Arrays of a plurality of different heterogeneous polymeric target compositions immobilized on the surface of a solid support are provided. In the subject arrays, the constituent polymeric targets of the heterogeneous target compositions are generally biopolymeric compounds, e.g., nucleic acids and proteins, where ribonucleic acids and proteins are the preferred polymeric targets in many embodiments. The subject arrays find use in a variety of different applications, including high throughput gene expression analysis applications.

The field of this invention is biopolymeric micro arrays.
xe2x80x9cBiochipsxe2x80x9d or micro arrays of binding agents, such as oligonucleotides, have become an increasingly important tool in the biotechnology industry and related fields. These binding agent micro arrays, in which a plurality of binding agents are deposited onto a solid support surface in the form of an array or pattern, find use in a variety of applications, including drug screening, oligonucleotide sequencing, and the like. One important use of biochips is in the analysis of differential gene expression, where the expression of genes in different cells, normally a cell of interest and a control, is compared and any discrepancies in expression are identified. In such assays, the presence of discrepancies indicates a difference in the classes of genes expressed in the cells being compared. Differential gene expression assays allow one to generate expression profiling data for several hundred or even thousand of genes simultaneously for a limited number of biological samples.
However, in certain instances it is desirable to analyze the expression level of a limited number of genes in hundreds or thousands of biological samples simultaneously. For example, such a xe2x80x9creverse approachxe2x80x9d would be critical not only for biological samples derived from healthy organisms but also for discovery of novel drug targets and diagnostic markers in large numbers of biological samples derived from different kinds of human pathological samples or model organisms like mouse, rat, etc. which imitate human diseases. The high throughput screening of disease-related biological samples requires the use of large numbers of samples in order to obtain statistically reliable data concerning specificity of gene expression changes in a particular disease state.
As such, there is continued interest in the development of array formats that can provide for high throughput expression analysis in a large number of different biological samples.
Patents and patent applications describing arrays of biopolymeric compounds and methods for their fabrication include: U.S. Pat. Nos. 5,242,974; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327; 5,445,934; 5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554,501; 5,556,752; 5,561,071; 5,599,895; 5,624,711; 5,639,603; 5,658,734; WO 93/17126; WO 95/11995; WO 95/35505; EP 742 287; and EP 799 897.
Patents and patent application describing methods of using arrays in various applications include: U.S. Pat. Nos. 5,143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806; 5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661,028; WO 95/21265; WO 96/31622; WO 97/10365; WO 97/27317; EP 373 203; and EP 785 280.
Other references providing a review of micro array technology, including formats for arrays and methods of their use include: Lockhart et al., Nature Biotechnology (December 1996) 14: 1675.
Clontech Catalogue, 97/98, (Clontech Laboratories, Inc. 1020 East Meadow Circle, Palo Alto Calif. 94303) p. 81 describes premade Northern blots.
Arrays of a plurality of different heterogeneous polymeric target compositions immobilized on the surface of a solid support are provided. In the subject arrays, the constituent polymeric targets of the heterogeneous target compositions are generally biopolymeric compounds, e.g., nucleic acids and peptides or proteins. The subject arrays find use in a variety of different applications, including high throughput gene expression analysis applications.
The term xe2x80x9cpeptidexe2x80x9d as used herein refers to any compound produced by amide formation between a carboxyl group of one amino acid and an amino group of another group.
The term xe2x80x9coligopeptidexe2x80x9d as used herein refers to peptides with fewer than about 10 to 20 residues, i.e. amino acid monomeric units.
The term xe2x80x9cpolypeptidexe2x80x9d as used herein refers to peptides with more than 10 to 20 residues.
The term xe2x80x9cproteinxe2x80x9d as used herein refers to polypeptides of specific sequence of more than about 50 residues.
The term xe2x80x9cnucleic acidxe2x80x9d as used herein means a polymer composed of nucleotides, e.g. deoxyribonucleotides or ribonucleotides.
The terms xe2x80x9cribonucleic acidxe2x80x9d and xe2x80x9cRNAxe2x80x9d as used herein means a polymer composed of ribonucleotides.
