • FIG. 3 A represents an expanded view of one spot on an array, where some of the immobilized molecules are each labeled with a tracer moiety (circles).
  • FIG. 3B represents the removal of the tracer moieties (circles) from the immobilized molecules and addition of target molecules that have been labeled with a tracer moiety (squares) onto the spot of FIG. 3 A.
  • FIG. 3C represents the same spot after addition of labeled target molecules, showing binding to immobilized molecules.
  • FIG. 4 A represents an expanded view of one spot on an array, as an example, where the immobilized molecules on one spot are.
  • chimeras each containing a "universal key" at the distal end that is common to all the immobilized molecules on that spot as well as for all the spots on the array, although all the immobilized molecules at a given spot may be unique to that spot.
  • FIG. 4B represents the same spot as FIG. 4A, showing the binding of target molecules to the chimeras, where the target molecules are shown as being labeled with a tracer moiety (squares).
  • FIG. 4C represents the same spot as in FIG. 4A and FIG. 4C, showing the labeling of the chimeras with another tracer moiety (circles) after removal of the target molecules .
  • FIG. 5 is a graphical representation of one example, showing the relationship between fractional occupancy and log concentration for a target molecule that is a 20-mer oligonucleotide.
  • FIG. 6 is a graphical representation of one example, showing the relationship between fractional occupancy of one target plotted against the fractional occupancy of a second target.
  • FIG. 7 is a graphical representation of one example showing the relationship between the concentration of one target plotted against the concentration of the second target, where the error bars for the measurements are also shown.
  • the present invention involves the labeling of the target molecules, reading the target signal in the presence or absence of the probe label or vice versa, determination of fractional occupancy of the immobilized molecules by the target molecules, and the use of K D> the dissociation constant for the dissociation of bound target molecules under equilibrium conditions, in the determination of target molecule concentration.
  • An “array” shall mean a collection of spots that can be one-dimensional, two- dimensional or three-dimensional, and that are supported by a solid substrate onto which immobilized molecules and target molecules are placed and reactions, such as binding, are allowed to occur.
  • a three-dimensional array includes an array of beads or particles.
  • immobilized molecule shall mean any molecule that can be immobilized on a substrate by any means conventional in the art.
  • a "molecule” shall mean a natural or synthetic, a biological or chemical molecule, particularly one that has significance in plants, human or animal health, bacteria, viruses, archebacteria, fungi, rickettsia, prions, mycoplasmas, and parasitic organisms.
  • Such molecules include, but are not limited to single molecules and synthetic or natural polymers, such as, nucleotides, polynucleotides, DNA, cDNA, RNA, mRNA, cRNA, peptide nucleic acids ("PNA"), oligonucleotides, polypeptides, antibodies, kinases, phosphatases and other enzymes, hormones, cytokines, antigens, cell surface receptors and other proteins, peptides displayed on a phage and other peptides, carbohydrates, polymers including those containing ⁇ -, ⁇ -, or ⁇ -amino acids, polymers such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, mixed polymers, small molecules (for example, drug candidates), analogs, fragments and optical isomers thereof, and combinations thereof that form chimeric molecules.
  • PNA peptid
  • a "molecular species" in reference to the labeled and unlabeled immobilized molecules shall refer to the nature of a molecule.”
  • the molecular species are the same when the underlying molecules are the same.
  • an unlabeled immobilized molecule that is an antibody to Her2 a breast cancer antigen, has the same molecular species as a labeled immobilized molecule that is a labeled antibody to Her2.
  • the molecular species are different when the underlying molecules are different.
  • an unlabeled immobilized molecule that is an oligonucleotide having a sequence complementary to a fragment of the Her2 gene has a different molecular species from a labeled immobilized molecule that is an oligonucleotide having a sequence complementary to a fragment of a prostate cancer gene.
  • a “probe” or “capture probe” shall mean any immobilized molecule or molecules that are capable of capturing one or more target molecules by specifically binding thereto or by specific hybridization thereto to create bound target molecules.
  • a probe that is labeled with a tracer moiety gives rise to a probe signal that is detectable.
  • Specific binding shall mean the inherent or artificially created property of a molecule to recognize and selectively bind another molecule ("its binding partner").
  • specific binding include, but are not limited to, antigen/antibody binding, biotin/avidin or strepavidin binding, receptor/ligand binding, hybridization of complementary oligonucleotides, polynucleotides, or nucleic acids, or synthetic molecules chemically synthesized to bind to other molecules, for example, peptoids.
  • Substrate shall mean any surface conventional in the art that supports an array and on which molecules are allowed to interact, and their interaction detected without degradation of or reaction with the surface.
  • the substrate can be planar, such as a glass slide or a nitrocellulose filter or in the form of beads or particles, such as microspheres or nanobeads.
  • Substrate can be made of glass, silicon or a synthetic, such as plastic. It can be permeable or impermeable. Additionally, the substrate can be, for example, a solid- phase synthesis support, a fiber, such as glass fiber, a capillary tube, or a silicon wafer.
  • target molecule shall mean any molecule that can be captured by an immobilized molecule, labeled directly or indirectly, detected and/or quantified.
  • target molecules may be of any origin including, but not limited to: plants, humans, other animals, other vertebrates or invertebrates, microbial, including bacterial, archebacterial, viral, or fungal, parasitic, or from mycoplasmas or prions.
  • a "tracer moiety” is a molecule that contains any detectable label that is conventional in the art.
  • the tracer moiety may be removable without damaging the molecule to which it binds.
  • the tracer moiety may further be directly or indirectly measurable.
  • directly measurable tracer moieties include, but are not limited to: radioactive isotopes, energy transfer dyes, enzymes, quantum dots (sometimes referred to as semiconductor nanocrystals) and luminescent labels.
  • Luminescent labels may be fluorescent labels, chemiluminescent labels, such as a phosphorescent label, bioluminescent labels such as phycobilisomes, colorimetric labels or combinations of such.
  • the label is a fluorescent label such as a fluorescein, a rhodamine, a polymethine dye derivative, a phosphor, an energy transfer dye, and the like.
  • the tracer moiety may be in the form of a bead or microsphere containing a directly measurable label, such as a fluorescent label.
  • the tracer moiety may be indirectly measurable, such that it is modified, chemically or otherwise, to associate with one member of a binding pair, with the opposite member of the binding pair being linked to a directly measurable moiety.
  • binding pairs include, but are not limited to: biotin/avidin or strepavidin; oligonucleotides, polynucleotides or nucleic acids for which a complementary molecule carrying a detectable label can be constructed to hybridize therewith; antibody/antigen; receptor/ligand; and molecules chemically synthesized to bind fo each other, such as by combinatorial chemistry.
  • sample shall mean a biological or other sample that is being assayed or tested for presence or for quantitation of "target molecules.”
  • the methods, kits and compositions of the present invention arise from the recognition that the concept of fractional occupancy, as determined by the Langmuir isotherm (see, for example, Kittel C, "Thermal Physics,” Wiley & Sons, 1969, pp. 341- 345), can be utilized to accurately determine the concentration of target molecules in solution, in an array-based assay in which a fraction of the target molecules are captured by immobilized molecules attached to a substrate supporting the array, under conditions in which the concentration of the immobilized molecules constitutes a small fraction of the concentration of the target molecules, and when provided with K D , the dissociation constant for the bound target molecules under equilibrium conditions.
  • any inconsistency or variation in the number of immobilized molecules deposited at each spot of an array can easily be taken into consideration, and comparisons between samples can be more accurately determined.
  • a special application of the present invention is in the area of diagnostics for diseases, infection or other medical conditions, detection of environmental hazards or conditions, SNP genotyping, small molecule drug discovery, discovery research, and determination and comparison of gene and protein expression in the areas of genomics and proteomics.
  • SNP genotyping small molecule drug discovery
  • discovery research determination and comparison of gene and protein expression in the areas of genomics and proteomics.
  • present invention may be applicable to other areas as well.
  • the present invention includes methods of determining target molecule concentration in an array-based assay, methods of determining expression of target molecules, methods of comparing expression of one or more target molecules, kits containing labeled or unlabeled arrays of molecules and information on dissociation constants for determination of concentration of target molecules, as well as compositions containing labeled arrays and labeled target molecules for determination of fractional occupancy of capture probes and, ultimately, concentration of target molecules through use of a dissociation constant, K D , which is unique to each bound target molecule.
  • the present invention enables calibration by measuring target signal, and separately or simultaneously, measuring probe signal to determine the ratio of the bound sample to the overall number of immobilized molecules at any spot, this ratio being known as the fractional occupancy.
  • This invention contains a variety of embodiments for determining the target concentration by measuring the fractional occupancy f, and also presents several means to obtain the dissociation constant K D that appears in the Langmuir equation.
  • a composition in one aspect of the present invention, contains an array supported by a substrate, where the array contains a number of spots. Usually, the spots are addressable spots. Immobilized molecules for the capture of target molecules are placed or made in situ in one or more spots of the array. One or more or all of the spots of the array may contain immobilized molecules that are either the same or different and that are directly attached to the substrate or, optionally, are attached to the substrate through a linker.
  • the present invention takes into consideration the amount of immobilized molecules attached at a given spot ("probes" for short) that are available for capture of target molecules. This may be achieved in a number of ways known to persons skilled in the art. For example, one way of determining the amount of probes at a spot is by direct or indirect attachment of a quantifiable marker to that spot that is in a known proportion to the immobilized molecules at that spot. In practice, a known or set portion of immobilized molecules labeled with tracer moieties may be mixed with a known or set portion of the same immobilized molecules but unlabeled prior to attaching the mixture of labeled and unlabeled immobilized molecules at a spot on the substrate.
  • the composition of molecules to be immobilized (i.e., probes) on a spot may be spiked with a different molecular species that has a detectable label and that is capable of immobilizing to the substrate with the same efficiency as the probes.
  • the spot will contain an amount of label that is proportional to the total number of immobilized molecules.
  • the amount of tracer moiety or detectable label at such a spot may be preset or predetermined to be a desired proportion relative to the amount of immobilized molecules at that spot.
  • the array therefore, may be constructed with different spots containing the same or different proportion of tracer moiety or detectable label as compared to the immobilized molecules.
  • Patent No. 6,245,518 issued June 12, 201 to Hyseq, Inc., entitled " Polynucleotide Arrays and Methods of Making and Using the Same.”
  • such proportions may be between about 0.16% to about 100%.
  • persons skilled in the art may utilize other ranges including: 0.16% to
  • the tracer moiety is attached to, incorporated within, or otherwise associated with the same type of molecules as that to be immobilized.
  • the molecules to be immobilized may be spiked with an amount of labeled molecules of the same type, forming labeled immobilized molecules.
  • microarrays may be constructed using a Biodot spotting apparatus and aldehyde-coated glass slides. Techniques for immobilizing the probes on the array are also well known to those skilled in the art as can be seen, for example, in Lindroos, K.
  • Protein arrays that are known and conventional in the art may also be used herein, as described in WO00/056926, entitled “Methods for detection of nucleic acid polymorphisms using peptide-labeled oligonucleotides and antibody arrays”; WO99/39210, entitled “High density arrays for proteome analysis and methods and compositions therefore”; and WO00/004389, entitled “Arrays of protein-capture agents and methods of use thereof.”
  • the arrays herein may contain "addressable" spots in that each spot can be located and/or identified by its coordinates or address on the array for a one-dimensional or two- dimensional array, such that by virtue of their positions in the array, the identity of the probes or targets will become known.
  • the array is a three-dimensional array containing beads or particles, such beads or particles may be coded for identification purposes or otherwise identified by its properties, such as fluorescence or size or magnetic properties or combinations of such.
  • the immobilized molecules herein are attached to a substrate directly or indirectly.
  • Direct attachment can be, for example, by covalent bonding to the substrate.
  • Indirect attachment can be, for example, covalent bonding to a linker affixed to the substrate.
  • the nature of the immobilized molecules is such that they are any molecules capable of acting as capture molecules for any target molecules. In a particular application, they are designed to bind specifically to the target molecules. They include, but are not limited to, molecules or sequences capable of specific binding to or hybridization with target molecules.
  • the immobilized molecules may be antibodies, including polyclonal antibodies, monoclonal antibodies, single chain antibodies, fragments or chimeras thereof, or they may be synthetic molecules, including peptides, peptoids or peptide nucleic acids, generated combinatorially or otherwise, to recognize and bind specific target molecules.
  • the immobilized molecules may be, for example, the complementary oligonucleotides, polynucleotides or nucleic acids, or hybridizing analogs or mimetics thereof, including nucleic acids in which the phosphodiester linkage has been replaced with a substitute linkage, such as phosphorothioate, methylimino, methylphosphonate, phosphoramidate, guanidine and the like; nucleic acids in which the ribose subunit has been substituted, such as hexose phosphodiester; and peptide nucleic acids.
  • immobilized molecules may include, but are not limited to: nucleotides, polynucleotides, DNA, cDNA, RNA, mRNA, cRNA, peptide nucleic acids, oligonucleotides, polypeptides, antibodies, enzymes, hormones, cytokines, antigens, other proteins, peptides displayed on phages and other peptides, carbohydrates, polymers containing alpha-, beta-, and omega-amino acids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, small molecule drugs, mixed polymers, fragments, analogs and optical isomers thereof, and combinations thereof that form chimeric molecules.
  • the length of the immobilized molecules in instances where they are nucleotides, polynucleotides, nucleic acids or similar polymers, will usually range between 5 to 1000 nucleotides, optionally 5 to 500 nucleotides, further optionally 5 to 250 nucleotides, still optionally, 5 to 100 nucleotides; still further optionally, 5 to 50 oligonucleotides.
  • the polynucleotide, oligonucleotide or nucleic acid probes may be double or single stranded, or PCR fragments amplified from cDNA.
  • the immobilized molecules may be tailored to specifically bind to or hybridize with specified target molecules. For example, if the array is used to determine expression of a particular gene from a cDNA library that has been reverse transcribed from mRNA molecules, the immobilized molecules will be constructed with a sequence complementary or otherwise capable of recognizing the gene, gene fragment or expression products of such gene or gene fragments.
  • the nucleic acids may be derived from any biological sources including, but is not limited to, human, animal, plants, bacterial, fungal, viral, environmental or other sources.
  • the substrate supporting the array may be any solid substrate of any suitable configuration that is conventional in the art for the construction of arrays.
  • the substrate may be substantially planar, such as * a glass slide, a silicon wafer, or a nitrocellulose membrane, or the substrate may be in the form of a bead or particle, such as a microsphere or a nanoparticle.
  • the array may be a one-dimensional, two- dimensional or three-dimensional array.
  • the substrate may be constructed of any suitable materials that provide the array with a solid support and yet is unreactive towards any of the components of the assay including the immobilized molecules, the tracer moieties, and the target molecules.
  • suitable materials for the substrate herein includes, but are not limited to: plastics, ceramics, metals, gels, membranes, glass, silicon and other synthetic materials.
  • Substrates of interest herein include, but are not limited to: solid-phase synthesis support, fibers, capillary tubes, silicon wafers, slides, membranes, and filters.
  • the immobilized molecules herein are usually spotted onto a substrate to form an array that have at 1 spot, optionally, at least 5 spots, optionally, at least 10 spots, further optionally, at least 100 spots.
  • the array of the present invention may contain at least 1000 spots, optionally, at least 10,000 spots and further optionally, at least 100,000 spots.
  • the substrate is in the form of beads or particles
  • immobilized molecules are attached to the beads and microparticles, each bead or particle being treated as a spot of the array.
  • the beads or microparticles herein may be coded for identification purposes or they may be identified through their chemical or physical properties.
  • the immobilized molecules herein may be situated at "addressable" spots in that the type of immobilized molecules at each spot is known or can be determined. Accordingly, expression of a certain gene or protein may be traced to an "address" on the array for identification of the gene or protein sequence being expressed.
  • immobilized molecules situated at different spots of the array may be the same.
  • the immobilized molecules situated at different spots of the array are different.
  • the immobilized molecules herein may be labeled directly, or optionally through a linker, with one or more tracer moieties, by covalent or non-covalent bonding. Such tracer moieties are conventional in the art as described, for example, U.S. Patent No. 4,366,241.
  • the tracer moiety that is suitable for use herein may be a directly measurable moiety or an indirectly measurable moiety.
  • the directly measurable moiety is a detectable or measurable label, which is conventional in the art.
  • Suitable tracer moieties are disclosed, for example, in U.S. Patent No. 5,800,992 issued September 1, 1998 to Fordor et al., entitled “Method of Detecting Nucleic Acids,” and include, but are not limited to: a radioactive isotope, an energy transfer dye, an enzyme, a luminescent label, a quantum dot, or a bead or particle containing such..
  • a luminescent label includes, for example, a fluorescent label, a chemiluminescent label such as a phosphorescent dye, a bioluminescent label such as a phycobilisome, or a colorimetric label.
  • Fluorescent molecules that are of interest herein include: fluorescein, rhodamine, Texas Red, cyanine dyes and the like. Radioisotopes that are of interest herein include: 35 S, 32 P, 3 H, 125 1, 14 C and the like.
  • the indirectly measurable moiety is a molecule that is not directly measurable on its own but that will interact with a directly measurable moiety to yield a detectable signal.
  • the immobilized molecule is linked to one member of a binding pair, such as, for example, a biotin/avidin or strepavidin pair, an antibody/antigen pair, a receptor/ligand pair, an enzyme/substrate pair, a hybridizing oligonucleotide, polynucleotide or nucleic acid pair, and the like.
  • a directly measurable moiety is then linked to the other member of the binding pair.
  • the immobilized molecule becomes measurable.
  • the tracer moiety herein may be attached to immobilized molecules through a detachable or removable linkage. This is advantageous in that either the probe signal from the labeled immobilized molecules or the target signal from the labeled target molecules may be read without interference from the other signal, whether the two tracer moieties are the same or different.
  • a removal tracer moiety may be attached in such a way that it is removable enzymatically, chemically, by light or other energy activation, or by a change in temperature.
  • linkages are conventional in the art. For example, a simple labeling procedure is offered by Pierce rwww.piercenet.com).
  • the target molecules of the present invention are any analytes that can be captured, labeled and measured.
  • these target molecules are biological or chemical molecules from natural sources, such as human, animals, plants, bacteria, viruses, fungi, prions, mycoplasmas, and rickettsia.
  • the target molecules may be synthetic molecules, for example, combinatorially synthesized chemical compounds that are small molecule drug candidates.
  • Target molecules from natural sources include, but are not limited to: oligonucleotides, polynucleotides, or nucleic acids, such as DNA, cDNA, RNA, mRNA, cRNA; proteins or polypeptides such as antibodies, antigens, enzymes, hormones, cytokines; carbohydrates; factors, cofactors, analogs, fragments or combinations thereof.
  • oligonucleotides, polynucleotides, or nucleic acids such as DNA, cDNA, RNA, mRNA, cRNA
  • proteins or polypeptides such as antibodies, antigens, enzymes, hormones, cytokines
  • carbohydrates factors, cofactors, analogs, fragments or combinations thereof.
  • Examples of-some target molecules suitable herein can be found in U.S. Patent No. 4,366,241 issued December 28, 1982 to Syva Company, entitled “Concentrating Zone Method in Heterogeneous Immunoassays.”
  • the target molecules herein may also be labeled with
  • the tracer moiety for labeling target molecules may be a directly or indirectly measurable moiety as described above.
  • the target molecule when the target molecule is labeled with an indirectly measurable moiety, the target molecule may be chemically modified to contain one member of a binding pair that can be recognized by the other member of the binding pair carrying a directly measurable moiety. Any such binding pair that is conventional in the art may be used herein. An example of such a binding pair is the biotin/avidin or strepavidin pair.
  • the present invention includes a method of determining target molecule concentration in an array-based assay.
  • the invention makes use of the measured fractional occupancy at each spot to obtain an absolute calibration of a target concentration, for example, to determine a gene expression profile, typically using cDNA molecules, reverse transcribed from mRNA, as target molecules.
  • two or more samples can be compared to one another by comparing the absolute calibration thereof, thereby obtaining, for example, differential comparison of gene expression among the samples.
  • a single sample will exhibit a range of fractional occupancies corresponding to the range of concentrations of each type of molecules in the sample.
  • a probe signal emitting from the labeled immobilized molecules can be first detected and quantified.
  • a composition containing the target molecules, labeled with a tracer moiety is allowed to contact the immobilized molecules of the array. After incubation or sufficient passage of time to allow specific binding between the target molecules and the immobilized molecules to occur, excess unbound target molecules are removed, the array washed, and the target signal is detected and measured. Alternatively, the probe and target signal can both be measured after hybridization of the target molecules and washing of the array.
  • the steps, reagents, and conditions for the conduct of an assay using an array such as the ones described herein are conventional in the art, depending on the immobilized molecules utilized and the target molecules to be captured. Such steps, reagents and conditions may be found in the patents cited herein.
  • the experimental conditions are such as to allow specific hybridization to occur, such as under high stringency conditions.
  • the immobilized molecules are antibodies and the target molecules are cancer antigens, for example, then experimental conditions are such as to allow antibody and antigen to form a complex.
  • the target molecules situated on any or all spots of an array may be the same or different and the target signals can be measured with or without removal of the tracer moiety from the labeled immobilized molecules, or in the presence or absence of the tracer moiety from the labeled or unlabeled immobilizes molecules.
  • the method herein may be useful in the determination of the expression and concentrations of each type of target molecule in the sample population.
  • the method herein is useful for the comparison of expression of the target molecules in the two or more sample populations.
  • a sample containing target molecules to be analyzed may contain a mixture of 2 samples, one taken from sample 1 containing target molecules 1 that are labeled with target tracer 1, and the other taken from sample 2 containing target molecules 2 that are labeled with target tracer 2.
  • target molecule 1 and target molecule 2 both contain the same molecular species and differ only in the tracer moiety used to label the targets.
  • the method of the present invention may be utilized to obtain a quantitation of probe signal, target molecule 1 signal and a target molecule 2 signal. The fractional occupancy and, thus, concentration of the target molecule 1 and target molecule 2 may be determined, provided that K D , the dissociation constant, for each of the bound target molecule 1 and 2 are known, provided or determined.
  • This method may be applied to the comparison of two samples, for example, by determination of target molecule concentration in each sample sequentially, one sample after another and, thus, obtaining two separate target signals in multiple steps or by combining aliquots of the two samples together to form one sample, in equal or known proportions, after labeling the target molecules in each sample in a distinguishing manner so as to obtain two different target signals upon binding between the respective target molecules to the immobilized molecules.
  • An example of this application is the comparison in gene or protein expression using genes and proteins, fragments thereof or molecules resulting from expression or reverse transcription of such genes, proteins and fragments.
  • Suitable sources of genes, proteins, fragments thereof or results of expression or reverse transcription thereof for application of the present methods include, but are not limited to: normal cells, tumor cells, diseased cells, infected cells, bacteria, viruses, prions, fungi, cells at different stages of division, cells at different stages of growth, cells at different stages of growth arrest, cells at different stages of cell death, and tissues at different stages of development from all biological sources including humans and other animals, plants and microorganisms.
  • Results of expression or reverse transcription of genes, proteins and fragments include, but is not limited to: mRNA, cDNA, cRNA, proteins and polypeptides and fragments thereof, including those resulting from natural post-transcriptional and/or post-translational processing.
  • the target composition may be a mixture containing target molecules taken from more than two sample populations, such as, up to N sample populations, where N is any positive integer greater than 1, and each sample population of target molecules are labeled with a distinguishing label .yielding distinguishing target signals.
  • target molecules from each of N sample populations may be detected and measured sequentially and the concentration of each is compared after separately determining the concentration thereof. Variations in the sequence of measuring probe signal and target signal are within the contemplation of the present invention. For example, if the tracer moiety for the immobilized molecules and the target molecules are the same, the probe signal may be first measured, then the combined probe and target signals are measured following binding of target molecules to immobilized molecules and removal of excess target molecules. The target signal is then obtained from subtracting the probe signal from the combined signal.
  • the probe and target signals are measured separately and/or independently.
  • the probe signal from the labeled immobilized molecules may be measured first and then removed. Then labeled target molecules are allowed to contact the immobilized molecules. Target signal from bound target molecules can then be measured without interference from the probe signal.
  • the tracer moiety labeling the immobilized molecules and target molecules may be the same or different.
  • a proportion of the immobilized molecules may be labeled with tracer moieties a second time, with or without removal of the target molecule, and the probe signal measured or determined again, either as a check for consistency or to obtain an average value for the probe signal.
  • the fracer moiety from the immobilized molecule is not removed and the target signal is measured in the presence of the probe signal.
  • the immobilized molecules are labeled with a tracer moiety that differs from the tracer moiety labeling the target molecule in its emission spectra or other measurable characteristics.
  • the combined probe and target signal following binding of target molecules to immobilized molecules may be measured first and the tracer moiety from either the immobilized molecules or the target molecules are then removed. The signal intensity of the remaining molecules is then measured.
  • signal intensity of the target molecules may be measured first after the composition containing target molecules is allowed to contact the immobilized molecules of the array, prior to the immobilized molecules being labeled. Then the immobilized molecules are labeled, followed by measurement of combined probe and target signal. Thereafter, through subtraction, the probe signal can be determined.
  • the target molecules may be allowed to interact with immobilized molecules before the immobilized molecules are labeled.
  • Target signal from bound target molecules may be read and the target molecules or their fracer moieties may be removed.
  • the immobilized molecules may be labeled with the same or different tracer moiety, and the probe signal measured without interference from the target molecules.
  • immobilized molecules may be attached to a universal linker.
  • a universal linker may be attached to all immobilized molecules, of the same or different specificities, on one or more or all spots of an array.
  • fracer moieties may be attached to the universal linker on the immobilized molecules and the probe signal measured on all the spots on the array, regardless of binding specificities of the immobilized molecules.
  • the resultant binding between the immobilized molecules and the tracer molecule or between the immobilized molecules and labeled target molecules may be visualized in a number of ways conventional in the art.
  • K D may be calculated based on a generally accepted formula (see Example 6 below).
  • K D may be determined experimentally, such as by conducting the method of the present invention for each of several different dilutions of a target molecule and plotting the target concenfration against the function f / (1-f ), where f is the fractional occupancy.
  • K D is the slope of the line through all points that minimizes the squared distance between the line and the points.
  • the present invention includes a kit that contains, in one aspect, an array of molecules immobilized on a substrate as described above and certain information relevant to the determination of target molecule concenfration, where the array contains a number of spots, at least one spot having more than one immobilized molecules, and where the molecules to be immobilized or already immobilized on the substrate of the array are labeled or as yet unlabeled.
  • the kit herein may further contain one or more of the following: (a) instructions for determining dissociation constant, K D , for any target molecules bound to immobilized molecules; (b) instructions for determination of gene or protein expression; (c) instructions for comparing gene or protein expression; (d) instructions for determination of target molecule concentration; (e) instructions regarding an appropriate tracer moiety to use for labeling target molecules so as to optimize the spectral separation between the target signal and probe signal.
  • the kit will contain instructions for more than one type of tracer moieties for labeling more than one type of target molecules.
  • the kit may contain a fracer moiety for labeling the immobilized molecules or a detectable label that is suitable for spiking the immobilized molecules.
  • the kit will contain a substrate suitable for attaching probes to produce an array, a solution of probes to be immobilized, a solution of labeled molecules for spiking the probes, and information regarding K D .
  • Example 1 Derivation of formula for determination of target molecule concenfration as a function of fractional occupancy, f.
  • Fractional occupancy is a ratio representing the total number of target molecules bound to immobilized molecules at a given spot on an array divided by the total number of immobilized molecules available at that spot.
  • the actual number of bound target molecules at a spot on average, will depend on the number of capture molecules available under a given set of experimental conditions.
  • the average number of bound target molecules is the fractional occupancy times the number of molecules located at the spot. Since arrays generally consist of many spots, with the immobilized molecules generally being different from one spot to another, the fractional occupancy and number of bound targets will generally differ from one spot to another.
  • [bound target] - [free molecule] + [target] the fractional occupancy, f , for bound target at a given spot equals [bound target] / ([bound target] + [free molecule]).
  • [bound target] is the concenfration of target molecules bound to the immobilized molecules at a spot
  • [free molecule] is the concenfration of unbound immobilized molecules at that spot
  • [target] is the concentration of the free target molecules in the sample solution.
  • the second te ⁇ n in the denominator is the dissociation constant K D for the reaction, so the fractional occupancy has the simple form:
  • the free target molecule concenfration in this formula changes if there are a large amount of immobilized molecules at a spot that can bind the free target molecules, it is usually convenient to have sufficiently large target sample concentration so that the number of target molecules is large compared to the number of immobilized molecules at the spot (or spots, if several spots are identical). In this limit, the concenfration of target does not change substantially as a result of binding events.
  • the target concenfration is determined based on this formula.
  • FIG. 1 a composition of the present invention is diagrammatically represented by the expanded view of one spot 7 on an array 8. Other spots are not shown here.
  • Labeled target molecules 1 are shown labeled with tracer moiety 2 (squares) and the labeled immobilized molecules 3 are shown labeled witi another fracer moiety 4 (circles), where . the immobilized molecules are attached to a substrate 5.
  • Unlabeled target molecules are not shown here, but unlabeled immobilized molecules 6 are shown without any fracer moieties attached.
  • FIGS. 1-4 This may be viewed, for example, in terms of cDNA molecules or oligonucleotides as target molecules 1, being hybridized to its complementary nucleic acid molecules 3 and 6 on the array 8.
  • Use of DNA in FIGS. 1-4 is not meant to limit the broad applicability of the present invention, since " these figures also apply to any other target molecules and the complementary immobilized molecules at a spot.
  • the immobilized molecules at the spot are shown as being partially labeled in FIG. 1. Since the immobilized molecules at a spot can be large in comparison to the bound target molecules, this partial labeling of the immobilized molecules can be useful so that the signal from the immobilized molecules does not mask the signal from the target molecules.
  • the total number of immobilized molecules at the spot is readily determined once the number of labeled immobilized molecules is known.
  • the target molecules in a sample to be assayed are labeled with a different tracer moiety than that used for the immobilized molecules.
  • the target molecules are shown as being completely labeled, since their concentration and corresponding assay signal strength will usually be low, although the target molecules can also be partially labeled. So long as the proportion of partial labeling of target molecules is known, then the total amount of target can be dete ⁇ nined from the amount of partially labeled targets.
  • the ratio of the bound target molecules to the total immobilized molecules at the spot, the fractional occupancy, is described by the Langmuir isotherm introduced above. As discussed in Example 1, it is convenient to work in the limit that the number of free target molecules is large compared to the number of immobilized molecules at the spot (or spots, if several spots are identical). Under such conditions, the concentration of target does not change substantially as a result of binding events.
  • the signal associated with the bound target molecules at a spot is measured. The probe signal can be measured either before or after the hybridization of the target molecules.
  • the fractional occupancy f is by definition the ratio of the bound target molecules divided by the total number of immobilized molecules.
  • the reading instrument does not necessarily need to be calibrated to separately determine the actual number of target and immobilized molecules. Instead, the instrument can be calibrated on a relative basis to adjust for efficiency differences in reading the signals associated with the target molecules versus reading the signals associated with the probes.
  • Calibration of a label signal intensity to number of molecules, starting from a standard sample with known amount of labeled molecules is a straight forward exercise in physical chemistry: The emission photon flux can be accurately measured, the fluorescent efficiency is known, and the numerical aperture and detection loss is part of any good imaging design.
  • the number of labeled probe molecules N p can be inferred from the signal intensity I p of the label used for the probe molecules, and the amount of labeled bound target N t can be infe ⁇ red from the intensity I t of the label associated with the bound target.
  • the concentration of target is then given by inserting this ratio into the formula for target concentration:
  • [target] K D f/ (l - f).
  • This embodiment of the invention has the probe label attached to the immobilized molecules that in turn can bind to the target molecules.
  • the label can be attached directly to the spot or indirectly to the spot by means of other molecules that are attached to the spot, where the other molecules do not interact with the target. In doing so, there needs to be a proportionality between amount of label at the spot and the amount of immobilized molecules that can interact with the target molecules.
  • a single tracer moiety may be used for labeling both the immobilized molecules and the target molecules. In doing so, the probe signal for a spot is measured first.
  • the target is hybridized to the immobilized molecules, excess target molecules are removed, and a composite signal consisting of the probe signal and the target signal at the spot is measured.
  • the actual target signal is then the incremental signal above the probe signal, that is, a simple difference of the second (composite) signal and the probe signal gives the target signal.
  • corrections need to be made to account for partial labeling of the immobilized molecules or of the target.
  • the target can be measured first and the probe labels are indirectly attached, in that they can be added after the prior measurement of the target signal.
  • the tracer moieties described in this example are conventional luminescent labels, both for the probes attached at each spot and for the target molecules.
  • FIG. 2 Another embodiment of the present invention is diagrammatically represented in FIG. 2 portraying one variation in the sequence of labeling of the probes.
  • FIG. 2 portraying one variation in the sequence of labeling of the probes.
  • the labeled target molecules 1 that are labeled with tracer moiety 2 are hybridized to unlabeled probes 6 immobilized on substrate 5 at a spot 7 of an array 8. After removal of excess target molecules, the target signal is measured. Following this, in FIG. 2B, tracer moieties 4 are added to label the probes, using a suitable linker mechanism, such as a biotin/avidin combination.
  • FIG. 2C the labeled target molecules 1 are shown hybridized to labeled immobilized molecules 3 and unlabeled immobilized molecules 6 on a substrate 5 at a spot 7 of the array 8. The probe signal from a spot 7 is then measured, the fractional occupancy is determined, and the target concenfration is determined as before through use of the Langmuir equation.
  • the fracer moieties for the target molecules and for the immobilized molecules can be the same or different.
  • the two signals may be read separately, preferably the target signal is measured first to avoid any noise background due to the probe signal.
  • a combined probe and target signal may be measured after labeling of the probe, and the probe signal can be arrived at by subfraction of target signal from the combined signal.
  • FIG. 3 shows use of the removable tracer moiety in connection with labeling of the immobilized molecules.
  • FIG. 3 A diagrammatically represents the labeling of the immobilized molecules 6 with a removable tracer moiety 4, to form labeled immobilized molecules 3 at a spot 7 (other spots not shown) of an array 8, for example, using a removable disulfide linker (Pierce EZ-Link Sulfo-NHS-SS-Biotin).
  • the probe signal from the labeled immobilized molecules 3 at the spot is measured first.
  • the removable tracer moieties 4 are then removed from the labeled immobilized molecules 3 to become unlabeled immobilized molecules 6.
  • the target molecules 1 with labels 2 are then brought into contact with the unlabeled immobilized molecules to allow binding. Excess target molecules (those that are not bound) are removed, and the target signal is then measured. Removing the fracer moieties from the probe following measurement of the probe signal permits use of the same tracer moieties for both probe and target molecules in this embodiment. Of course, two different tracer moieties may be used here as well. Apart from the detail of using a removable tracer moiety for labeling the immobilized molecules, this embodiment is very similar to the earlier two embodiments, including any corrections that are needed for partial probe labeling prior to computation of the target concenfration. This variation in labeling and measuring target and probe signals flips around the process described in Example 3 above, where the probe signal is measured first in this Example 4, then the tracer moiety is removed from the probe, then the labeled target molecules are added.
  • FIG. 4 is a diagrammatic representation of another embodiment of the present invention in which an intermediate molecule 10 is used for labeling of the immobilized molecules attached to the substrate 5 to form composite molecules 9.
  • FIG. 4A shows immobilized composite molecules in which a portion of each composite molecule 9 is complementary to the target molecule and is made to be unique for one or more spots of the array 8 (Other spots not shown herein.). Another portion of the composite molecule 9 is made to be identical or "universal" for all the spots of the array.
  • the composite molecules 9 may be referred to as chimeric probes.
  • FIG. 4B shows labeled target molecules 1 being brought into contact with composite molecules 9and binding thereto. In practice, excess target molecules are removed. The target signal at the spot is then measured. The number of immobilized molecules at a spot is then dete ⁇ nined by bringing the universal portion of the immobilized composite molecules in contact with a solution that contains the labeled complement molecule to the universal portion of the immobilized molecules. If the amount of labeled complement molecules are in significant excess, compared to the number of immobilized molecules, experimental conditions including the temperature can be adjusted so that virtually all of the immobilized molecules will be bound to their labeled complement. The signal from the labeled complement is then measured.
  • the concentration of target can be calculated as before.
  • One variation to this fourth embodiment is to reverse the order of measuring the target signal and the probe signal.
  • the same fracer moiety can be used for both the target and the immobilized composite molecules.
  • only a fraction of the immobilized molecules have a universal portion, so a correction needs to be made as discussed before.
  • only a portion of the targets may be labeled. In such a case a correction here also would be necessary.
  • the present invention has been described in which single target molecule populations are measured separately and compared to the signal from the probes for absolute calibration, in determining the target concentration. Often the interest is in a differential comparison of the two concentrations.
  • the method described herein can equally well be extended to include several target populations that are run simultaneously, each of which uses a different tracer moiety and for each of which the calibration is achieved using the absolute calibration provided by the immobilized molecules at each spot (which in turn have is labeled by another fracer moiety which differs from the fracer moieties used for labeling the target molecules). By running two or more populations simultaneously and calibrating them to the immobilized molecules, they can subsequently be compared to each other to arrive at the differential comparison.
  • the invention is applicable to analysis of many polymers, including polypeptides, carbohydrates, and synthetic polymers, including alpha-, beta-, and omega-amino acids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, proteins, antibodies, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, and mixed polymers.
  • polymers including polypeptides, carbohydrates, and synthetic polymers, including alpha-, beta-, and omega-amino acids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, proteins, antibodies, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, and mixed polymers.
  • Various optical isomers e.g., various D- and L- forms of the monomers, may be used.
  • antibody probes will be generated which specifically recognize particular subsequences found on a polymer.
  • antibodies may be generated which are specific for recognizing a three contiguous amino acid sequence, and monoclonal antibodies may be preferred. Optimally, these antibodies would not recognize any sequences other than the specific three amino acid stretch desired and the binding affinity should be insensitive to flanking or remote sequences found on a target molecule.
  • antibodies specific for particular carbohydrate linkages or sequences may be generated.
  • a similar approach may be used for preparing specific reagents that recognize other polymer subunit sequences. These reagents would typically be site specifically localized to a substrate matrix pattern where the regions are closely packed.
  • the specific reagents will often be polypeptides. These polypeptides may be protein binding domains from enzymes or other proteins that display specificity for binding. Usually an antibody molecule may be used, and monoclonal antibodies may be particularly desired.
  • K D dissociation constant
  • the K D in moles can be expressed as a function of the free energy G:
  • the free energy G can be related to the well-measured melting temperature T m by noting that T m is the temperature at which the free energy is zero.
  • the free energy G equals H - TS, where H and S are the enthalpy and entropy, respectively.
  • the melting temperature of oligonucleotides is closely approximated by an increase of 3 deg Celsius for every nucleotide (Strachan T, Human Molecular Genetics):
  • the enthalpy is calculated from the average number (2.5) of hydrogen bonds (approx. 3 Kcal/mole) for each Watson-Crick DNA pair:
  • K D More precise calculations of K D are readily available.
  • a variety of calculations express the melting temperature T m in terms of enthalpy, entropy, sodium content, and GC content including nearest neighbor interactions for a given molar concentration of the strands (see for example the melting temperature calculator in www.genosys.com, also for DNA see Breslauer et al, Proc. Natl. Acad. Sci USA 83: 3746-3750 (1986)). Since the melting temperature is by definition when the fractional occupancy equals one-half, this is when K D equals the strand concenfration. Therefore, replacing the strand concenfration with K D in the chosen melting temperature equation and inverting this equation gives K D to sufficiently high precision for many applications.