The term xe2x80x9cmRNAxe2x80x9d as used herein refers to only that fraction of the total ribonucleic acids found in a biological source which are capable of being translated into proteins, and therefore includes messenger RNA and polyA+ RNA, but does not include ribosomal RNAs or tRNAs.
The terms xe2x80x9cdeoxyribonucleic acidxe2x80x9d and xe2x80x9cDNAxe2x80x9d as used herein means a polymer composed of deoxyribonucleotides.
The term xe2x80x9coligonucleotidexe2x80x9d as used herein denotes single stranded nucleotide multimers of from about 10 to 100 nucleotides and up to 200 nucleotides in length.
The term xe2x80x9cpolynucleotidexe2x80x9d as used herein refers to single or double stranded polymer composed of nucleotide monomers of generally greater than 100 nucleotides in length.
Arrays of a plurality of different heterogeneous polymeric target compositions immobilized on the surface of a solid support are provided. In the subject arrays, the constituent polymeric targets of the heterogeneous target compositions are generally biopolymeric compounds, e.g., nucleic acids and peptides or proteins. The subject arrays find use in a variety of different applications, including high throughput gene expression analysis applications. In further describing the subject invention, the subject arrays will be described first, followed by a review of representative methods of using the arrays and kits for use in practicing such methods.
Before the subject invention is further described, it is to be understood that the invention is not limited to the particular embodiments of the invention described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.
In this specification and the appended claims, the singular forms xe2x80x9ca,xe2x80x9d xe2x80x9can,xe2x80x9d and xe2x80x9cthexe2x80x9d include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.
Arrays
As summarized above, the subject arrays are arrays of polymeric target compositions that are immobilized on a solid support. The polymeric target constituents of the different polymeric target compositions of the subject arrays, i.e., the individual molecules that make up each target composition on the array, are typically biopolymeric, by which is meant that they are naturally occurring polymeric compounds or at least mimetics or analogues of naturally occurring polymeric compounds. Biopolymeric compounds of particular interest are nucleic acids, including ribonucleic acids, as well as ribonucleic and deoxyribonucleic acid derivatives thereof generated through a variety of processes (usually enzymatic processes) such as reverse transcription, amplification, transcription, etc., e.g. cDNA amplified from RNA (both single and double stranded), cDNA inserts from cDNA libraries, and the like, where in certain embodiments the targets are cDNA molecules generated using the target generation protocols described in U.S. Pat. No. 5,962,272 (e.g., the SMART(trademark) technology) which disclosure is incorporated herein by reference; and peptides, such as oligopeptides, polypeptides and proteins.
In certain preferred embodiments, the polymeric targets are ribonucleic acids. Ribonucleic acids of interest as polymeric targets include total RNA, polyA+RNA, polyAxe2x88x92RNA, snRNA (small nuclear), hnRNA (heterogeneous nuclear), cytoplasmic RNA, pre mRNA, mRNA, cRNA (complementary), and the like. In yet other embodiments, the targets of each of the polymeric target compositions on the array are deoxyribonucleic acids, e.g., cDNA amplified from RNA (both single and double stranded), cDNA inserts from cDNA libraries, and the like, where in certain embodiments the targets are cDNA molecules generated using the target generation protocols described in U.S. Pat. No. 5,962,272 (e.g., the SMART(trademark) technology) which disclosure is incorporated herein by reference. In yet other preferred embodiments, the target constituents of each polymeric target composition on the array are peptides. Peptides of interest as polymeric targets include naturally occurring proteins or fragments thereof, e.g., whole cell or tissue extracts, extracts of particular fractions thereof, such as cytoplasmic proteins, nuclear proteins, extracellular proteins and the like, where such proteins include: antibodies, receptors, hormones, and the like. The peptide/protein targets may be in their native state, or denatured prior to laying down on the substrate surface.