  • Example 7 Fractional occupancy versus concentration of target cDNAs in solution binding to 20-mer oligo probes.
  • Example 1 The K D obtained in Example 1 is used to generate the results shown herein in Table 1. These results are also graphically represented in FIG. 5. Note a target typically has a range of cDNA concentrations, to which correspond to a range of fractional occupancies.
  • the fractional occupancies are independently determined by, for example, labeling the target molecules and the probes at each spot and calculating the ratio of the respective signal intensities, the point to this invention, then the concentration of the target corresponding to the probes at that spot is readily determined.
  • samples from different populations are compared to a given sample as a reference sample. In such cases, only one reference sample needs to be run and different populations are run separately thereafter. The total number of runs is therefore quite close to the total number of runs needed for competitive hybridization as described in U.S. Patent 5,800,992, that is, there is no need to run the reference sample every time a new target sample is mn.
  • Example 8 Comparison of gene expression between two different targets and determination of the error bars associated with this comparison.
  • Example 7 was for only one type of labeled target molecule in solution while the other label is associated with the complementary probes at a spot.
  • “competitive hybridization” for example, U.S. Patent 5,800,992
  • differential gene expression measurements separate targets are measured using two different arrays, and comparison is made by using normalization control genes or other target molecules.
  • the present invention affords an absolute calibration of two or more types of target molecules, and comparison of each of the at least two types of target molecules against this absolute calibration, and provides a means to then compare the concenfration of each target molecule type against each other, thereby, obtaining differential gene expression.
  • Table 2 2 samples of 5 cDNA. Sample 2 loaded at 1.5 x sample 1. Third cDNA has 2x expression.
  • ⁇ f (f * (l ' -f) /probe#) ⁇ 0.5 for binomial distribution (One standard deviation.)
  • fractional occupancy depends strictly on the number of fixed molecules of each type, whether the molecules are at one array site or many, or on one bead or many.
  • the statistics for fractional occupancy are quite robust: The total number of hybridized cDNA of a given type is divided by the total number of available oligos for that type. In the case where the same oligo or cDNA is located at multiple microarray sites or beads and all of the measurement data is "good,” this model states that the best answer is to take the total signal from all the hybridized cDNA and divide it by the total signal from all the fixed oligos or cDNA of that type, rather than (for example) computing a ratio for each site or bead and averaging the ratios from any replicates.
  • the data goodness can be analyzed through an initial processing step in which, for each cDNA type, a regression fit is generated of the hybridized cDNA signal versus the calibration signal from the fixed oligos or cDNA. (Each of the relevant sites or beads contributes a data point to this regression fit.) This allows any data outliers from the regression fit to be rejected before summing up the total signal as indicated above.
  • Example 9 Cancellation of dissociation constant K D errors in differential comparisons.
  • the target concentration is linear in the dissociation constant
  • fr_occ2 and fr_occl can be used to form the respective f / (1- f ) value corresponding to each of the expression concenfrations shown in the rows.
  • the ratio of these values gives a differential expression value of 1.5.
  • the ratio of the f / (1- f ) values gives a value of 3.0. This is consistent with the concentration assumptions used in this example.
  • the ratio of the f / (1- f ) values results in the concentration ratios, or differential expression ratio, independent of K D .
  • the following example describes an antibody as the immobilized molecule being bound to a solid support, and a protein interacting with the immobilized antibody as the target molecule.
  • the antibody can be any molecule having the characteristics of an antibody, including a monoclonal or polyclonal antibody, and can be derived from human (e.g.: IgG), non-human (e.g.: IgY) or mixed (humanized Ab) sources.
  • Antibodies have been well characterized and are known to those skilled in the art. They consist of two chains: a light chain and a heavy chain, and resemble in structure a "Y". The variable domains inside the "V of the "Y" contain complementary determining regions (CDRs) which bind to antigens.
  • the binding capacity is substantially increased by covalent binding.
  • high binding levels are achieved at much lower concentrations than with passive binding. This is the result of the irreversible nature of the covalent binding process; monolayer coverage can be achieved at low concenfrations, limited only by the diffusion rate of the antibodies to the surface. Monolayer coverage is usually about 300 ng. per square centimeter, and can be achieved with concentrations containing twice that amount of protein in agitated systems.
  • the presence of surfactant in a protein solution does not inhibit covalent binding- with XENOBINDTM plates as it does with passive binding.
  • Antibody-antigen dissociation constants vary due to the specificity of the variable domain. Typical values range between 10 "6 - 10 "7 . Protein affinity determinations, or 1/ K D , can be found in www.biacore.com, or see also NHRC Publication 84-37 (Griswold,
  • the fractional occupancy can be detected by any of the methods described above.
  • the immobilized antibody can be labeled with one dye and the target antigen protein labeled with a spectrally dissimilar dye.
  • the same process can be used to label both the antibody and the protein since the antibody is a protein and contains numerous N-termini.
  • a simple labeling procedure is offered by Pierce (www.piercenet.com).
  • the EZ-LinkTM Suylfo-NHS-LC-Biotinylation Kit allows biotin labeling of proteins or antibodies in 30 minutes at room temperature.
  • the biotin labeled proteins or antibodies can be reacted with streptavidin-conjugated dyes (e.g.:
  • the probe signal from the dye attached to the antibody must be calibrated to the number of dye molecules.
  • the signal from the spectrally dissimilar dye attached to the target protein (antigen) must be calibrated to the number of protein molecules. This can easily be done by one skilled in the art by measuring the signal associated with known concentrations of antibodies and proteins, respectively.
  • the fractional occupancy f is calculated by dividing the number of bound protein molecules by the number of antibody molecules.
  • K D can easily be measured by those skilled in the art.
  • One method is to use varying concenfrations of protein, measure the associated fractional occupancy f and then plot the protein concenfration as a function of the fraction f/(l-f), analogous to Example 10
  • the slope of a line through the origin minimizing the squared difference between the line and the concentration is the best estimate of K D .
  • SNPs Single Nucleotide Polymo ⁇ hisms
  • the concentration calculated for one spot is plotted on the x- axis, and the other on the y-axis, and this is done for a population of individuals, there will tend to be a scatter plot between the positive x and y axes due to the noise sources.
  • the variation in concenfrations can be large, and at times it is hard to tell which are the homozygotes and which are the heterozygotes.
  • this invention improves precision by removing variability due to spot size. Therefore, the scatter plots are reduced in their spread, allowing easy identification of SNP type.

Abstract

The present invention relates to methods, kits and compositions for determining any target molecule concentrations in array-based assays utilizing the principle of fractional occupancy as governed by the Langmuir isotherm.

Description

New Method in Quantitative Analysis of Gene and Protein Expression This application claims the benefit of the Provisional Patent Application entitled "Method in Quantitative Gene Expression," Serial No. 60/246,336, filed Nov. 7, 2000, the contents of which are incorporated herein in their entireties.
I. FIELD OF THE INVENTION
The present invention relates to improved methods, compositions and arrays for detection and quantitation of biological or chemical molecules, whether natural or synthetic, including but not limited to areas such as diagnostics of diseases, infection, or other medical conditions, detection of environment hazards, SNP genotyping, search for specific binding partners, such as small molecule drug discovery, determination of gene and protein expression, etc., all relevant to the field of plants, human and animal health, bacteria, viruses, archebacteria, fungi, rickettsia, prions, mycoplasmas, and parasitic organisms. II. BACKGROUND OF THE INVENTION
Arrays such as DNA and protein arrays have gained much popularity in the recent years especially for the conduct of genomics, proteomics and diagnostics research. Arrays are used, for example, in comparing the gene expression between normal cells and tumor cells. Nucleic acids from a normal cell sample may carry one detectable label, for example, a fluorescent label, and nucleic acids from a tumor cell sample may carry a different detectable fluorescent label. The two samples are mixed and hybridized to a set of oligonucleotide probes on an array. One hybridization signal from a given spot on the array may indicate hybridization mostly with nucleic acid from normal cells, another hybridization signal may indicate hybridization mostly with nucleic acids from tumor cells, and a mixed hybridization signal may indicate hybridization with both normal and tumor cells. Assays of this type, are qualitative at best and assay results represent only an approximate indicator of gene expression. In particular, when hybridization measurements for each sample are carried out using separate arrays, variation in spot sizes and, thus, the number of probes at each spot, renders any comparisons between relative expression levels even more difficult to interpret.
An attempt has been made to improve the quality control of such arrays, for example, in U.S. Patent No. 6,245,518, entitled "Polynucleotide Arrays and Methods of Making and Using the Same." Such attempts, however, have only been met by limited success, probably because of technical difficulties. There is, thus, a need for a better array or a better method of making and using an array to accommodate these types of variations, and to better quantitate the sample molecules being measured.
U.S. patents 6,040,138 and 6,004,755 also attempt to improve the measurement quantitation in differential expression. In both of these patents, a relatively small number of normalization controls are introduced. Normalization controls are oligonucleotide probes that are perfectly complementary to labeled reference oligonucleotides that are added to the nucleic acid sample. The signals obtained from the normalization controls after hybridization provide a control for variations in hybridization conditions, label intensity, "reading" efficiency and other factors that may cause the hybridization signal to vary between arrays. The signals (e.g., fluorescence intensity) read from all other probes in the array are calibrated by the signal (e.g., fluorescence intensity) from the control probes thereby normalizing the measurements. In contrast, our method does not rely on extrapolation from a relatively small set of normalization control probes, with potential attendant extrapolation errors, but rather provides quantitative measurements (i.e. absolute calibration) for each and every site or bead as desired.
Other U.S. patents that address the measurement of analyte concentrations include U.S. patent 5,516,635 (EP 0608370B1) and U.S. patent 5,837,551. These involve both a capture binding agent and a developing binding agent. Related patents in this series include U.S. 5,599,720 (EP 0134215), US 5,171,695 (EP 0271974), U.S. 5,834,319 (EP
0737175B1), and U.S. 5,807,755.
III. SUMMARY OF THE INVENTION The invention described herein addresses the unmet needs in the art for accurate detection and determination of concentration of a variety of compounds or molecules in solution, using an array-based assay.
In accordance with one of the objects of the present invention, there is provided a method of determining target molecule concentration in an array-based assay, where the assay includes the steps of:
(a) providing a first composition having an array of molecules immobilized on a substrate, the array having a plurality of spots, at least one spot containing more than one immobilized molecule, where the immobilized molecules are attached to the substrate, and at least one of the immobilized molecules is labeled with a first tracer moiety that yields a probe signal; (b) providing a second composition having a plurality of target molecules, where at least one target molecule is labeled with a second tracer moiety that yields a target signal;
(c) allowing the first composition to contact the second composition under conditions to allow binding of one or more target molecules to one or more immobilized molecules and to allow determination of fractional occupancy;
(d) measuring the probe signal;
(e) measuring the target signal; and
(f) determining concentration of target molecules. In another embodiment of the present invention, there is provided the method as described herein, where the array contains immobilized molecules and target molecules that are oligonucleotides or nucleic acids.
In another embodiment of the present invention, there is provided the method as described herein, where the substrate is in the form of a planar surface such as a sheet, or in the form of a bead or a microsphere.
In a further embodiment of the present invention, there is provided the method as described herein, where the array contains immobilized molecules that are situated at different spots of the array, and the immobilized molecules at different spots are the same or different. Optionally, in the methods of the present invention described herein, the target molecule is labeled with a second tracer moiety that is indirectly measurable. Further optionally, the labeled immobilized molecules and the unlabeled immobilized molecules herein may be the same or different molecular species.
In a further embodiment of the present invention, there is provided a method of determining target molecule concentration in an array-based assay, further including the step of providing or determining a dissociation constant for the bound target molecules In an alternative embodiment, there is optionally provided a method as described herein, where the second tracer moiety contains an indirectly measurable moiety. In another embodiment, there is optionally provided a method as described herein, where the labeled immobilized molecules form a first amount and the immobilized molecules form a second amount, and where the first amount is proportional to the second amount.
Further, in such instances, the method of the present invention optionally includes the step of determining fractional occupancy of the immobilized molecules by the bound target molecules. In a further embodiment, there is provided a method as described herein, where the steps of labeling probe or target molecules and/or measuring probe and target signals are repeated sequentially for each target molecule concentration to be determined, and the target molecule concentrations obtained thereby are compared. In a further embodiment of the present invention, there is provided a method as described herein, where target molecules of different dilutions are employed in a series of concentration determinations to arrive at a dissociation constant for the target molecules.
In yet another embodiment of the present invention, there is provided a. method of determining expression of a target molecule in a sample including the steps of: (a) providing a first composition having an array of molecules immobilized on a substrate, where the array contains a plurality of spots, at least one spot having more than one immobilized molecule, where the immobilized molecules are attached to the substrate, and at least one of the immobilized molecules is labeled with a first tracer moiety to form a labeled immobilized molecule that yields a probe signal; (b) providing a second composition having a plurality of target molecules, where at least one target molecule is directly labeled with a second tracer moiety that yields a target signal;
(c) allowing the first composition to contact the second composition under conditions to allow binding of one or more target molecules to one or more immobilized molecules and to allow determination of fractional occupancy;
(d) measuring the probe signal;
(e) measuring the target signal; and
(f) determining concentration of the target molecules; where the target molecules are genes, gene fragments or molecules resulting from expression or reverse transcription of genes or gene fragments; and where concentration of target molecules is related to expression thereof.
In still another embodiment of the present invention, there is provided a method of comparing expression of a first target molecule with expression of a second target molecule including the steps of: (a) providing a first composition having an array of molecules immobilized on a substrate, where the array contains a plurality of spots, at least one spot having more than one immobilized molecule, where the immobilized molecules are attached to the
) substrate, and at least one of the immobilized molecule is labeled with a first tracer moiety to form a labeled immobilized molecule that yields a probe signal; (b) providing a second composition that contains a plurality of first target molecules and a plurality of second target molecules, where at least one first target molecule is labeled with a second tracer moiety to form a first labeled target molecule that yields a first target signal and at least one second target molecule is labeled with a third tracer moiety to form a second labeled target molecule that yields a second target signal;
(c) allowing the first composition to contact the second composition under conditions to allow binding of first and/or second target molecules to immobilized molecules and to allow determination of fractional occupancy of first target molecules and second target molecules;
(d) measuring probe signals;
(e) measuring target signals;
(f) determining and/or a first concentration of the first target molecules and a second concentration of the second target molecules; and (g) comparing the first concentration and the second concentration; where the first and second target molecules are genes, gene fragments or molecules resulting from expression or reverse transcription of genes or gene fragments; and where concentration of target molecules is related to expression thereof.
In yet another embodiment of the present invention, there is provided a method of comparing expression of target molecules from N different sample populations, where N is a positive integer greater than one, including the steps of:
(a) providing a first composition comprising an array of molecules immobilized on a substrate, where the array comprises a plurality of spots, at least one spot comprising more than one immobilized molecule, where the immobilized molecules are attached to the substrate, and at least one of the immobilized molecule is labeled with a first tracer moiety to form a labeled a labeled immobilized molecule that yields a probe signal;
(b) providing a second composition containing a plurality of target molecules from N different sample populations, where at least one target molecule of at least one target type in a plurality of the N sample populations is labeled with a distinguishing tracer moiety to form a distinguishing labeled target molecule that yields a distinguishing target signal that is distinguishing for the at least one target type; (c) allowing the first composition to contact the second composition under conditions to allow binding of target molecules to immobilized molecules and to allow determination of fractional occupancy;
(d) measuring the probe signal;
(e) measuring the target signals; (f) determining and/or comparing concentration of target molecules of different target types from the N sample populations, where the target molecules are genes, gene fragments or molecules resulting from expression or reverse transcription of genes or gene fragments; and wherein concentration of target molecules is related to expression thereof. In a further embodiment of the present invention, there is provided a method as described herein where, optionally, the immobilized molecules and target molecules are each oligonucleotides or nucleic acids. Further, optionally, such methods are applicable to the determination of gene or protein expressions. Still optionally, the immobilized molecules situated at different spots of the array may be the same or different, such as in their specificities. Additionally, still optionally, the immobilized molecules that are different from spot to spot are all labeled with the same tracer moiety.
Moreover, there is provided in a further embodiment a method as described herein, where the plurality of target molecules in the second composition may be the same or different. Also in the methods herein, the tracer moieties for labeling the immobilized molecules and target molecules are also the same or different.
In another embodiment of the present invention, there is provided a method as described herein for an array-based assay, where the array is supported by a substrate and the substrate has a planar surface, or is a bead or a microsphere.
In yet another embodiment of the present invention, there is provided a method as described herein, where at least one dissociation constant for dissociation of at least one type of bound target molecules to free target molecules is provided or is determined.
In a further embodiment of the present invention, there is provided a method as described herein, where the second composition contains target molecules from more than one sample population, where the tracer moiety for labeling the target molecules from one sample population are different from the tracer moiety for labeling target molecules from another sample population, and concenfration of the target molecules from the one sample population is compared with the concentration of target molecules from the other sample population. In another embodiment of the present invention, there is provided a method of comparing gene or protein expression of two or more target molecules from two or more target sample populations, where the target molecules from the different target population contains the same molecular species, where at least the target molecules from one of the sample populations are directly or indirectly labeled with a second tracer moiety. In a further embodiment, in the methods of determining or comparing gene or protein expression of target molecules from different sample populations, there is optionally provided that at least one type of target molecules are labeled with a tracer moiety that carries a directly measurable or indirectly measurable moiety. In the embodiment where the second tracer moiety carries an indirectly measurable moiety, tlie target molecules are modified to link, by covalent bonding, to the indirectly measurable moiety.
In accordance to another object of the present invention, there is provided in one embodiment a kit that contains: (a) a composition having an array of molecules immobilized on a substrate, where the array contains a plurality of spots, at least one spot having more than one immobilized molecules, where the immobilized molecules are attached to the substrate; and (b) information on at least one dissociation constant for target molecules of one specificity bound to the immobilized molecules.
In another embodiment of the present invention, there is provided a kit as described herein, where the kit optionally includes a first tracer moiety for labeling or spiking the immobilized molecules, and/or instructions for at least a second tracer moiety for labeling the target molecules. Moreover, in another embodiment, the kit includes a plurality of different tracer moieties for labeling target molecules of different specificities. In a further embodiment, the kit as described herein includes instructions for determining dissociation constants for target molecules of different specificities bound to immobilized molecules, and/or instructions for determining gene or polypeptide expression.
In one embodiment of the present invention, there is provided a kit containing an array as described herein, where the array has for a substrate a planar surface, such as, for example, a sheet, a slide, a silicon wafer, or the substrate may be in the form of beads or microspheres, with one bead or microsphere representing one spot, for example. In a further embodiment of the present invention, there is provided a kit containing: (a) a composition having an array of molecules immobilized on a substrate, where the array contains a plurality of spots, at least one spot having more than one immobilized molecules, where the immobilized molecules are attached to the substrate; and (b) instructions on determining dissociation constant for any target molecules bound to the immobilized molecules.
In yet another embodiment of the present invention, there is provided a kit having: (a) a composition that contains an array of molecules immobilized on a substrate, where the array includes a plurality of spots, at least one spot having more than one immobilized molecule, where the immobilized molecules are attached to the substrate; (b) information on at least one dissociation constant for one kind of target molecules bound to the immobilized molecules; (c) a first tracer moiety for labeling or spiking the immobilized molecules; (d) instructions for at least a second tracer moiety for labeling target molecules; (e) instructions for determination of dissociation constant for any target molecules; and (f) instructions for determination of target concentration or gene or protein expression.
In a further embodiment of the present invention, there is provided a kit as described herein where the immobilized molecules and target molecules are nucleic acids or oligonucleotides. Alternatively, the kit herein contains immobilized molecules that are polypeptides or proteins.
In yet another embodiment of the present invention, there is provided a kit that contains: (a) a composition having an array of molecules immobilized on a substrate, where the array includes a plurality of spots, at least one spot having more than one immobilized molecule, where the immobilized molecules are attached to the substrate, and at least one immobilized molecule is labeled with a first tracer moiety to form a labeled immobilized molecule; (b) information on at least one dissociation constant for one kind of target molecules bound to the immobilized molecules; (c) instructions for at least a second tracer moiety for labeling target molecules; (d) instructions for determination of dissociation constant for any target molecules; and (e) instructions for determination of target concentration or gene expression.
In a further embodiment of the present invention, there is provided a kit containing: (a) a composition having an array of molecules immobilized on a substrate, where the array includes a plurality of spots, at least one spot containing more than one immobilized molecules, where the immobilized molecules are attached to the substrate, and at least one immobilized molecule is labeled with a first tracer moiety; and (b) information on at least one dissociation constant for target molecules of one specificity bound to the immobilized molecules. In yet another embodiment of the present invention, there is provided the kit as described herein, further including instructions for determination of expression of a target molecule, where the target molecule is a gene or gene fragment, or result of expression or reverse transcription of a gene or gene fragment. In another embodiment of the present invention, there is provided a kit that contains: (a) a composition having an array of molecules immobilized on a substrate, where the array contains a plurality of spots, at least one spot having more than one immobilized molecules, where the immobilized molecules are attached to the substrate, and at least one immobilized molecule is labeled with a first tracer moiety; and (b) instructions on determining dissociation constant for target molecules bound to the immobilized molecules.
In a further embodiment of the present invention, there is provided the kit as described herein, where the immobilized molecules are any suitable probe for capturing a target molecule that is conventional in the art. Examples of such probes include: nucleotides, polynucleotides, DNA, cDNA, RNA, mRNA, cRNA, peptide nucleic acids, oligonucleotides, polypeptides, antibodies, enzymes, hormones, cytokines, antigens, other proteins, peptides displayed on phages and other peptides, carbohydrates, polymers containing alpha-, beta-, and omega-amino acids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, mixed polymers, small molecule drugs, fragments, analogs and optical isomers thereof, or combinations thereof that form chimeric molecules.
In accordance to another object of the present invention, there is provided a composition having an array of molecules immobilized on a substrate, where the array contains a plurality of spots, at least one spot having:
(a) more than one immobilized molecule, where the immobilized molecules are directly attached to the substrate or optionally tlirough a first linker, and at least one immobilized molecule is labeled with a first tracer moiety, directly or optionally through a second linker, to form a labeled immobilized molecule; and (b) at least one target molecule that binds to at least one labeled or unlabeled immobilized molecule; where the at least one target molecule is labeled with a second tracer moiety to form a labeled target molecule; and where the labeled immobilized molecule forms a first amount and the immobilized molecules form a second amount, the first amount being proportional to the second amount at the at least one spot, and the proportion is in the range of about 0.16% to about 100%.
In one embodiment of the present invention, there is provided the composition described herein, where the proportion is any proportion greater than about 0.16%. For example, proportions suitable herein may be one selected from the group of ranges of: about 0.16% to about 1%, about 1% to about 2%, about 2% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 50%, about 50% to about 70%, or about 70% to about 100%.
In a further embodiment of the present invention, there is provided a composition as described herein, where the proportion of the first amount to the second amount can be the same or different for different spots of the array.
In as yet another embodiment, the array is supported by a substrate that conventional in the art. For example, the substrate may be planar in surface, such as on a glass slide or a flat sheet of nitrocellulose, or the substrate may be in the form of a bead or a particle such as a microsphere. Further, the substrate may be glass or silicon or a synthetic material. Examples of substrates include solid-phase synthesis supports, fibers, capillary tubes, silicon wafers, slides, membranes, filters or other sheets.
In an embodiment of the present invention, there is provided a composition as described herein where the immobilized molecules may be attached to the substrate by covalent or non-covalent bonding, either directly or indirectly through a linker.
Moreover, the first tracer moiety may be attached to the immobilized molecules also by covalent or non-covalent bonding, either directly or indirectly.
In another embodiment of the present invention, there is provided a composition as described herein where the first tracer moiety is removable from the immobilized molecules after attachment. This facilitates reading of the target signal without interference from the probe signal. Optionally, the first tracer moiety remains attached to the immobilized molecule, and the probe signal can be read in the presence or absence of the target signal. Where the first tracer moiety is the same as the second tracer moiety, and the target signal can be determined by subtraction. In as yet another embodiment of the present invention, there is provided a composition as described herein, where the population of target molecules is a population containing target molecules of different types (that is, different "binding specificities" for example). In a further embodiment of the present invention, the first or second tracer moiety may be the same or different and are each directly measurable. Optionally, the first or second tracer moiety may be indirectly measurable.
In another embodiment of the present invention, the tracer moiety is optionally directly or indirectly measurable and can be any label that is conventional in the art. For example, the first and second tracer moiety may be a radioactive isotope, an enzyme including one catalyzing light emission such as luciferase , a luminescent label, or a bead or microsphere containing one or more of such. Examples of luminescent labels include quantum dots, fluorescent labels, energy transfer dyes, chemiluminescent labels such as phosphorescent dyes, bioluminescent labels such as phycobilisomes, colorimetric labels, and combinations thereof. In as yet another embodiment of the present invention, the tracer moiety may be indirectly measurable using any indirectly measurable molecules conventional in the art, such as conventional binding pairs, one of which can carry a directly measurable moiety. For example, an indirectly measurable molecule may be one of a pair of binding partners, including but not limited to: an antibody/antigen pair, a biotin/avidin or strepavidin pair, a digoxigenin/anti-digoxigenin pair, a carbohydrate/lectin pair, a pair of complementary oligonucleotides or nucleic acids that hybridize to each other, a receptor/ligand pair, or a synthetic pair that is chemically synthesized to bind to each other with specificity. Nucleic acid derivatives that are capable of hybridizing to a complementary molecule, such as peptide nucleic acids, may also be used as indirectly measurable moieties.
Optionally, in one embodiment, only those binding pairs are used where the molecule to be labeled is chemically modified to comprise one member of the binding pair, the other member of the binding pair being linked or otherwise associated with a directly measurable moiety.
In a further embodiment of the present invention, there is provided a composition as described herein, where the first tracer moiety is removable from the immobilized molecules and the removal is effected enzymatically, chemically, by light activation or other energy activation, or by a change in temperature. In another embodiment of the present invention, there is provided a composition as described herein, where the immobilized molecule is any molecule that is suitable for capturing a target molecule. For example, suitable immobilized molecules include, but is not limited to nucleotides, polynucleotides, DNA, cDNA, RNA, mRNA, cRNA, peptide nucleic acids, oligonucleotides, polypeptides, antibodies, enzymes, hormones, cytokines, other antigens or proteins, peptides displayed on phages or other peptides, carbohydrates, polymers containing alpha-, beta-, and omega-amino acids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, mixed polymers, fragments, analogs or optical isomers thereof, or combinations thereof that form chimeric probes.
In as yet another embodiment of the present invention, there is provided a composition as described herein, where the immobilized molecules situated at different spots of the array are the same or different in the specificities in recognizing or capturing different target molecules. In a further embodiment of the present invention, there is provided a composition as described herein, where the target molecules situated at one spot may be the same or different in their specificities in binding to the immobilized molecules.
Optionally, in one embodiment of the present invention, there is provided a composition as described herein where the labeled and unlabeled immobilized molecules comprise the same molecular species and the target molecule is labeled with a second tracer moiety that is an indirectly measurable moiety, and the target molecule is chemically modified to comprise the second tracer moiety.
In accordance to another one of the objects of the present invention, there is provided a method of determining target concentration in a sample in an array-based assay having the steps of:
(a) performing an array-based assay using one of the kits of the present invention as described above;
(b) measuring a probe signal from labeled immobilized molecules;
(c) measuring at least a first target signal from the at least one kind of bound labeled target molecules, before or after labeling the immobilized molecules, in the presence or absence of the first tracer moiety from the labeled immobilized molecules; and
(d) determining target concentration.
In another embodiment of the present invention, there is provided a method of determining target concentration having the steps of:
(a) performing an array-based assay using one of the compositions the present invention as described herein;
(b) measuring a probe signal from labeled immobilized molecules; (c) measuring at least a first target signal from the at least one bound labeled target molecules, before or after labeling the immobilized molecules, in the presence or absence of the first tracer moiety from the labeled immobilized molecules; and
(d) determining target concentration. In as yet another embodiment of the present invention, there is provided a method as described herein, where the target molecules are oligonucleotides or nucleic acids.
In a further embodiment of the present invention, there is provided a method as described herein, wherein the target molecules are any target or analytes conventional in the art. For example, the target molecules may be nucleotides, polynucleotides, DNA, cDNA, RNA, mRNA, cRNA, peptide nucleic acids, oligonucleotides, polypeptides, antibodies, enzymes, hormones, cytokines, other antigens and proteins', peptides displayed on phages and other peptides, carbohydrates, polymers containing alpha-, beta-, and omega-amino acids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, mixed polymers, fragments, analogs and optical isomers thereof, and combinations thereof that form chimeric probes.
In as yet another embodiment of the present invention, there is provided a method as described herein, where the target molecules may have originated from human or other animal host sources and may be derived from humans or other animals or from infectious or parasitic organisms, such as bacteria, viruses, fungi, prions, etc.
In a further embodiment of the present invention, there is provided a method as described herein, where the method is applied to the determination of concentration of a number of target molecules at one spot, where the target molecules may have the same or different binding specificities to different immobilized molecules. In another embodiment of the present invention, there is provided a composition as described herein, where the first tracer moiety is directly or indirectly attached to the immobilized molecule. Where the first tracer moiety is indirectly attached to the immobilized molecules, such attachment is through an intermediate molecule. In one embodiment of the present invention, the method herein provides an intermediate molecule that is commonly shared among all the immobilized molecules for all the spots of the array.
In a further embodiment of the present invention, there is provided a method as described herein, where the first tracer moiety is attached to an intermediate molecule, before or after a target molecule has contacted the immobilized molecules, and the probe signal is measured in the presence or absence of the target molecule.
Further objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description, while indicating certain embodiments of the invention, is given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
IV. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a diagrammatic representation of an array of the present invention showing an expanded view of one spot on an array, as an example, showing target molecules (two represented here) that have been labeled with a tracer moiety (squares) binding to immobilized probes that have been labeled (4 immobilized probes out of 6 are shown labeled) with another tracer moiety (circles). FIG. 2 is a diagrammatic representation of a process for measuring target signal and probe signal, as an example. FIG. 2A is a- diagrammatic representation of an expanded view of one spot on an array, as an example, showing binding of target molecules that have been each labeled with a tracer moiety (squares) to immobilized molecules that have not been labeled. FIG. 2B is a diagrammatic representation of an expanded view of one spot on an aπ-ay, as an example, showing the addition of a plurality of tracer moieties (circles) to the array for the labeling of the immobilized molecules.
FIG. 2C is a diagrammatic representation of an expanded view of one spot on an array, as an example, showing the bound target molecules each labeled with a tracer moiety (squares) binding to immobilized molecules, some of which have been labeled with another tracer moiety (circles).
FIG. 3 is a diagrammatic representation of a process for measuring target signal and probe signal, as an example. FIG. 3 A represents an expanded view of one spot on an array, where some of the immobilized molecules are each labeled with a tracer moiety (circles). FIG. 3B represents the removal of the tracer moieties (circles) from the immobilized molecules and addition of target molecules that have been labeled with a tracer moiety (squares) onto the spot of FIG. 3 A. FIG. 3C represents the same spot after addition of labeled target molecules, showing binding to immobilized molecules. FIG. 4 A represents an expanded view of one spot on an array, as an example, where the immobilized molecules on one spot are. shown as chimeras, each containing a "universal key" at the distal end that is common to all the immobilized molecules on that spot as well as for all the spots on the array, although all the immobilized molecules at a given spot may be unique to that spot.
FIG. 4B represents the same spot as FIG. 4A, showing the binding of target molecules to the chimeras, where the target molecules are shown as being labeled with a tracer moiety (squares).
FIG. 4C represents the same spot as in FIG. 4A and FIG. 4C, showing the labeling of the chimeras with another tracer moiety (circles) after removal of the target molecules .
FIG. 5 is a graphical representation of one example, showing the relationship between fractional occupancy and log concentration for a target molecule that is a 20-mer oligonucleotide.
FIG. 6 is a graphical representation of one example, showing the relationship between fractional occupancy of one target plotted against the fractional occupancy of a second target.
FIG. 7 is a graphical representation of one example showing the relationship between the concentration of one target plotted against the concentration of the second target, where the error bars for the measurements are also shown. V. DETAILED DESCRIPTION OF THE INVENTION
The inventors herein have discovered that the prior art problems with quality control of arrays, such as microarrays, can be addressed by the present invention of methods, kits and compositions for absolute calibration of target concentration, regardless of the variation in the number of immobilized molecules deposited from spot to spot in the microarray, where the immobilized molecules act as capture probes for the targets.
The present invention, described in greater detail below, involves the labeling of the target molecules, reading the target signal in the presence or absence of the probe label or vice versa, determination of fractional occupancy of the immobilized molecules by the target molecules, and the use of KD> the dissociation constant for the dissociation of bound target molecules under equilibrium conditions, in the determination of target molecule concentration.
Definitions: For purposes herein, the following terms shall have the meanings defined below. It is to be noted that the use of the singular herein includes the plural unless clearly specified otherwise. Further, all technical and scientific terms shall have the meaning as commonly understood by persons skilled in the art, unless other defined.
An "array" shall mean a collection of spots that can be one-dimensional, two- dimensional or three-dimensional, and that are supported by a solid substrate onto which immobilized molecules and target molecules are placed and reactions, such as binding, are allowed to occur. A three-dimensional array includes an array of beads or particles.
An "immobilized molecule" shall mean any molecule that can be immobilized on a substrate by any means conventional in the art.
A "molecule" shall mean a natural or synthetic, a biological or chemical molecule, particularly one that has significance in plants, human or animal health, bacteria, viruses, archebacteria, fungi, rickettsia, prions, mycoplasmas, and parasitic organisms. Such molecules include, but are not limited to single molecules and synthetic or natural polymers, such as, nucleotides, polynucleotides, DNA, cDNA, RNA, mRNA, cRNA, peptide nucleic acids ("PNA"), oligonucleotides, polypeptides, antibodies, kinases, phosphatases and other enzymes, hormones, cytokines, antigens, cell surface receptors and other proteins, peptides displayed on a phage and other peptides, carbohydrates, polymers including those containing α-, β-, or ω-amino acids, polymers such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, mixed polymers, small molecules (for example, drug candidates), analogs, fragments and optical isomers thereof, and combinations thereof that form chimeric molecules.
A "molecular species" in reference to the labeled and unlabeled immobilized molecules shall refer to the nature of a molecule." The molecular species are the same when the underlying molecules are the same. For example, an unlabeled immobilized molecule that is an antibody to Her2, a breast cancer antigen, has the same molecular species as a labeled immobilized molecule that is a labeled antibody to Her2. The molecular species are different when the underlying molecules are different. For example, an unlabeled immobilized molecule that is an oligonucleotide having a sequence complementary to a fragment of the Her2 gene, has a different molecular species from a labeled immobilized molecule that is an oligonucleotide having a sequence complementary to a fragment of a prostate cancer gene.
A "probe" or "capture probe" shall mean any immobilized molecule or molecules that are capable of capturing one or more target molecules by specifically binding thereto or by specific hybridization thereto to create bound target molecules. A probe that is labeled with a tracer moiety gives rise to a probe signal that is detectable.
"Specific binding" shall mean the inherent or artificially created property of a molecule to recognize and selectively bind another molecule ("its binding partner"). Examples of specific binding include, but are not limited to, antigen/antibody binding, biotin/avidin or strepavidin binding, receptor/ligand binding, hybridization of complementary oligonucleotides, polynucleotides, or nucleic acids, or synthetic molecules chemically synthesized to bind to other molecules, for example, peptoids.
"Substrate" shall mean any surface conventional in the art that supports an array and on which molecules are allowed to interact, and their interaction detected without degradation of or reaction with the surface. The substrate can be planar, such as a glass slide or a nitrocellulose filter or in the form of beads or particles, such as microspheres or nanobeads. Substrate can be made of glass, silicon or a synthetic, such as plastic. It can be permeable or impermeable. Additionally, the substrate can be, for example, a solid- phase synthesis support, a fiber, such as glass fiber, a capillary tube, or a silicon wafer.
A "target molecule" shall mean any molecule that can be captured by an immobilized molecule, labeled directly or indirectly, detected and/or quantified. Such target molecules may be of any origin including, but not limited to: plants, humans, other animals, other vertebrates or invertebrates, microbial, including bacterial, archebacterial, viral, or fungal, parasitic, or from mycoplasmas or prions.
A "tracer moiety" is a molecule that contains any detectable label that is conventional in the art. The tracer moiety may be removable without damaging the molecule to which it binds. The tracer moiety may further be directly or indirectly measurable. Examples of directly measurable tracer moieties include, but are not limited to: radioactive isotopes, energy transfer dyes, enzymes, quantum dots (sometimes referred to as semiconductor nanocrystals) and luminescent labels. Luminescent labels may be fluorescent labels, chemiluminescent labels, such as a phosphorescent label, bioluminescent labels such as phycobilisomes, colorimetric labels or combinations of such. Preferably, the label is a fluorescent label such as a fluorescein, a rhodamine, a polymethine dye derivative, a phosphor, an energy transfer dye, and the like.
Commercially available fluorescent labels include, inter alia, fluorescein phosphoramidites such as Fluoreprime (Pharmacia, Piscataway, N.J.), Fluoredite (Millipore, Bedford, Mass.) and FAM (ABI, Foster City, Calif.). Optionally, the tracer moiety may be in the form of a bead or microsphere containing a directly measurable label, such as a fluorescent label. Alternatively, the tracer moiety may be indirectly measurable, such that it is modified, chemically or otherwise, to associate with one member of a binding pair, with the opposite member of the binding pair being linked to a directly measurable moiety. Examples of such binding pairs include, but are not limited to: biotin/avidin or strepavidin; oligonucleotides, polynucleotides or nucleic acids for which a complementary molecule carrying a detectable label can be constructed to hybridize therewith; antibody/antigen; receptor/ligand; and molecules chemically synthesized to bind fo each other, such as by combinatorial chemistry.
A "sample" shall mean a biological or other sample that is being assayed or tested for presence or for quantitation of "target molecules."
The methods, kits and compositions of the present invention arise from the recognition that the concept of fractional occupancy, as determined by the Langmuir isotherm (see, for example, Kittel C, "Thermal Physics," Wiley & Sons, 1969, pp. 341- 345), can be utilized to accurately determine the concentration of target molecules in solution, in an array-based assay in which a fraction of the target molecules are captured by immobilized molecules attached to a substrate supporting the array, under conditions in which the concentration of the immobilized molecules constitutes a small fraction of the concentration of the target molecules, and when provided with KD, the dissociation constant for the bound target molecules under equilibrium conditions. According to the present invention, any inconsistency or variation in the number of immobilized molecules deposited at each spot of an array can easily be taken into consideration, and comparisons between samples can be more accurately determined.
A special application of the present invention is in the area of diagnostics for diseases, infection or other medical conditions, detection of environmental hazards or conditions, SNP genotyping, small molecule drug discovery, discovery research, and determination and comparison of gene and protein expression in the areas of genomics and proteomics. However, a person skilled in the art would understand that the present invention may be applicable to other areas as well.