The above described targets may be obtained or derived from naturally occurring biological sources, particularly mammalian sources and more particularly mouse, rat or human sources, where such sources include: fetal tissues, such as whole fetus or subsections thereof, e.g. fetal brain or subsections thereof, fetal heart, fetal kidney, fetal liver, fetal lung, fetal spleen, fetal thymus, fetal intestine, fetal bone marrow; adult tissues, such as whole brain and subsections thereof, e.g. amygdala, caudate nucleus, corpus callosum, hippocampus, hypothalamus, substantia nigra, subthalamic nucleus, thalamus, cerebellum, cerebral cortex, medula oblongata, occipital pole, frontal lobe, temporal lobe, putamen, adrenal cortex, adrenal medula, nucleus accumbens, pituitary gland, adrenal gland and subsections thereof, such as the adrenal cortex and adrenal medulla, aorta, appendix, bladder, bone marrow, colon, colon proximal with out mucosa, heart, kidney, liver, lung, lymph node, mammary gland, ovary, pancreas, peripheral leukocytes, placental, prostate, retina, salivary gland, small intestine, skeletal muscle, skin, spinal cord, spleen, stomach, testis, thymus, thyroid gland, trachae, uterus, uterus without endometrium; cell lines, such as breast carcinoma T-47D, colorectal adenocarcinoma SW480, HeLa, leukemia chronic myelogenous K-562, leukemia lymphoblastic MOLT-4, leukemia promyelocytic HL-60, lung carcinoma A549, lumphoma Burkitt""s Daudi, Lymphoma Burkitt""s Raji, Melanoma G361, teratocarcinoma PA-1, leukemia Jurkat; and the like. The target, e.g. RNA, may be used directly from its source when present in relatively great abundance or amplified when possible/desired, e.g. by PCR, transcription or cloning procedures, where necessary, e.g. when relatively rare, such as RNA from rare tissue, etc.
Where the targets are derived from naturally occurring sources, such as mammalian tissues as described above, the targets may be derived from the same or different organisms. As such, in certain embodiments the targets that make up each of the different compositions displayed on the array surface are derived from the same organism. In other embodiments, the targets that make up each of the different compositions displayed on the array surface are derived from different organisms.
In addition, the target samples arrayed on the plate can be derived from normal and disease or condition states of the same organism or multiple organisms, like cancer, stroke, heart failure, aging, infectious diseases, inflammation, exposure to toxic, drug or other agents, conditional treatment, such as heat shock, sleep deprivation, physical activity, etc., different developmental stages, and the like.
In certain embodiments, the plurality of different target compositions that is displayed on the surface of the array, i.e., the set of different target compositions of the array, includes at least one normal/disease pair, where the number of different normal/disease pairs of target compositions present on the array may be much greater, and in many embodiments is at least 5, usually at least 10 and more usually at least 20, where the number may be 50, 100, 200 or higher, depending on the particular array. By xe2x80x9cnormal/disease pair of target compositionsxe2x80x9d is meant a collection or set of two or more different target compositions which includes at least one target composition derived from a normal tissue and at least one target composition derived from a disease tissue. In other words, in any given set or collection of target compositions that make up a normal/disease pair of target compositions, at least one target composition will be a composition obtained from a normal sample and at least one target composition will be a composition obtained from a disease sample. As such, any given normal/disease pair of target compositions includes, at a minimum two different compositionsxe2x80x94one normal and one disease. In certain embodiments, a given normal/disease pair of target compositions includes more than one disease and/or target composition, where in these embodiments, the number of different disease compositions in the pair ranges from about 20 to 10,000, usually from about 50 to 5,000 and more usually from about 100 to 2,000, where the number is, in many embodiments, a number that provides for a statistically relevant number of samples displayed on the array, by which is meant at least 5 disease compositions for each type of disease displayed on the array. The number of different diseases displayed on the array can be as great as about 1000 or greater, but usually is less than about 300 and more usually less than about 100. The subject arrays can include one particular type of disease on the array or several different types of diseases. The number of control or normal samples or compositions in a given pair may range from about 2 to 500 usually from about 10 to 100. In those embodiments where a given normal/disease pair includes two or more different disease target compositions, the different disease compositions of the pair all represent the same disease, e.g., they are taken from multiple different organisms all suffering from the same disease, such that only one disease is represented in any given normal/disease pair of target compositions. The normal/disease pair can be derived from the same tissue/cell type or from different tissue/cell types for each particular disease state. In general, normal target is derived from a healthy organism, i.e., one that does not have any symptoms of disease, particularly the disease with which it will be paired. The number of normal target compositions is usually less than the number of disease targets, and may be at least 2, usually at least 5 and more usually at least 10, but can be 50 or higher.
The normal/disease pairs described above may represent a variety of different diseases. Diseases of interest include: (a) Infectious and Parasitic Diseases, e.g., Intestinal Infectious Diseases, i.e. Cholera, Typhoid Fever, Salmonella Infections; Tuberculosis; Bacterial Diseases, i.e. Diphtheria; Human Immunodeficiency Virus (HIV) Infection; Poliomyelitis; Viral Diseases, i.e. Herpes Simplex, Measles, Rubella, Yellow Fever; Viral Hepatitis; Arthropod-Bome Diseases, i.e. Malaria; Venereal Diseases, i.e. Syphilis; Mycoses; (b) Neoplasms, e.g., Malignant Neoplasms; Benign Neoplasms; Carcinoma in situ; (c) Endocrine, Nutritional, and Metabolic Diseases and Immunity Disorders; e.g., Diabetes Mellitus, subtypes; Thiamine and Niacin Deficiency States, i.e. Beriberi; Vitamin Deficiency States; Disorders of Amino-Acid Transport and metabolism, i.e. Phenylketonuria; (d) Diseases of the Blood and Blood-Forming Organs, e.g., Anemias; Thalassemias; Coagulation Defects, i.e. Factor VIII Deficiency, von Willebrand""s Disease; Systemic Lupus Erythematosis; Leucemia, ie. Acute Promyelocytic Leucemia; Chronic Myeloleukemia; Lymphogranulomatasis; Lymphoblastosis; Hemophilia A and B; Autoimmune Thrombocytopenia; (e) Mental Disorders, e.g., Psychoses, i.e. Dementia; Arteriosclerotic Dementia; Alcoholic Psychoses; Drug Psychoses; Alzheimer Disease; Epilepsy; Huntington""s Disease; Jacob-Creutzfeldt Disease; Multiple Sclerosis; Schizophrenia; Neurotic Disorders, i.e. Anxiety, Multiple Personality, Phobic Disorders; Neurosis; (f) Diseases of the Nervous System and Sense Organs; Inflammatory Diseases, i.e. Meningitis, Encephalitis; Gaucher Disease; Alzheimer""s Disease; Parkinson""s Disease; (g) Diseases of the Circulatory System and Cardiovascular Diseases; Arthritis; Rheumatic Fever; Rheumatic Heart Disease; Hypertensive Diseases; Ischemic Heart Disease, i.e. Arteriosclerosis; Arteritis; Cardiomyopathy; Dysfunction of Glucose Metabolism; Dysfunction of Cellular Iron Channels; Dysfunction of Lipid Metabolism; (h) Diseases of the Respiratory System, e.g., Bronchitis; Pneumonia and Influenza; (i) Diseases of the Digestive System, e.g., Ulcer; Gastritis; Necrosis; Chronic Liver Diseases and Cirrhosis, i.e. Chronic Hepatitis; (j) Diseases of the Genitourinary System, i.e., Prostatitis; Benign Mammary Dysplasias, i.e. Fibrosclerosis; (k) Diseases of the Skin and Subcutaneous Tissue, e.g., Cellulitis; Dermatitis; (l) Diseases of the Musculoskeletal System and Connective Tissue; e.g., Systemic Lupus erythematosus; Arteriosclerosis; Arthritis; Osteoarthrosis; etc.
In the subject arrays, the targets are stably associated with the surface of a solid support. By stably associated is meant that the targets maintain their position relative to the support surface under hybridization and washing conditions. In other words, the targets are immobilized on the support (i.e., substrate) surface. The targets can be non-covalently or covalently stably associated with the rigid support surface. Examples of non-covalent association include non-specific adsorption, specific binding through a specific binding pair member covalently attached to the support surface, and entrapment in a matrix material, e.g., a hydrated or dried separation medium, which presents the target in a manner sufficient for binding, e.g. hybridization, to occur. Examples of covalent binding include covalent bonds formed between the target and a functional group present on the surface of the rigid support, e.g., xe2x80x94OH, where the functional group may be naturally occurring or present as a member of an introduced linking group, as described in greater detail below.