It is to be noted in the first instance that the embodiments described below are for the purpose of illustrating the present invention, but the present invention is not limited to these particular embodiments. Variations from these embodiments may be made and still fall within the scope of the present invention. Further, the terminology employed is for the purpose of describing the invention and is not intended to be limiting. The scope of the present invention is to be determined by the claims herein. The present invention includes methods of determining target molecule concentration in an array-based assay, methods of determining expression of target molecules, methods of comparing expression of one or more target molecules, kits containing labeled or unlabeled arrays of molecules and information on dissociation constants for determination of concentration of target molecules, as well as compositions containing labeled arrays and labeled target molecules for determination of fractional occupancy of capture probes and, ultimately, concentration of target molecules through use of a dissociation constant, KD, which is unique to each bound target molecule. The present invention enables calibration by measuring target signal, and separately or simultaneously, measuring probe signal to determine the ratio of the bound sample to the overall number of immobilized molecules at any spot, this ratio being known as the fractional occupancy. This invention contains a variety of embodiments for determining the target concentration by measuring the fractional occupancy f, and also presents several means to obtain the dissociation constant KD that appears in the Langmuir equation.
In one aspect of the present invention, a composition is provided that contains an array supported by a substrate, where the array contains a number of spots. Usually, the spots are addressable spots. Immobilized molecules for the capture of target molecules are placed or made in situ in one or more spots of the array. One or more or all of the spots of the array may contain immobilized molecules that are either the same or different and that are directly attached to the substrate or, optionally, are attached to the substrate through a linker.
The present invention takes into consideration the amount of immobilized molecules attached at a given spot ("probes" for short) that are available for capture of target molecules. This may be achieved in a number of ways known to persons skilled in the art. For example, one way of determining the amount of probes at a spot is by direct or indirect attachment of a quantifiable marker to that spot that is in a known proportion to the immobilized molecules at that spot. In practice, a known or set portion of immobilized molecules labeled with tracer moieties may be mixed with a known or set portion of the same immobilized molecules but unlabeled prior to attaching the mixture of labeled and unlabeled immobilized molecules at a spot on the substrate.
Optionally, in lieu of mixing labeled and unlabeled immobilized molecules as above, the composition of molecules to be immobilized (i.e., probes) on a spot may be spiked with a different molecular species that has a detectable label and that is capable of immobilizing to the substrate with the same efficiency as the probes. Following immobilization of all labeled and unlabeled molecules to the spot, the spot will contain an amount of label that is proportional to the total number of immobilized molecules. The amount of tracer moiety or detectable label at such a spot may be preset or predetermined to be a desired proportion relative to the amount of immobilized molecules at that spot.
The array, therefore, may be constructed with different spots containing the same or different proportion of tracer moiety or detectable label as compared to the immobilized molecules.
By virtue of the method of the present invention, it is possible to use proportions of tracer moiety to immobilized molecules in ranges higher than that disclosed in U.S.
Patent No. 6,245,518 issued June 12, 201 to Hyseq, Inc., entitled " Polynucleotide Arrays and Methods of Making and Using the Same." Notably, in the present invention, such proportions may be between about 0.16% to about 100%. Optionally, depending on the quality of the array made, the target molecules, the immobilized molecules utilized, and the tracer moieties, persons skilled in the art may utilize other ranges including: 0.16% to
1%, 1% to 2%, 2% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 50%, 50% to 70%, 70% to 100%.
Thus, in one embodiment of the present invention, the tracer moiety is attached to, incorporated within, or otherwise associated with the same type of molecules as that to be immobilized. In such an instance, the molecules to be immobilized may be spiked with an amount of labeled molecules of the same type, forming labeled immobilized molecules.
In the practice of the present invention, a variety of arrays and immobilized molecules of different configuration can be made- and used herein. Suitable methods of constructing the present arrays and immobilized molecules, as capture probes, and methods of immobilizing the capture probes onto solid substrates are conventional in the art. Examples of such can be found in several issued patents including: U.S. 6,045,996 issued April 4, 2000 to Affymetrix, Inc., entitled "Hybridization Assays on Oligonucleotide Arrays"; U.S. Patent No. 6,040,138 issued March 21, 2000 to Affymetrix, Inc., entitled "Expression Monitoring by Hybridization to High Density
Oligonucleotide Arrays"; U.S. Patent No. 6,004,755 issued December 21, 1999 to Incyte Pharmaceuticals, Inc., entitled "Quantitative Microarray Hybridization Assays"; U.S. Patent No. 5,800,992 issued September 1, 1998 to Fodor et al., entitled "Methods of Detecting Nucleic Acids." As a further example, microarrays may be constructed using a Biodot spotting apparatus and aldehyde-coated glass slides. Techniques for immobilizing the probes on the array are also well known to those skilled in the art as can be seen, for example, in Lindroos, K. et al., Minisequencing on oligonucleotide microarrays: comparison of immobilization chemistries," Nucleic Acids Res, 29: p69 (2001). Additional information regarding suitable methods, substrates, arrays, probes, and other related reagents for making arrays can be accessed from the Amersham Biosciences website, www.apbiotech.com. and particularly, www.apbiotech.com/application/microarrav.
Protein arrays that are known and conventional in the art may also be used herein, as described in WO00/056926, entitled "Methods for detection of nucleic acid polymorphisms using peptide-labeled oligonucleotides and antibody arrays"; WO99/39210, entitled "High density arrays for proteome analysis and methods and compositions therefore"; and WO00/004389, entitled "Arrays of protein-capture agents and methods of use thereof." The arrays herein may contain "addressable" spots in that each spot can be located and/or identified by its coordinates or address on the array for a one-dimensional or two- dimensional array, such that by virtue of their positions in the array, the identity of the probes or targets will become known. Optionally, if the array is a three-dimensional array containing beads or particles, such beads or particles may be coded for identification purposes or otherwise identified by its properties, such as fluorescence or size or magnetic properties or combinations of such.
Briefly, the immobilized molecules herein are attached to a substrate directly or indirectly. Direct attachment can be, for example, by covalent bonding to the substrate. Indirect attachment can be, for example, covalent bonding to a linker affixed to the substrate.
The nature of the immobilized molecules is such that they are any molecules capable of acting as capture molecules for any target molecules. In a particular application, they are designed to bind specifically to the target molecules. They include, but are not limited to, molecules or sequences capable of specific binding to or hybridization with target molecules. In case of protein targets, the immobilized molecules may be antibodies, including polyclonal antibodies, monoclonal antibodies, single chain antibodies, fragments or chimeras thereof, or they may be synthetic molecules, including peptides, peptoids or peptide nucleic acids, generated combinatorially or otherwise, to recognize and bind specific target molecules. In the case of hybridizing oligonucleotide, polynucleotide, or nucleic acid targets, the immobilized molecules may be, for example, the complementary oligonucleotides, polynucleotides or nucleic acids, or hybridizing analogs or mimetics thereof, including nucleic acids in which the phosphodiester linkage has been replaced with a substitute linkage, such as phosphorothioate, methylimino, methylphosphonate, phosphoramidate, guanidine and the like; nucleic acids in which the ribose subunit has been substituted, such as hexose phosphodiester; and peptide nucleic acids.
Thus, immobilized molecules may include, but are not limited to: nucleotides, polynucleotides, DNA, cDNA, RNA, mRNA, cRNA, peptide nucleic acids, oligonucleotides, polypeptides, antibodies, enzymes, hormones, cytokines, antigens, other proteins, peptides displayed on phages and other peptides, carbohydrates, polymers containing alpha-, beta-, and omega-amino acids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, small molecule drugs, mixed polymers, fragments, analogs and optical isomers thereof, and combinations thereof that form chimeric molecules.
The length of the immobilized molecules, in instances where they are nucleotides, polynucleotides, nucleic acids or similar polymers, will usually range between 5 to 1000 nucleotides, optionally 5 to 500 nucleotides, further optionally 5 to 250 nucleotides, still optionally, 5 to 100 nucleotides; still further optionally, 5 to 50 oligonucleotides. The polynucleotide, oligonucleotide or nucleic acid probes may be double or single stranded, or PCR fragments amplified from cDNA.
The immobilized molecules may be tailored to specifically bind to or hybridize with specified target molecules. For example, if the array is used to determine expression of a particular gene from a cDNA library that has been reverse transcribed from mRNA molecules, the immobilized molecules will be constructed with a sequence complementary or otherwise capable of recognizing the gene, gene fragment or expression products of such gene or gene fragments. In this context, the nucleic acids may be derived from any biological sources including, but is not limited to, human, animal, plants, bacterial, fungal, viral, environmental or other sources.
The substrate supporting the array may be any solid substrate of any suitable configuration that is conventional in the art for the construction of arrays. For example, the substrate may be substantially planar, such as* a glass slide, a silicon wafer, or a nitrocellulose membrane, or the substrate may be in the form of a bead or particle, such as a microsphere or a nanoparticle. Accordingly, the array may be a one-dimensional, two- dimensional or three-dimensional array. Further, the substrate may be constructed of any suitable materials that provide the array with a solid support and yet is unreactive towards any of the components of the assay including the immobilized molecules, the tracer moieties, and the target molecules. Examples of suitable materials for the substrate herein includes, but are not limited to: plastics, ceramics, metals, gels, membranes, glass, silicon and other synthetic materials. Substrates of interest herein include, but are not limited to: solid-phase synthesis support, fibers, capillary tubes, silicon wafers, slides, membranes, and filters. The immobilized molecules herein are usually spotted onto a substrate to form an array that have at 1 spot, optionally, at least 5 spots, optionally, at least 10 spots, further optionally, at least 100 spots. Alternatively, the array of the present invention may contain at least 1000 spots, optionally, at least 10,000 spots and further optionally, at least 100,000 spots. Where the substrate is in the form of beads or particles, immobilized molecules are attached to the beads and microparticles, each bead or particle being treated as a spot of the array. The beads or microparticles herein may be coded for identification purposes or they may be identified through their chemical or physical properties.
The immobilized molecules herein may be situated at "addressable" spots in that the type of immobilized molecules at each spot is known or can be determined. Accordingly, expression of a certain gene or protein may be traced to an "address" on the array for identification of the gene or protein sequence being expressed. In one embodiment of the present invention, immobilized molecules situated at different spots of the array may be the same. In another embodiment of the present invention, the immobilized molecules situated at different spots of the array are different. The immobilized molecules herein may be labeled directly, or optionally through a linker, with one or more tracer moieties, by covalent or non-covalent bonding. Such tracer moieties are conventional in the art as described, for example, U.S. Patent No. 4,366,241.
Notably, the tracer moiety that is suitable for use herein may be a directly measurable moiety or an indirectly measurable moiety. The directly measurable moiety is a detectable or measurable label, which is conventional in the art. Suitable tracer moieties are disclosed, for example, in U.S. Patent No. 5,800,992 issued September 1, 1998 to Fordor et al., entitled "Method of Detecting Nucleic Acids," and include, but are not limited to: a radioactive isotope, an energy transfer dye, an enzyme, a luminescent label, a quantum dot, or a bead or particle containing such.. A luminescent label includes, for example, a fluorescent label, a chemiluminescent label such as a phosphorescent dye, a bioluminescent label such as a phycobilisome, or a colorimetric label. Fluorescent molecules that are of interest herein include: fluorescein, rhodamine, Texas Red, cyanine dyes and the like. Radioisotopes that are of interest herein include: 35S, 32P, 3H, 1251, 14C and the like.
The indirectly measurable moiety is a molecule that is not directly measurable on its own but that will interact with a directly measurable moiety to yield a detectable signal. Thus, for example, in reference to an immobilized molecule being labeled with a tracer moiety that is indirectly measurable, the immobilized molecule is linked to one member of a binding pair, such as, for example, a biotin/avidin or strepavidin pair, an antibody/antigen pair, a receptor/ligand pair, an enzyme/substrate pair, a hybridizing oligonucleotide, polynucleotide or nucleic acid pair, and the like. A directly measurable moiety is then linked to the other member of the binding pair. Upon interaction between the immobilized molecule linked to one member of a binding pair and the directly measurable moiety linked to the other member of the binding pair, the immobilized molecule becomes measurable.
In one embodiment of the present invention, the tracer moiety herein may be attached to immobilized molecules through a detachable or removable linkage. This is advantageous in that either the probe signal from the labeled immobilized molecules or the target signal from the labeled target molecules may be read without interference from the other signal, whether the two tracer moieties are the same or different. A removal tracer moiety may be attached in such a way that it is removable enzymatically, chemically, by light or other energy activation, or by a change in temperature. Such linkages are conventional in the art. For example, a simple labeling procedure is offered by Pierce rwww.piercenet.com).
The target molecules of the present invention are any analytes that can be captured, labeled and measured. Typically, these target molecules are biological or chemical molecules from natural sources, such as human, animals, plants, bacteria, viruses, fungi, prions, mycoplasmas, and rickettsia. Alternatively, the target molecules may be synthetic molecules, for example, combinatorially synthesized chemical compounds that are small molecule drug candidates. Target molecules from natural sources include, but are not limited to: oligonucleotides, polynucleotides, or nucleic acids, such as DNA, cDNA, RNA, mRNA, cRNA; proteins or polypeptides such as antibodies, antigens, enzymes, hormones, cytokines; carbohydrates; factors, cofactors, analogs, fragments or combinations thereof. Examples of-some target molecules suitable herein can be found in U.S. Patent No. 4,366,241 issued December 28, 1982 to Syva Company, entitled "Concentrating Zone Method in Heterogeneous Immunoassays." The target molecules herein may also be labeled with tracer moieties directly or optionally through a linker, such as by covalent bonding. The tracer moiety for labeling target molecules may be a directly or indirectly measurable moiety as described above. In one embodiment, when the target molecule is labeled with an indirectly measurable moiety, the target molecule may be chemically modified to contain one member of a binding pair that can be recognized by the other member of the binding pair carrying a directly measurable moiety. Any such binding pair that is conventional in the art may be used herein. An example of such a binding pair is the biotin/avidin or strepavidin pair.
The present invention includes a method of determining target molecule concentration in an array-based assay. The invention makes use of the measured fractional occupancy at each spot to obtain an absolute calibration of a target concentration, for example, to determine a gene expression profile, typically using cDNA molecules, reverse transcribed from mRNA, as target molecules. Moreover, two or more samples can be compared to one another by comparing the absolute calibration thereof, thereby obtaining, for example, differential comparison of gene expression among the samples. In this context, a single sample will exhibit a range of fractional occupancies corresponding to the range of concentrations of each type of molecules in the sample. In the practice of the present invention, utilizing an array as described above, a probe signal emitting from the labeled immobilized molecules can be first detected and quantified. Next, a composition containing the target molecules, labeled with a tracer moiety, is allowed to contact the immobilized molecules of the array. After incubation or sufficient passage of time to allow specific binding between the target molecules and the immobilized molecules to occur, excess unbound target molecules are removed, the array washed, and the target signal is detected and measured. Alternatively, the probe and target signal can both be measured after hybridization of the target molecules and washing of the array.
The steps, reagents, and conditions for the conduct of an assay using an array such as the ones described herein are conventional in the art, depending on the immobilized molecules utilized and the target molecules to be captured. Such steps, reagents and conditions may be found in the patents cited herein. For example, if the immobilized molecules and target molecules are both oligonucleotides, the experimental conditions are such as to allow specific hybridization to occur, such as under high stringency conditions. If the immobilized molecules are antibodies and the target molecules are cancer antigens, for example, then experimental conditions are such as to allow antibody and antigen to form a complex.
In the present invention, the target molecules situated on any or all spots of an array may be the same or different and the target signals can be measured with or without removal of the tracer moiety from the labeled immobilized molecules, or in the presence or absence of the tracer moiety from the labeled or unlabeled immobilizes molecules. In instances in which all the target molecules are taken from one sample population, the method herein may be useful in the determination of the expression and concentrations of each type of target molecule in the sample population. In instances in which the target molecules are taken from two or more sample populations, the method herein is useful for the comparison of expression of the target molecules in the two or more sample populations.
As an example, a sample containing target molecules to be analyzed may contain a mixture of 2 samples, one taken from sample 1 containing target molecules 1 that are labeled with target tracer 1, and the other taken from sample 2 containing target molecules 2 that are labeled with target tracer 2. In this example, target molecule 1 and target molecule 2 both contain the same molecular species and differ only in the tracer moiety used to label the targets. The method of the present invention may be utilized to obtain a quantitation of probe signal, target molecule 1 signal and a target molecule 2 signal. The fractional occupancy and, thus, concentration of the target molecule 1 and target molecule 2 may be determined, provided that KD, the dissociation constant, for each of the bound target molecule 1 and 2 are known, provided or determined.
This method may be applied to the comparison of two samples, for example, by determination of target molecule concentration in each sample sequentially, one sample after another and, thus, obtaining two separate target signals in multiple steps or by combining aliquots of the two samples together to form one sample, in equal or known proportions, after labeling the target molecules in each sample in a distinguishing manner so as to obtain two different target signals upon binding between the respective target molecules to the immobilized molecules. An example of this application is the comparison in gene or protein expression using genes and proteins, fragments thereof or molecules resulting from expression or reverse transcription of such genes, proteins and fragments.
Suitable sources of genes, proteins, fragments thereof or results of expression or reverse transcription thereof for application of the present methods include, but are not limited to: normal cells, tumor cells, diseased cells, infected cells, bacteria, viruses, prions, fungi, cells at different stages of division, cells at different stages of growth, cells at different stages of growth arrest, cells at different stages of cell death, and tissues at different stages of development from all biological sources including humans and other animals, plants and microorganisms. Results of expression or reverse transcription of genes, proteins and fragments include, but is not limited to: mRNA, cDNA, cRNA, proteins and polypeptides and fragments thereof, including those resulting from natural post-transcriptional and/or post-translational processing.
Optionally, the target composition may be a mixture containing target molecules taken from more than two sample populations, such as, up to N sample populations, where N is any positive integer greater than 1, and each sample population of target molecules are labeled with a distinguishing label .yielding distinguishing target signals. Alternatively, target molecules from each of N sample populations may be detected and measured sequentially and the concentration of each is compared after separately determining the concentration thereof. Variations in the sequence of measuring probe signal and target signal are within the contemplation of the present invention. For example, if the tracer moiety for the immobilized molecules and the target molecules are the same, the probe signal may be first measured, then the combined probe and target signals are measured following binding of target molecules to immobilized molecules and removal of excess target molecules. The target signal is then obtained from subtracting the probe signal from the combined signal.
In another aspect of the present invention, the probe and target signals are measured separately and/or independently. For example, the probe signal from the labeled immobilized molecules may be measured first and then removed. Then labeled target molecules are allowed to contact the immobilized molecules. Target signal from bound target molecules can then be measured without interference from the probe signal. In this situation, the tracer moiety labeling the immobilized molecules and target molecules may be the same or different. Optionally, a proportion of the immobilized molecules may be labeled with tracer moieties a second time, with or without removal of the target molecule, and the probe signal measured or determined again, either as a check for consistency or to obtain an average value for the probe signal.
Alternatively, the fracer moiety from the immobilized molecule is not removed and the target signal is measured in the presence of the probe signal. In this situation, the immobilized molecules are labeled with a tracer moiety that differs from the tracer moiety labeling the target molecule in its emission spectra or other measurable characteristics. In another aspect of the present invention, the combined probe and target signal following binding of target molecules to immobilized molecules may be measured first and the tracer moiety from either the immobilized molecules or the target molecules are then removed. The signal intensity of the remaining molecules is then measured.
In a further variation, signal intensity of the target molecules may be measured first after the composition containing target molecules is allowed to contact the immobilized molecules of the array, prior to the immobilized molecules being labeled. Then the immobilized molecules are labeled, followed by measurement of combined probe and target signal. Thereafter, through subtraction, the probe signal can be determined.
In a variation of the embodiment described above, the target molecules may be allowed to interact with immobilized molecules before the immobilized molecules are labeled. Target signal from bound target molecules may be read and the target molecules or their fracer moieties may be removed. Subsequent thereto, the immobilized molecules may be labeled with the same or different tracer moiety, and the probe signal measured without interference from the target molecules.
Alternatively, immobilized molecules may be attached to a universal linker. Such a universal linker may be attached to all immobilized molecules, of the same or different specificities, on one or more or all spots of an array. Before or after interaction with target molecules, with or without removal of the target molecules, or in the presence or absence of the target molecules, fracer moieties may be attached to the universal linker on the immobilized molecules and the probe signal measured on all the spots on the array, regardless of binding specificities of the immobilized molecules. The resultant binding between the immobilized molecules and the tracer molecule or between the immobilized molecules and labeled target molecules may be visualized in a number of ways conventional in the art. The particular manner of detection depends on the tracer moiety or label utilized, such as scintillation counting, autoradiography, fluorescence measurement, colorimetric measurement, light emission measurement and the like. Based on the intensity of the signals, fractional occupancy and concentration of the target molecules may be determined or calculated. Apart from corrections for partial labeling of the probes or target molecules, and issues of instrument calibration, the fractional occupancy f is given by f = It / Ip , where It refers to the target intensity and Ip refers to the probe intensity. The examples provide more detail.
In one aspect of the present invention, KD may be calculated based on a generally accepted formula (see Example 6 below). In another aspect of the present invention, KD may be determined experimentally, such as by conducting the method of the present invention for each of several different dilutions of a target molecule and plotting the target concenfration against the function f / (1-f ), where f is the fractional occupancy.
As shown in Example 10, KD is the slope of the line through all points that minimizes the squared distance between the line and the points. The present invention includes a kit that contains, in one aspect, an array of molecules immobilized on a substrate as described above and certain information relevant to the determination of target molecule concenfration, where the array contains a number of spots, at least one spot having more than one immobilized molecules, and where the molecules to be immobilized or already immobilized on the substrate of the array are labeled or as yet unlabeled.
In the present invention, the kit herein may further contain one or more of the following: (a) instructions for determining dissociation constant, KD, for any target molecules bound to immobilized molecules; (b) instructions for determination of gene or protein expression; (c) instructions for comparing gene or protein expression; (d) instructions for determination of target molecule concentration; (e) instructions regarding an appropriate tracer moiety to use for labeling target molecules so as to optimize the spectral separation between the target signal and probe signal. Usually, the kit will contain instructions for more than one type of tracer moieties for labeling more than one type of target molecules. Further optionally, the kit may contain a fracer moiety for labeling the immobilized molecules or a detectable label that is suitable for spiking the immobilized molecules. In a variation of this embodiment, the kit will contain a substrate suitable for attaching probes to produce an array, a solution of probes to be immobilized, a solution of labeled molecules for spiking the probes, and information regarding KD.
The following examples are given by way of illustration to facilitate a better understanding of the invention herein, and is not to be interpreted as limiting the invention in any way. Example 1. Derivation of formula for determination of target molecule concenfration as a function of fractional occupancy, f.
Fractional occupancy is a ratio representing the total number of target molecules bound to immobilized molecules at a given spot on an array divided by the total number of immobilized molecules available at that spot. The actual number of bound target molecules at a spot, on average, will depend on the number of capture molecules available under a given set of experimental conditions. The average number of bound target molecules is the fractional occupancy times the number of molecules located at the spot. Since arrays generally consist of many spots, with the immobilized molecules generally being different from one spot to another, the fractional occupancy and number of bound targets will generally differ from one spot to another.
The problem at hand has been analyzed for both solutions and gases and follows the general form called a Langmuir isotherm (Kittel, C, "Thermal Physics", Wiley & Sons, 1969, pg. 341 - 345). For the general reaction: -
[bound target] - [free molecule] + [target], the fractional occupancy, f , for bound target at a given spot equals [bound target] / ([bound target] + [free molecule]). Here, [bound target] is the concenfration of target molecules bound to the immobilized molecules at a spot, [free molecule] is the concenfration of unbound immobilized molecules at that spot, and [target] is the concentration of the free target molecules in the sample solution. By multiplying the numerator and denominator by the product of [target]/[bound target], the fractional occupancy, f, can be rewritten as:
f = [target] / ([target] + [target] [ free molecule]/[bound target]).
Here, the second teπn in the denominator is the dissociation constant KD for the reaction, so the fractional occupancy has the simple form:
f = [target] / ([target] + KD).
Since the free target molecule concenfration in this formula changes if there are a large amount of immobilized molecules at a spot that can bind the free target molecules, it is usually convenient to have sufficiently large target sample concentration so that the number of target molecules is large compared to the number of immobilized molecules at the spot (or spots, if several spots are identical). In this limit, the concenfration of target does not change substantially as a result of binding events.
This formula can be inverted to give the target concentration as a function of the fractional occupancy:
[Target] = KD * f / (l-f ).
The target concenfration is determined based on this formula.
Example 2. Hybridization of Target Molecules, cDNA herein, to Immobilized Molecules that are Complementary Nucleic Acid Molecules*
The present invention will now be illustrated by way of the figures. In FIG. 1, a composition of the present invention is diagrammatically represented by the expanded view of one spot 7 on an array 8. Other spots are not shown here. Labeled target molecules 1 are shown labeled with tracer moiety 2 (squares) and the labeled immobilized molecules 3 are shown labeled witi another fracer moiety 4 (circles), where . the immobilized molecules are attached to a substrate 5. Unlabeled target molecules are not shown here, but unlabeled immobilized molecules 6 are shown without any fracer moieties attached. This may be viewed, for example, in terms of cDNA molecules or oligonucleotides as target molecules 1, being hybridized to its complementary nucleic acid molecules 3 and 6 on the array 8. Use of DNA in FIGS. 1-4 is not meant to limit the broad applicability of the present invention, since" these figures also apply to any other target molecules and the complementary immobilized molecules at a spot. The immobilized molecules at the spot are shown as being partially labeled in FIG. 1. Since the immobilized molecules at a spot can be large in comparison to the bound target molecules, this partial labeling of the immobilized molecules can be useful so that the signal from the immobilized molecules does not mask the signal from the target molecules. So long as the partial labeling of the immobilized molecules is carried out such that it is in a known proportion to the overall number of immobilized molecules at a spot, the total number of immobilized molecules at the spot is readily determined once the number of labeled immobilized molecules is known.
In this example, the target molecules in a sample to be assayed are labeled with a different tracer moiety than that used for the immobilized molecules. The target molecules are shown as being completely labeled, since their concentration and corresponding assay signal strength will usually be low, although the target molecules can also be partially labeled. So long as the proportion of partial labeling of target molecules is known, then the total amount of target can be deteπnined from the amount of partially labeled targets.
The ratio of the bound target molecules to the total immobilized molecules at the spot, the fractional occupancy, is described by the Langmuir isotherm introduced above. As discussed in Example 1, it is convenient to work in the limit that the number of free target molecules is large compared to the number of immobilized molecules at the spot (or spots, if several spots are identical). Under such conditions, the concentration of target does not change substantially as a result of binding events. After hybridization and removal of the excess target sample, the signal associated with the bound target molecules at a spot is measured. The probe signal can be measured either before or after the hybridization of the target molecules. The fractional occupancy f is by definition the ratio of the bound target molecules divided by the total number of immobilized molecules. Since this is a ratio, the reading instrument does not necessarily need to be calibrated to separately determine the actual number of target and immobilized molecules. Instead, the instrument can be calibrated on a relative basis to adjust for efficiency differences in reading the signals associated with the target molecules versus reading the signals associated with the probes.
Calibration of a label signal intensity to number of molecules, starting from a standard sample with known amount of labeled molecules is a straight forward exercise in physical chemistry: The emission photon flux can be accurately measured, the fluorescent efficiency is known, and the numerical aperture and detection loss is part of any good imaging design. Once calibrated, the number of labeled probe molecules Np can be inferred from the signal intensity Ip of the label used for the probe molecules, and the amount of labeled bound target Nt can be infeϊred from the intensity It of the label associated with the bound target.
The ratio of number of target molecules to probes is thus given by the ratio Nt / Np where the subscripts refer to target and probe respectively. If the probes are partially labeled, the probe number Np needs to be divided by the proportion of probes that are labeled to reflect the actual number of probe molecules at a spot that are available to bind a target molecule. In the event the target molecules are partially labeled, a similar correction needs to be made. Apart from such corrections, the fractional occupancy is given by f =Nt / Np.
The concentration of target is then given by inserting this ratio into the formula for target concentration:
[target] = KD f/ (l - f). This embodiment of the invention has the probe label attached to the immobilized molecules that in turn can bind to the target molecules. In a variation to this embodiment, the label can be attached directly to the spot or indirectly to the spot by means of other molecules that are attached to the spot, where the other molecules do not interact with the target. In doing so, there needs to be a proportionality between amount of label at the spot and the amount of immobilized molecules that can interact with the target molecules. In a variation of this embodiment, a single tracer moiety may be used for labeling both the immobilized molecules and the target molecules. In doing so, the probe signal for a spot is measured first. Following this, the target is hybridized to the immobilized molecules, excess target molecules are removed, and a composite signal consisting of the probe signal and the target signal at the spot is measured. The actual target signal is then the incremental signal above the probe signal, that is, a simple difference of the second (composite) signal and the probe signal gives the target signal. Here also, corrections need to be made to account for partial labeling of the immobilized molecules or of the target. In a variation to this embodiment, the target can be measured first and the probe labels are indirectly attached, in that they can be added after the prior measurement of the target signal.
The tracer moieties described in this example are conventional luminescent labels, both for the probes attached at each spot and for the target molecules.
Example 3. Variations in the Sequence of Labeling and Measuring Target and Probe Molecules are Contemplated.
Another embodiment of the present invention is diagrammatically represented in FIG. 2 portraying one variation in the sequence of labeling of the probes. Here, in FIG.
2A, the labeled target molecules 1 that are labeled with tracer moiety 2 (squares) are hybridized to unlabeled probes 6 immobilized on substrate 5 at a spot 7 of an array 8. After removal of excess target molecules, the target signal is measured. Following this, in FIG. 2B, tracer moieties 4 are added to label the probes, using a suitable linker mechanism, such as a biotin/avidin combination. In FIG. 2C, the labeled target molecules 1 are shown hybridized to labeled immobilized molecules 3 and unlabeled immobilized molecules 6 on a substrate 5 at a spot 7 of the array 8. The probe signal from a spot 7 is then measured, the fractional occupancy is determined, and the target concenfration is determined as before through use of the Langmuir equation.
Generally, the fracer moieties for the target molecules and for the immobilized molecules can be the same or different. In this example, where the tracer moiety labeling the probe is different from the fracer moiety labeling the target molecules, the two signals may be read separately, preferably the target signal is measured first to avoid any noise background due to the probe signal. Alternatively, if the fracer moieties for labeling the target molecules and the probe are the same, a combined probe and target signal may be measured after labeling of the probe, and the probe signal can be arrived at by subfraction of target signal from the combined signal.
The variation of attaching the probe label'directly or indirectly to the spot via a linker also applies, where a proportionality to the immobilized molecules must be maintained. As discussed, probes and target measurements need to be corrected if partial labeling of either the immobilized molecules or of the target is implemented.
Example 4. Another Variation in the Labeling of Target Molecules and Immobilized Molecules and Measuring of Target and Probe Signals by Use of Removable Tracer
Moieties.
A removable tracer moiety may be used for labeling of the target molecules or the immobilized molecules. FIG. 3 shows use of the removable tracer moiety in connection with labeling of the immobilized molecules. Here, FIG. 3 A diagrammatically represents the labeling of the immobilized molecules 6 with a removable tracer moiety 4, to form labeled immobilized molecules 3 at a spot 7 (other spots not shown) of an array 8, for example, using a removable disulfide linker (Pierce EZ-Link Sulfo-NHS-SS-Biotin). The probe signal from the labeled immobilized molecules 3 at the spot is measured first. The removable tracer moieties 4 are then removed from the labeled immobilized molecules 3 to become unlabeled immobilized molecules 6. The target molecules 1 with labels 2 are then brought into contact with the unlabeled immobilized molecules to allow binding. Excess target molecules (those that are not bound) are removed, and the target signal is then measured. Removing the fracer moieties from the probe following measurement of the probe signal permits use of the same tracer moieties for both probe and target molecules in this embodiment. Of course, two different tracer moieties may be used here as well. Apart from the detail of using a removable tracer moiety for labeling the immobilized molecules, this embodiment is very similar to the earlier two embodiments, including any corrections that are needed for partial probe labeling prior to computation of the target concenfration. This variation in labeling and measuring target and probe signals flips around the process described in Example 3 above, where the probe signal is measured first in this Example 4, then the tracer moiety is removed from the probe, then the labeled target molecules are added.
Example 5. Use of an Intermediate Molecule for all Spots on an Array to Facilitate Measurement of Probe Signal from Immobilized Molecules of Different Molecular Species. FIG. 4 is a diagrammatic representation of another embodiment of the present invention in which an intermediate molecule 10 is used for labeling of the immobilized molecules attached to the substrate 5 to form composite molecules 9. FIG. 4A shows immobilized composite molecules in which a portion of each composite molecule 9 is complementary to the target molecule and is made to be unique for one or more spots of the array 8 (Other spots not shown herein.). Another portion of the composite molecule 9 is made to be identical or "universal" for all the spots of the array. In the case of nucleic acids, the composite molecules 9 may be referred to as chimeric probes. FIG. 4B shows labeled target molecules 1 being brought into contact with composite molecules 9and binding thereto. In practice, excess target molecules are removed. The target signal at the spot is then measured. The number of immobilized molecules at a spot is then deteπnined by bringing the universal portion of the immobilized composite molecules in contact with a solution that contains the labeled complement molecule to the universal portion of the immobilized molecules. If the amount of labeled complement molecules are in significant excess, compared to the number of immobilized molecules, experimental conditions including the temperature can be adjusted so that virtually all of the immobilized molecules will be bound to their labeled complement. The signal from the labeled complement is then measured. With KD being known, the concentration of target can be calculated as before. One variation to this fourth embodiment is to reverse the order of measuring the target signal and the probe signal. In another variation, the same fracer moiety can be used for both the target and the immobilized composite molecules. In another variation, only a fraction of the immobilized molecules have a universal portion, so a correction needs to be made as discussed before. Similarly, only a portion of the targets may be labeled. In such a case a correction here also would be necessary.
The present invention has been described in which single target molecule populations are measured separately and compared to the signal from the probes for absolute calibration, in determining the target concentration. Often the interest is in a differential comparison of the two concentrations. The method described herein can equally well be extended to include several target populations that are run simultaneously, each of which uses a different tracer moiety and for each of which the calibration is achieved using the absolute calibration provided by the immobilized molecules at each spot (which in turn have is labeled by another fracer moiety which differs from the fracer moieties used for labeling the target molecules). By running two or more populations simultaneously and calibrating them to the immobilized molecules, they can subsequently be compared to each other to arrive at the differential comparison.
The invention is applicable to analysis of many polymers, including polypeptides, carbohydrates, and synthetic polymers, including alpha-, beta-, and omega-amino acids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, proteins, antibodies, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, and mixed polymers. Various optical isomers, e.g., various D- and L- forms of the monomers, may be used.
In other embodiments, antibody probes will be generated which specifically recognize particular subsequences found on a polymer. For example, antibodies may be generated which are specific for recognizing a three contiguous amino acid sequence, and monoclonal antibodies may be preferred. Optimally, these antibodies would not recognize any sequences other than the specific three amino acid stretch desired and the binding affinity should be insensitive to flanking or remote sequences found on a target molecule. Likewise, antibodies specific for particular carbohydrate linkages or sequences may be generated. A similar approach may be used for preparing specific reagents that recognize other polymer subunit sequences. These reagents would typically be site specifically localized to a substrate matrix pattern where the regions are closely packed. For other polymer targets, the specific reagents will often be polypeptides. These polypeptides may be protein binding domains from enzymes or other proteins that display specificity for binding. Usually an antibody molecule may be used, and monoclonal antibodies may be particularly desired.
Example 6. Approximation to KD for polynucleotides
It is useful to have an approximation to Kp for use the other examples to follow. In the case of polynucleotides the dissociation constant KD generally depends on GC content, GC context, and a variety of other factors in a hybridization experiment. In this example a very simple calculation of KD is presented. More precise methods for the determination of KD are also cited.
To solve for the KD of a typical 20-mer oligonucleotide the KD in moles can be expressed as a function of the free energy G:
KD = eΛ(-G RT),
where eΛx is defined to be ex, and R is the gas constant, which has a value of .001987 Kcal/(mole deg K). The free energy G can be related to the well-measured melting temperature Tm by noting that Tm is the temperature at which the free energy is zero. The free energy G equals H - TS, where H and S are the enthalpy and entropy, respectively. The melting temperature of oligonucleotides is closely approximated by an increase of 3 deg Celsius for every nucleotide (Strachan T, Human Molecular Genetics):
Tm = 273 + 20*3 = 333°K.
The enthalpy is calculated from the average number (2.5) of hydrogen bonds (approx. 3 Kcal/mole) for each Watson-Crick DNA pair:
H = 3 Kcal/mole * 2.5 * 20 = 150 Kcal mole
The entropy is calculated from the melting temperature: S = H / Tm = 150 / 333 = .45045 Kcal (mole deg K)
Meanwhile, hybridization usually occurs at 37° C, or 310° K. The free energy at 37° C is: G = H - TS = 10.36 Kcal/mole
The corresponding KD = eΛ(-10.36/(.001987 *310) = 4.96 E -8 molar. Note that this constant depends only on absolute temperature, a result found by Langmuir in developing the fractional occupancy formula. Most examples below use this value of 310° K. Apart from the temperature dependence, we note that the free energy and thus the fractional occupancy will vary somewhat from one probe type to the next. Such minor variations will not alter the results in examples that follow.
More precise calculations of KD are readily available. A variety of calculations express the melting temperature Tm in terms of enthalpy, entropy, sodium content, and GC content including nearest neighbor interactions for a given molar concentration of the strands (see for example the melting temperature calculator in www.genosys.com, also for DNA see Breslauer et al, Proc. Natl. Acad. Sci USA 83: 3746-3750 (1986)). Since the melting temperature is by definition when the fractional occupancy equals one-half, this is when KD equals the strand concenfration. Therefore, replacing the strand concenfration with KD in the chosen melting temperature equation and inverting this equation gives KD to sufficiently high precision for many applications.
Example 7: Fractional occupancy versus concentration of target cDNAs in solution binding to 20-mer oligo probes.
The KD obtained in Example 1 is used to generate the results shown herein in Table 1. These results are also graphically represented in FIG. 5. Note a target typically has a range of cDNA concentrations, to which correspond to a range of fractional occupancies.
Table 1 : Fractional Occupancy, f, versus log concentration.
log cone. cone.
-10 1E-10 0.002013 -9 0.000000001 0.019769 -8 0.00000001 0.167829
-7 0.0000001 0.668518
-6 0.000001 0.952758 *
-5 0.00001 0.995066
-4 • 0.0001 0.999504
Conversely, if the fractional occupancies are independently determined by, for example, labeling the target molecules and the probes at each spot and calculating the ratio of the respective signal intensities, the point to this invention, then the concentration of the target corresponding to the probes at that spot is readily determined. Often samples from different populations are compared to a given sample as a reference sample. In such cases, only one reference sample needs to be run and different populations are run separately thereafter. The total number of runs is therefore quite close to the total number of runs needed for competitive hybridization as described in U.S. Patent 5,800,992, that is, there is no need to run the reference sample every time a new target sample is mn.
Use of this absolute calibration method, involving a single array only, would be invaluable in a variety of gene or protein expression profile applications, determination of viral loads, etc.
Example 8: Comparison of gene expression between two different targets and determination of the error bars associated with this comparison.
Table 1 of Example 7 was for only one type of labeled target molecule in solution while the other label is associated with the complementary probes at a spot. In contrast, in "competitive hybridization" (for example, U.S. Patent 5,800,992), there are often two differently labeled types of target molecules in solution for which the gene expressions profiles are to be compared. Alternatively, in differential gene expression measurements, separate targets are measured using two different arrays, and comparison is made by using normalization control genes or other target molecules. The present invention affords an absolute calibration of two or more types of target molecules, and comparison of each of the at least two types of target molecules against this absolute calibration, and provides a means to then compare the concenfration of each target molecule type against each other, thereby, obtaining differential gene expression. This can be done using separate microarrays for determining the concentration of each target molecule type, and the method can be extended to include a separate tracer moiety for differentially labeling each of two or more targets in solution using the same microarray, with a different tracer moiety reserved for labeling the probes. The concenfration of each target can be .independently determined, and comparison of the target concentrations so obtained yields their differential expression. In these comparisons, the anticipated error bars associated with the measurements need to be small relative to the differential expression that is being analyzed. Otherwise, the error noise will mask the differential expression outliers. To demonstrate that the errors are acceptably small, consider two samples 1 and 2 with expression concentrations concl and conc2. Further, sample 2 happens to be loaded at 1.5 times the concentration of sample 1, and also the third cDNA of sample 2 has twice the expression of the third cDNA of sample 1. Starting with these known concentrations, we want to walk through the formalism and display the results including anticipated errors.
In considering the measurement statistics there will be a variation in the fractional occupancy at a spot since a cDNA in solution can randomly attach to any one of the available complementary probes, and the standard error δf associated with this variation is δf = square root [ (f *(l-f )/probe#) ] , where probe# is the number of probes that are available at a given spot. In actual measurements we are comparing the concentrations directly, so the formula for f can be inverted to give the concentration c as c = f * KD / (l-f), while a variation in f gives a variation in c called δc: δc = δf * KD * (l/(l-f)Λ2 ). The table below gives example results for the above considerations, where we have used twice the computed value of δf to correspond to two standard errors, and we have assumed that, for each cDNA type in solution, there are one million fixed probes available at the corresponding spot on the array.
Table 2: 2 samples of 5 cDNA. Sample 2 loaded at 1.5 x sample 1. Third cDNA has 2x expression.
δf = (f * (l'-f) /probe#)Λ0.5 for binomial distribution (One standard deviation.) c = f * KD / (l-f) δc = δf * KD (l/(l-f)Λ2)
Cone 1 Cone 2 fr occl fr occ2 2*δfl 2*δf2
1.0.0E-08 1.50E-08 1.68E-01 2.32E-01 7.47E-04 8.45E-04
5.00E-08 7.50E-08 5.02E-01 6.02E-01 1.00E-03 9.79E-04
1.00E-07 3.00E-07 6.69E-01 8.58E-01 9.41E-04 6.98E-04
5.00E-07 7.50E-07 9.10E-01 9.38E-01 5.73E-04 4.82E-04
1.00E-06 1.50E-06 9.53E-01 9.68E-01 4.24E-04 3.52E-04
δcl δc2 fr_high2 fr_low2 conc_high2 conc_low2
5.35E-11 7.10E-11 2.33E-01 2.31E-01 1.51E-08 1.49E-08
2.00E-10 3.06E-10 6.03E-01 6.01E-01 7.53E-08 7.47E-08
4.25E-10 1.72E-09 8.59E-01 8.57E-01 3.02E-07 2.98E-07
3.49E-09 6.22E-09 9.38E-01 9.38E-01 7.56E-07 7.44E-07
9.43E-09 1.70E-08 9.68E-01 9.68E-01 1.52E-06 1.48E-06
The data from Table 2 are used to plot the fractional occupancy of sample 2 against sample 1 (shown in FIG. 6, "Fractional occupancy of 2 samples, 2σ"). On this scale, the error bars associated with plus and minus 2 standard deviations (conc_high2 and conc_low2 respectively) are too close to distinguish or to be shown.
Next, we show the concentration of target molecules in sample 2 plotted against the concentration of target molecules in sample 1, as shown in FIG. 7 (Graph titled, "Concentration of two samples, 5% errors). On this graph as well, the error bars were initially too close to distinguish. However, we added a high and low error term that was plus and minus 5 % of each sample 2 concentration data point (actually, this error term was computed in quadrature. The differential expression results in FIG. 7 show a linear relationship between concentration of target molecules in the two samples, except for the outlier associated with the third cDNA. This outlier is readily distinguishable, in particular for the low concentrations of most interest. (Empirically most genes do not differentially express, so a linear regression fit is readily established for a practical case of dozens or hundreds of genes, and the outliers are thus readily distinguishable even in the presence of normal noise levels.)
The calculation of fractional occupancy depends strictly on the number of fixed molecules of each type, whether the molecules are at one array site or many, or on one bead or many. The statistics for fractional occupancy are quite robust: The total number of hybridized cDNA of a given type is divided by the total number of available oligos for that type. In the case where the same oligo or cDNA is located at multiple microarray sites or beads and all of the measurement data is "good," this model states that the best answer is to take the total signal from all the hybridized cDNA and divide it by the total signal from all the fixed oligos or cDNA of that type, rather than (for example) computing a ratio for each site or bead and averaging the ratios from any replicates. The data goodness can be analyzed through an initial processing step in which, for each cDNA type, a regression fit is generated of the hybridized cDNA signal versus the calibration signal from the fixed oligos or cDNA. (Each of the relevant sites or beads contributes a data point to this regression fit.) This allows any data outliers from the regression fit to be rejected before summing up the total signal as indicated above.
Example 9. Cancellation of dissociation constant KD errors in differential comparisons. In applications involving determination of target concentration, such as gene or protein expression profiling, the target concentration is linear in the dissociation constant
KD. Therefore any errors in KD are propagated directly into the concentration error. On the other hand detecting a differential expression outlier depends on the ratio of the two target concentrations being compared. Each concenfration is linear in the same dissociation constant KD, and therefore the ratio results in the constant KD being divided out. In short, the differential expression is based on the ratio of the respective f / (1-f) values for each target as measured in an experiment.
This is consistent with Table 2 of the previous example. The two columns fr_occ2 and fr_occl can be used to form the respective f / (1- f ) value corresponding to each of the expression concenfrations shown in the rows. For each of the rows except for the third, the ratio of these values gives a differential expression value of 1.5. However, in the third row the ratio of the f / (1- f ) values gives a value of 3.0. This is consistent with the concentration assumptions used in this example. The ratio of the f / (1- f ) values results in the concentration ratios, or differential expression ratio, independent of KD.
Example 10: Calibration procedure for precise specification of KD
The equation developed for the target concentration as a function of fractional occupancy, [target] = KD * f / (1- f ), treats the target concentration as the unknown while KD and f are known. If the target molecule concentration [target] and are known, KD can be calibrated. Consider, for example, the case of cDNA. We are typically interested in identifying specific cDNA types and amounts in an unknown target sample. In turning the process around, we can start with a cDNA library with known concentrations of each cDNA component in the library. The known concenfrations can easily be determined from absorbance measurements at high concenfrations. We then dilute the cDNA library, for example, by half, and half again, until we obtain very low concentrations. If we measure the fractional occupancy for each of these known concentrations, and plot the concenfration as a function of (f / (1 - f )), the slope of a straight line. through the origin that minimizes the squared difference between the line and measured values is the best estimate of KD. Moreover, the residual errors in this linear fit to KD provide an estimate of the error in KD- Foπnulas to compute the slope of such a line and the associated errors are well known in the art (for example, see: Press, W. et al, Numerical Recipes in C, 2nd edition, Cambridge University Press, 1992).
Example 11. Application of the Method of the Present Invention to Proteins
The following example describes an antibody as the immobilized molecule being bound to a solid support, and a protein interacting with the immobilized antibody as the target molecule. The antibody can be any molecule having the characteristics of an antibody, including a monoclonal or polyclonal antibody, and can be derived from human (e.g.: IgG), non-human (e.g.: IgY) or mixed (humanized Ab) sources. Antibodies have been well characterized and are known to those skilled in the art. They consist of two chains: a light chain and a heavy chain, and resemble in structure a "Y". The variable domains inside the "V of the "Y" contain complementary determining regions (CDRs) which bind to antigens. Techniques for creating monoclonal and polyclonal antibodies specific to a particular antigen are well known to those skilled in the art. For example, one simple kit that produces large amounts of chicken antibodies (IgY) is called EGGstract™ from Promega. Glass slides are prepared according to standard procedures (e.g.: XENOBIND™ from Xenocore, www.xenocore.com.) The antibody to be bound is dissolved in a buffer solution at a pH above the isoelecfric point of the antibody (important because the binding takes place through the amine groups on the protein, and these must be in the free form for binding to take place); the protein solution is incubated for 2-3 hours at 37° C. This results in the formation of a covalent bond between the glass surface and the protein.
In most cases the binding capacity is substantially increased by covalent binding. For typical antibodies high binding levels are achieved at much lower concentrations than with passive binding. This is the result of the irreversible nature of the covalent binding process; monolayer coverage can be achieved at low concenfrations, limited only by the diffusion rate of the antibodies to the surface. Monolayer coverage is usually about 300 ng. per square centimeter, and can be achieved with concentrations containing twice that amount of protein in agitated systems. In most cases the presence of surfactant in a protein solution does not inhibit covalent binding- with XENOBIND™ plates as it does with passive binding.
Once the antibodies have been covalently attached to the glass surface, target proteins molecules are introduced in buffer to a small volume surrounding antibodies. Antibody-antigen dissociation constants vary due to the specificity of the variable domain. Typical values range between 10"6 - 10"7. Protein affinity determinations, or 1/ KD, can be found in www.biacore.com, or see also NHRC Publication 84-37 (Griswold,
W.R. & D.P. Nelson, Immunology Letters, 9, pages 15-18 (1985).The associated concentration of protein [prot] is calculated in the manner as described earlier using, for example, a KD of 10"7:
[prot] = KD = 10"'
1 - f 1 - f
For fractional occupancies between .001 and 0.9 this would give the following values for the concenfration of the bound protein:
Figure imgf000046_0001
The fractional occupancy can be detected by any of the methods described above. For example, the immobilized antibody can be labeled with one dye and the target antigen protein labeled with a spectrally dissimilar dye. The same process can be used to label both the antibody and the protein since the antibody is a protein and contains numerous N-termini. For example, a simple labeling procedure is offered by Pierce (www.piercenet.com). The EZ-LinkTM Suylfo-NHS-LC-Biotinylation Kit allows biotin labeling of proteins or antibodies in 30 minutes at room temperature. The biotin labeled proteins or antibodies can be reacted with streptavidin-conjugated dyes (e.g.:
ImmunoPure Streptavidin Fluorescein Conjugated from Pierce) in another 30 minutes. The probe signal from the dye attached to the antibody must be calibrated to the number of dye molecules. Similarly, the signal from the spectrally dissimilar dye attached to the target protein (antigen) must be calibrated to the number of protein molecules. This can easily be done by one skilled in the art by measuring the signal associated with known concentrations of antibodies and proteins, respectively. The fractional occupancy f is calculated by dividing the number of bound protein molecules by the number of antibody molecules.
KD can easily be measured by those skilled in the art. One method is to use varying concenfrations of protein, measure the associated fractional occupancy f and then plot the protein concenfration as a function of the fraction f/(l-f), analogous to Example 10 The slope of a line through the origin minimizing the squared difference between the line and the concentration is the best estimate of KD.
Once KD is known, subsequent measurements of f can produce accurate estimates of protein concenfration using the formula:
[prot] = KD
1 - f
Example 12. Detection of Mutations and Single Nucleotide Polymoφhisms ("SNPs") This invention is useful in detecting possible genomic mutations in an individual, where the nucleic acid sequence for a mutation of interest is known in advance. Existence of the mutation is declared when there is an adequately sfrong target signal and associated concenfration corresponding to the immobilized molecules on a spot of an array complementary to the mutation.
Closely related to this application of the present invention is the typing of SNPs from single individuals. As is well known, genomic DNA contains two chromosomes, and most SNPs have only two alleles. In this invention, the spots with nucleic acids complementary to each of the alleles would yield information on the concentration of each SNP. For homozygous individuals, only one spot would result in a target signal, whereas for heterozygote individuals both spots would yield a target signal. Sample preparation, buffer conditions, and hybridization constraints are never perfect. This results, for example, in mismatches n many techniques, where the wrong allele hybridizes to a spot. If the concentration calculated for one spot is plotted on the x- axis, and the other on the y-axis, and this is done for a population of individuals, there will tend to be a scatter plot between the positive x and y axes due to the noise sources. At times, the variation in concenfrations can be large, and at times it is hard to tell which are the homozygotes and which are the heterozygotes. In contrast, this invention improves precision by removing variability due to spot size. Therefore, the scatter plots are reduced in their spread, allowing easy identification of SNP type.
All publications and patents cited herein are incoφorated by reference in their entireties, including all publications and patents cited therein, as if each were specifically and individually indicated to be incoφorated by reference. The citation of any publication and patent is for its disclosure prior tQ the filing date of this application and any benefit to which it is entitled, and should not be construed as an admission that the present invention is not entitled to antedate such publication or patent by virtue of prior invention.

Claims

What Is Claimed Is:
1. A method of determining target molecule concenfration in an array-based assay comprising the steps of:
(a) providing a first composition comprising an array of molecules immobilized on a substrate, wherein the array comprises a plurality of spots, at least one spot comprising more than one immobilized molecule, wherein the immobilized molecules are attached to the subsfrate, and at least one of the immobilized molecules is labeled with a first tracer moiety to form a labeled immobilized molecule that yields a probe signal; (b) providing a second composition comprising a plurality of target molecules, wherein at least one target molecule is labeled with a second tracer moiety to form a labeled target molecule that yields a target signal;
(c) allowing the first composition to contact the second composition under conditions to allow binding of one or more target molecules to one or more immobilized molecules, labeled or unlabeled, and to allow determination of fractional occupancy;
(d) measuring the probe signal;
(e) measuring the target signal; and
(f) determining concenfration of target molecules.
2. The method of claims 1, 3-10, 69, 70, 75, 76, 78 and 79, wherein the immobilized molecules and target molecules are each oligonucleotides or nucleic acids.
3. The method of claims 1, 4-10, 69, 70, 75,-76, 78 and 79, wherein the subsfrate is in the form of a planar surface, a bead or a microsphere.
4. The method of claims 1, 5-10, 69, 70, 75, 76, and 79, wherein the immobilized molecules situated at different spots of the array are the same or different.
5. The method of claims 1, 6-8, 69, 70, 75, 76, and 79, wherein the plurality of target molecules in the second composition are the same or different.
6. A method of determining target molecule concenfration in an array-based assay comprising the steps of:
(a) providing a first composition comprising an array of molecules immobilized on a substrate, wherein the array comprises a plurality of spots, at least one spot comprising more than one immobilized molecule, wherein the immobilized molecules are attached to the substrate, and at least one of the immobilized molecules is labeled with a first fracer moiety to form a labeled immobilized molecule that yields a probe signal; (b) providing a second composition comprising a plurality of target molecules, wherein at least one target molecule is labeled with a second tracer moiety to foπn a labeled target molecule that yields a target signal;
(c) allowing the first composition to contact the second composition under conditions to allow binding of one or more target molecules to one or more immobilized molecules and to allow determination of fractional occupancy;
(d) measuring the probe signal;
(e) measuring the target signal;
(f) providing a dissociation constant for bound target molecules; and (g) determining concenfration of target molecules.
7. A method of determining target molecule concenfration in an array-based assay comprising the steps of:
(a) providing a first composition comprising an array of molecules immobilized on a subsfrate, wherein the array comprises a plurality of spots, at least one spot comprising more than one immobilized molecule, wherein the immobilized molecules are attached to the substrate, and at least one of the immobilized molecules is labeled with a first tracer moiety to form a labeled immobilized molecule that yields a probe signal;
(b) providing a second composition comprising a plurality of target molecules, wherein at least one target molecule is labeled with a second fracer moiety to form a labeled target molecule that yields a target signal;
(c) allowing the first composition to contact the second composition under conditions to allow binding of one or more target molecules to one or more immobilized molecules and to allow determination of fractional occupancy; (d) measuring the probe signal;
(e) measuring the target signal;
(f) determining dissociation constant for bound target molecules; and
(g) determining concentration of target molecules.
8 A method of determining expression of a target molecule in a sample comprising the steps of:
(a) providing a first composition comprising an array of molecules immobilized on a subsfrate, wherein the array comprises a plurality of spots, at least one spot comprising more than one immobilized molecule, wherein the immobilized molecules are attached to the substrate, and at least one of the immobilized molecules is labeled with a first fracer moiety to form a labeled immobilized molecule that yields a probe signal;
(b) providing a second composition comprising a plurality of target molecules, wherein at least one target molecules is labeled with a second tracer moiety to form a labeled target molecule that yields a target signal;
(c) allowing the first composition to contact the second composition under conditions to allow binding of one or more target molecules to one or more immobilized molecules and to allow determination of fractional occupancy;
(d) measuring the probe signal; (e) measuring the target signal; and
(f) determining concenfration of target molecules; wherein the target molecules are genes, gene fragments or molecules resulting from expression or reverse transcription of genes or gene fragments; and wherein concenfration of target molecules is related to expression thereof.
9. A method of comparing expression of a first target molecule with expression of a second target molecule comprising the steps of:
(a) providing a first composition comprising an array of molecules immobilized on a subsfrate, wherein the array comprises a plurality of spots, at least one spot comprising more than one immobilized molecule, wherein the immobilized molecules are attached to the substrate, and at least one of the immobilized molecules is labeled with a first fracer moiety to form a labeled immobilized molecule that yields a probe signal;
(b) providing a second composition comprising a plurality of first target molecules and a plurality of second target molecules, wherein at least one first target molecule is labeled with a second tracer moiety to form a first labeled target molecule that yields a first target signal and at least one second target molecule is labeled with a third tracer moiety to form a second labeled target molecule that yields a second target signal;
(c) allowing the first composition to contact the second composition' under conditions to allow binding of first and/or second target molecules to immobilized molecules and to allow determination of fractional occupancy of the first target molecules and fractional occupancy of the second target molecules;
(d) measuring the probe signal;
(e) measuring the target signals; (f) determining a first concenfration of the first target molecules and a second concenfration of the second target molecules; and
(g) comparing the first concenfration and tlie second concenfration, wherein the first and second target molecules are genes, gene fragments or molecules resulting from expression or reverse transcription of genes or gene fragments; and wherein concenfration of target molecules is related to expression thereof.
10. A method of comparing expression of target molecules from N different sample populations, wherein N is a positive integer greater than one, comprising the steps of:
(a) providing a first composition comprising an array of molecules immobilized on a subsfrate, wherein the array comprises a plurality of spots, at least one spot comprising more than one immobilized molecule, wherein the immobilized molecules are attached to the substrate, and at least one of the immobilized molecules is labeled with a first fracer moiety to form a labeled immobilized molecule that yields a probe signal; (b) providing a second composition comprising a plurality of a target molecules from N different sample populations, wherein at least one target molecule of at least one type in each of the N sample population is labeled with a distinguishing fracer moiety to form a distinguishing labeled target molecule that yields a distinguishing target signal that is distinguishing for the at least one type of target molecules in each of the N sample populations;
(c) allowing the first composition to contact the second composition under conditions to allow binding of target molecules to immobilized molecules and to allow determination of fractional occupancy;
(d) measuring the probe signal; (e) measuring the target signals;
(f) determining and/or comparing concentration of target molecules from the N sample populations, wherein the target molecules are genes, gene fragments or molecules resulting from expression or reverse transcription of genes" or gene fragments; wherein concentration of target molecules is related to expression thereof.
11. A kit comprising: (a) a composition that comprises an array of molecules immobilized on a subsfrate, wherein the array comprises a plurality of spots, at least one spot comprising more than one immobilized molecules, wherein the immobilized molecules are attached to the substrate; and (b) information on at least one dissociation constant for one kind of target molecules bound to the immobilized molecules.
12. The kit of claim 11 , further comprising a first fracer moiety for labeling or spiking the immobilized molecules.
13. The kit of claim 12, further comprising instructions for at least a second tracer moiety for labeling at least one type of target molecules.
14. The kit of claim 11 , wherein the substrate is in the form of a planar surface, a bead or a microsphere.
15. The kit of claim 11 , further comprising instructions for determining dissociation constant for any target molecules bound to immobilized molecules.
16. The kit of claim 11 , further comprising instructions for determination of gene expression.
17. A kit comprising: (a) a composition that comprises an array of molecules immobilized on a subsfrate, wherein the array comprises a plurality of spots, at least one spot comprising more than one immobilized molecules, wherein the immobilized molecules are attached to the subsfrate; and (b) instructions on determining dissociation constant for any target molecules bound to the immobilized molecules.
18. The kit of claim 17, further comprising a first fracer moiety for labeling or spiking the immobilized molecules.
19. The kit of claim 18, further comprising instructions for at least a second fracer moiety for labeling target molecules.
20. The kit of claim 17, wherein the substrate-is in the form of a planar surface, a bead or a microsphere.
21. The kit of claim 17, further comprising instructions for determination of gene expression.
22. A kit comprising: (a) a composition that comprises an array of molecules immobilized on a subsfrate, wherein the array comprises a plurality of spots, at least one spot comprising more than one immobilized molecule, wherein the immobilized molecules are attached to the substrate; (b) information on at least one dissociation constant for one kind of target molecules bound to the immobilized molecules; (c) a first fracer moiety for labeling or spiking the immobilized molecules; (d) instructions for at least a second fracer moiety for labeling target molecules; (e) instructions for determination of dissociation constant for any target molecules; and (f) instructions for determination of target concenfration or gene or protein expression.
23. The kit of claim 22, wherein the subsfrate is in the form of a planar surface, a bead or a microparticle.
24. The kit of claim 22, wherein the immobilized molecules and target molecules are nucleic acids or oligonucleotides.
25. A kit comprising: (a) a composition that comprises an array of molecules immobilized on a subsfrate, wherein the array comprises a plurality of spots, at least one spot comprising more than one immobilized molecules, wherein the immobilized molecules are attached to the subsfrate, and at least one of the immobilized molecules is labeled with a first tracer moiety to form a labeled immobilized molecule; (b) information on at least one dissociation constant for one kind Of target molecules bound to the immobilized molecules; (c) instructions for at least a second tracer moiety for labeling target molecules; (d) instructions for determination of dissociation constant for any target molecules; and (e) instructions for determination of target concentration or gene expression.
26. The kit of claim 25, wherein the subsfrate is in the form of a planar surface, a bead or a microsphere.
27. The kit of claim 25, wherein the immobilized molecules and target molecules are nucleic acids or oligonucleotides.
28. A kit comprising: (a) a composition that comprises an array of molecules immobilized on a substrate, wherein the array comprises a plurality of spots, at least one spot comprising more than one immobilized molecules, wherein the immobilized molecules are attached to the substrate, and at least one of the immobilized molecules is labeled with a first fracer moiety to form a labeled immobilized molecule; and (b) information on at least one dissociation constant for one kind of target molecules bound to the immobilized molecules.
29. The kit of claim 28, further comprising instructions for at least a second fracer moiety for labeling target molecules.
30. The kit of claim 28, further comprising instructions for determining dissociation constant for any target molecules bound to immobilized molecules.
31. The kit of claim 28, wherein the subsfrate is in the form of a planar surface, a bead or a microsphere.
32. The kit of claim 28, further comprising instructions for determination of target molecule expression, wherein the target molecule is a gene or gene fragment, or result of expression or reverse transcription of a gene or gene fragment.
33. A kit comprising: (a) a composition that comprises an array of molecules immobilized on a substrate, wherein the array comprises a plurality of spots, at least one spot comprising more than one immobilized molecule, wherein the immobilized molecules are attached to the substrate, and at least one of the immobilized molecules is labeled with a first fracer moiety to form a labeled immobilized molecule; and (b) instructions on determining dissociation constant for target molecules bound to the immobilized molecules.
34. The kit of claim 33, further comprising instructions for at least a second fracer moiety for labeling target molecules.
35. The kit of claim 33, wherein the substrate is in the form of a planar surface, a bead or a microsphere.
36. The kit of claim 33 , further comprising instructions for determination of gene expression.
37. The kit of any one of claims 11, 17, 22, 25, 28, and 33, wherein the immobilized molecules are selected from the group consisting of nucleotides, polynucleotides, DNA, cDNA, RNA, mRNA, cRNA, peptide nucleic acids, oligonucleotides, polypeptides, antibodies, enzymes, hormones, cytokines, antigens, other proteins, peptides displayed on phages and other peptides, carbohydrates, polymers containing alpha-, beta-, and omega- amino acids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, mixed polymers, small molecule drugs, fragments, analogs and optical isomers thereof, and combinations thereof that form chimeric molecules.
38. A composition comprising an array of molecules immobilized on a subsfrate, wherein the array comprises a plurality of spots, at least one spot comprising: (a) more than one immobilized molecule, wherein the immobilized molecules are directly attached to the substrate or optionally through a first linker, and at least one of the immobilized molecules is labeled with a first fracer moiety, directly or optionally through a second linker, to form a labeled immobilized molecule; and
(b) at least one target molecule that binds to at least one labeled or unlabeled immobilized molecule; wherein H e at least one target molecule is labeled with a second tracer moiety to form a labeled target molecule, wherein the second tracer moiety is a directly measurable moiety; and wherein the labeled immobilized molecule forms a first amount and the immobilized molecules form a second amount, the first amount being proportional to the second amount at the at least one spot, and the proportion is in the range of about 0.16% to about 100%.
39. The composition of claim 38, wherein the proportion is selected from the group of ranges consisting of: 0.16% to 1%, 1% to 2%, 2% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 50%, 50% to 70%, and 70% to 100%.
40. The composition of claim 38, wherein the proportion of the first amount to the second amount is the same or different for at least two spots in the array.
41. The composition of claim 38, wherein the substrate is in the form of a planar surface, a bead or a particle.
42. The composition of claim 38, wherein the substrate is glass or silicon or a synthetic material.
43. The composition of claim 38, wherein the substrate is selected from the group consisting of: a solid-phase synthesis support, a fiber, a capillary tube, a silicon wafer, a slide, a membrane, a filter and other sheets.
44. The composition of claim 38, wherein the immobilized molecules are attached to the subsfrate by covalent or non-covalent bonding, directly or indirectly.
45. The composition of claim 38, wherein the first tracer moiety is attached to the immobilized molecule by covalent bonding, directly or indirectly.
46. The composition of claim 38, wherein the" first fracer moiety is removable from the immobilized molecule.
47. The composition of claim 38, wherein the composition comprises a plurality of target molecules of different types.
48. The composition of claim 38, wherein the first or second fracer moiety is a directly measurable moiety.
49. The composition of claim 38, wherein the first tracer moiety is an indirectly measurable moiety.
50. The composition of claim 38, wherein the directly measurable moiety comprises a radioactive isotope, an enzyme, a luminescent label, or a bead or microsphere containing one or more of such.
51. The composition of claim 50, wherein the luminescent label is selected from the group consisting of a fluorescent label, an energy fransfer dye, a chemiluminescent label, a bioluminescent label, a colorimetric label, a quantum dot and a combination thereof.
52. The composition of claim 38, wherein the directly measurable moiety is selected from the group consisting of: a phycobilisome, a phosphorescent dye, an enzyme catalyzing light emission, and a combination thereof.
53. The composition of claim 38, wherein the second fracer moiety is an indirectly measurable moiety and the target molecule is chemically modified to comprise the indirectly measurable moiety.
54. The composition of claim 49, wherein the indirectly measurable moiety is an oligonucleotide or nucleic acid.
55. The composition of claim 49, wherein the indirectly measurable moiety comprises one member of a binding pair selected from the group consisting of: an antibody/antigen pair, a digoxigenin anti-digoxigenin pair, a receptor/ligand pair, a biotin/sfrepavidin or avidin pair, a carbohydrate/lectin pair, a hybridizing oligonucleotide, polynucleotide, or nucleic acid pair, and a chemically synthesized pair.
56. The composition of claim 38, wherein the first fracer moiety is removable.
57. The composition of claim 38, wherein the immobilized molecule is selected from the group consisting of: nucleotides, polynucleotides, DNA, cDNA, RNA, mRNA, cRNA, peptide nucleic acids, oligonucleotides, polypeptides, antibodies, enzymes, hormones, cytokines, other antigens and proteins, peptides displayed on phages and other peptides, carbohydrates, polymers containing alpha-, beta-, and omega-amino acids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, mixed polymers, fragments, analogs and optical isomers thereof, and combinations thereof that form chimeric probes.
58. The composition of claim 38, wherein immobilized molecules situated at different spots of the array are the same or different.
59. The composition of claim 38, wherein the composition comprises a plurality of target molecules situated at at least one spot, wherein the plurality of target molecules are the same or different.
60. A method of determining target concenfration in a sample in an array-based assay comprising the steps of:
(a) performing an array-based assay using the kit that comprises: (i) a composition that comprises an array of molecules immobilized on a subsfrate, wherein the array comprises a plurality of spots, at least one spot comprising more than one immobilized molecule, wherein the immobilized molecules are attached to the subsfrate; (ii) information on at least one dissociation constant for one type of target molecules bound to the immobilized molecules; (iii) a first fracer moiety for labeling or spiking the immobilized molecules to form labeled immobilized molecules that yield a probe signal; (iv) instructions for at least a second fracer moiety for labeling at least one type of target molecules to form labeled target molecules that yield a target signal; (v) instructions for determination of dissociation constant for any target molecules; and (vi) instructions for determination of target concentration or gene expression;
(b) measuring a probe signal from labeled immobilized molecules;
(c) measuring at least a first target signal from the at least one kind of bound labeled target molecules, before or after labeling the immobilized molecules, in the presence or absence of the first fracer moiety from the labeled immobilized molecules; and
(d) determining target concentration.
61. A method of determining target concentration in a sample in an array-based assay comprising the steps of:
(a) performing an array-based assay using the kit that comprises: (i) a composition that comprises an array of molecules immobilized on a substrate, wherein the array comprises a plurality of spots, at least one spot comprising more than one immobilized molecules, wherein the immobilized molecules are attached to the subsfrate, and at least one of the immobilized molecules is labeled with a first fracer moiety to form a labeled immobilized molecule that yields a probe signal; (ii) information on at least one dissociation constant for one type of target molecules bound to the immobilized molecules; (iii) instructions for at least a second fracer moiety for labeling at least one type of target molecules to form labeled target molecules that yield a target signal; (iv) instructions for determination of dissociation constant for target molecules; and (v) instructions for determination of target concenfration or gene expression;
(b) measuring a probe signal from labeled immobilized molecules;
(c) measuring at least a first target signal from the at least one kind of bound labeled target molecules, before or after labeling the immobilized molecules, and in the presence or absence of the first tracer moiety from the labeled immobilized molecules; and
(d) determining target concentration.
62. A method of determining target concenfration comprising the steps of: (a) performing an array-based assay to form the composition comprising: an array of molecules immobilized on a substrate, wherein the array comprises a plurality of spots, at least one spot comprising:
(i) more than one immobilized molecule, wherein the immobilized molecules are directly attached to the substrate or optionally through a first linker, and at least one of the immobilized molecules is labeled with a first fracer moiety, directly or optionally through a second linker, to form a labeled immobilized molecule, wherein the labeled immobilized molecule and unlabeled immobilized molecule, if any, comprise the same or different molecular species; and (ii) at least one target molecule that binds to at least one labeled or unlabeled immobilized molecule; wherein the at least one target molecule is labeled with a second tracer moiety to form a labeled target molecule, wherein the second fracer moiety is a directly measurable moiety; wherein the labeled immobilized molecule forms a first amount and the labeled and unlabeled immobilized molecules form a second amount, the first amount being proportional to the second amount at the at least one spot, and the proportion is in the range of about 0.16% to about 100%; (b) measuring a probe signal from labeled immobilized molecules;
(c) measuring at least a first target signal from the at least one bound labeled target molecules, before or after labeling the immobilized molecules, in the presence or absence of the first tracer moiety from the labeled immobilized molecules; and
(d) determining target concentration.
63. The method of claim 62, wherein the target molecules are oligonucleotides, polynucleotides or nucleic acids and the method is applicable to determination of gene expression.
64. The method of claim 62, wherein the target molecules are proteins or antigens, and the method is applicable to determination of protein expression.
65. The method of claim 62, wherein the method is applied to determination of concenfration of a plurality of target molecules at one spot, wherein the plurality of target molecules are the same or different.
66. The method of claim 62, wherein the first tracer moiety is indirectly attached to the immobilized molecule through an intermediate molecule.
67. The method of claim 66, wherein the intermediate molecule is the same for two or more spots in the array.
68. The method of claim 67, wherein the first fracer moiety is attached to the intermediate molecule before or after a target molecule has contacted the immobilized molecules, and the probe signal is measured in the presence or absence of the target molecule.
69. A method of determining target molecule concenfration in an array-based assay comprising the steps of:
(a) providing a first composition comprising an array of molecules immobilized on a subsfrate, wherein the array comprises a plurality of spots, at least one spot comprising more than one immobilized molecule, wherein the immobilized molecules are attached to the substrate, and at least one of the immobilized molecules is labeled with a first fracer moiety that yields a probe signal;
(b) providing a second composition comprising a plurality of target molecules, wherein at least one target molecule is labeled with a second tracer moiety that yields a target signal, wherein the second fracer moiety is.an indirectly measurable moiety; (c) allowing the first composition to contact the second composition under conditions to allow binding of one or more target molecules to one or more immobilized molecules, labeled or unlabeled, and to allow determination of fractional occupancy;
(d) measuring the probe signal;
(e) measuring the target signal; and (f) determining concenfration of target molecules, wherein the labeled immobilized molecule and unlabeled immobilized molecule, if any, comprises the same or different molecular species.
70. The method of claim 1, wherein the first fracer moiety and the second tracer moiety are the same or different.
71. A composition comprising an array of molecules immobilized on a substrate, wherein the array comprises a plurality of spots, at least one spot comprising:
(a) more than one immobilized molecule, wherein the immobilized molecules are directly attached to the subsfrate or optionally through a first linker, and at least one of the immobilized molecules is labeled with a first tracer moiety, directly or optionally through a second linker, to form a labeled immobilized molecule, wherein the labeled immobilized molecule and unlabeled immobilized molecule, if any, comprise the same molecular species; and
(b) at least one target molecule that binds to at least one labeled or unlabeled immobilized molecule; wherein the at least one target molecule is labeled with a second tracer moiety to form a labeled target molecule, wherein the second tracer moiety comprises a directly measurable moiety; wherein the labeled immobilized molecule forms a first amount and the immobilized molecules form a second amount, the first amount being proportional to the second amount at the at least one spot, and the proportion is in the range of about 0.16% to about 100%.
72. A composition comprising an array of molecules immobilized on a subsfrate, wherein the array comprises a plurality of spots, at least one spot comprising:
(a) more than one immobilized molecule, wherein the immobilized molecules are directly attached to the substrate or optionally through a first linker, and at least one of the immobilized molecules is labeled with a first tracer moiety, directly or optionally through a second linker, to form a labeled immobilized molecule, wherein the labeled immobilized molecule and unlabeled immobilized molecule, if any, comprise the same molecular species; and (b) at least one target molecule that binds to at least one labeled or unlabeled immobilized molecule; wherein the at least one target molecule is chemically modified to comprise a second tracer moiety to form a labeled target molecule; wherein the labeled immobilized molecule forms a first amount and the immobilized molecules form a second amount, the first amount being proportional to the second amount at the at least one spot, and the proportion is in the range of about 0.16% to about 100%.
73. The composition of claim 72, wherein the at least one target molecule is chemically modified to comprise a biotin molecule, an avidin molecule, a strepavidin molecule or a digoxigenin molecule.
74. A composition comprising an array of molecules immobilized on a subsfrate, wherein the array comprises a plurality of spots, at least one spot comprising:
(a) more than one immobilized molecule, wherein the immobilized molecules are directly attached to the subsfrate or optionally through a first linker, and at least one of the immobilized molecules is labeled with a first fracer moiety, directly or optionally through a second linker, to form a labeled immobilized molecule, wherein the labeled immobilized molecule and unlabeled immobilized molecule, if any, comprise different molecular species; and
(b) at least one target molecule that binds to at least one labeled or unlabeled immobilized molecule; wherein the at least one target molecule is labeled with a second tracer moiety to form a labeled target molecule, wherein the second fracer moiety is a directly or indirectly measurable moiety; wherein the labeled immobilized molecule forms a first amount and the immobilized molecules form a second amount, the first amount being proportional to the second amount at the at least one spot, and the proportion is in the range of about 0.16% to about 100%.
75. A method of determining target molecule concenfration in an array-based assay comprising the steps of:
(a) providing a first composition comprising an array of molecules immobilized on a subsfrate, wherein the array comprises a plurality of spots, at least one spot comprising more than one immobilized molecule, wherein the immobilized molecules are attached to the substrate, and at least one of the immobilized molecules is labeled with a first tracer moiety to form a labeled immobilized molecule that yields a probe signal; (b) providing a second composition comprising a plurality of target molecules, wherein at least one target molecule is labeled with a second tracer moiety that yields a target signal;
(c) allowing the first composition to contact the second composition under conditions to allow binding of one or more target molecules to one or more immobilized molecules, labeled or unlabeled, and to allow determination of fractional occupancy;
(d) measuring the probe signal;
(e) measuring the target signal; and
(f) determining concentration of target molecules, wherein the labeled immobilized molecules form a first amount and the immobilized molecules form a second amount, and the first amount is proportional to the second amount.
76. A method of determining target molecule concenfration in an array-based assay comprising the steps of:
(a) providing a first composition comprising an array of molecules immobilized on a subsfrate, wherein the array comprises a plurality of spots, at least one spot comprising more than one immobilized molecule, wherein the immobilized molecules are attached to the substrate, and at least one of the immobilized molecules is labeled with a first tracer moiety to form a labeled immobilized molecule that yields a probe signal; (b) providing a second composition comprising a plurality of target molecules, wherein at least one target molecule is labeled with a second tracer moiety that yields a target signal;
(c) allowing the first composition to contact the second composition under conditions to allow binding of one or more target molecules to one or more immobilized molecules, labeled or unlabeled, and to allow determination of fractional occupancy;
(d) measuring the probe signal;
(e) measuring the target signal;
(f) determining fractional occupancy of immobilized molecules; and (g) determining concentration of target molecules, wherein the labeled immobilized molecules form a first amount and the immobilized molecules form a second amount, and the first amount is proportional to the second amount.
77. The composition of claim 38, wherein the first fracer moiety and second fracer moiety are the same or different.
78. A method of comparing expression of a first target molecule with expression of a second target molecule comprising the steps of:
(a) providing a first composition comprising an array of molecules immobilized on a subsfrate, wherein the array comprises a plurality of spots, at least one spot comprising more than one immobilized molecule, wherein the immobilized molecules are attached to the substrate, and at least one of the immobilized molecules is labeled with a first fracer moiety to form a labeled immobilized molecule that yields a probe signal;
(b) providing a second composition comprising a plurality of first target molecules and a plurality of second target molecules, wherein at least one first target molecule is directly labeled with a second tracer moiety that yields a first target signal and at least one second target molecule is labeled with a third fracer moiety that yields a second target signal;
(c) allowing the first composition to contact the second composition under conditions to allow binding of first and/or second target molecules to immobilized molecules and to allow determination of fractional occupancy of each first target molecules and second target molecules;
(d) measuring the probe signal;
(e) measuring the target signals; (f) determining the fractional occupancy of the first and second target molecules;
(g) determining a first concentration of the first molecules and a second concenfration of the second target molecules; and (h) comparing the first concentration and the second concentration, wherein the first and second target molecules are genes, gene fragments or molecules resulting from expression or reverse franscription of genes or gene fragments; and wherein concentration of target molecules is related to expression thereof.
79. The method of claim 1, wherein steps (a) to (f) are performed for determination of a first target molecule concentration and the same steps (a) to (f) are repeated for determination of a second target molecule concentration, and the first and second target molecule concentrations are compared.
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