The solid support may be a rigid or flexible support or substrate. Therefore, in certain embodiments the solid support of the array is a rigid substrate. By rigid is meant that the support is solid and does not readily bend, i.e. the support is not flexible. Examples of solid materials which are not rigid supports with respect to the present invention include membranes, flexible plastic films, and the like. As such, the rigid substrates of the subject arrays are sufficient to provide physical support and structure to the polymeric targets present thereon under the assay conditions in which the array is employed, particularly under high throughput handling conditions.
The rigid substrates of the subject arrays may be fabricated from a variety of materials. The materials from which the substrate is fabricated should ideally exhibit a low level of non-specific binding of probe during hybridization or specific binding events. In many situations, it will also be preferable to employ a material that is transparent to visible and/or UV light. Specific materials of interest include: glass; plastics, e.g. polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof, and the like; metals, e.g. gold, platinum, and the like; etc.
As indicated above, in other embodiments of the subject invention, the substrates or supports are flexible. By flexible is meant that the support is capable of being bent, folded or similarly manipulated without breakage. Examples of solid materials which are flexible solid supports with respect to the present invention include membranes, e.g., nylon membranes, flexible plastic films, and the like.
The substrates upon which the subject patterns of targets are presented in the subject arrays may take a variety of configurations ranging from simple to complex, depending on the intended use of the array. Thus, the substrate could have an overall slide or plate configuration, such as a rectangular or disc configuration, where an overall rectangular configuration, as found in standard microtiter plates and microscope slides, is preferred. Generally, the length of the rigid substrates will be at least about 1 cm and may be as great as 40 cm or more, but will usually not exceed about 30 cm and may often not exceed about 15 cm. The width of rigid substrate will generally be at least about 1 cm and may be as great as 30 cm, but will usually not exceed 20 cm and will often not exceed 10 cm. The height of the rigid substrate will generally range from 0.01 mm to 10 mm, depending at least in part on the material from which the rigid substrate is fabricated and the thickness of the material required to provide the requisite rigidity. The substrate can be used directly in hybridization assays or can be used in a cartridge or chips, e.g., it can be surrounded by a rigid material, like plastic, for easy manipulation during hybridization, washing and collection steps.
The substrate of the subject arrays comprises at least one surface on which a pattern of target compositions is present, where the surface may be smooth or substantially planar, or have irregularities, such as depressions or elevations. The surface on which the pattern of target compositions, e.g., spots, is presented may be modified with one or more different layers of compounds that serve to modulate the properties of the surface in a desirable manner. Such modification layers, when present, will generally range in thickness from a monomolecular thickness to about 1 mm, usually from a monomolecular thickness to about 0.1 mm and more usually from a monomolecular thickness to about 0.001 mm. Modification layers of interest include: inorganic and organic layers such as metals, metal oxides, polymers, small organic molecules and the like. Polymeric layers of interest include layers of: peptides, proteins, polynucleic acids or mimetics thereof, e.g. peptide nucleic acids and the like; polysaccharides, phospholipids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneamines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, and the like, where the is polymers may be hetero- or homopolymeric, and may or may not have separate functional moieties attached thereto, e.g. conjugated.
As mentioned above, each different target composition on the arrays is a heterogeneous mixture of different molecules, e.g., nucleic acids, proteins, etc., where the number of different or distinct molecules present in a given target composition is generally at least about 100, usually at least about 1000 and more usually at least about 5,000. Any two molecules are considered different or distinct if they have a molecular structure that differs, e.g., they differ from each other by nucleotide or amino acid sequence. The mass of each different target composition on the array may range from about 1 pg to 10 xcexcg, usually from about 100 pg to 5 xcexcg and more usually from about 1 ng to 1 xcexcg.
In certain embodiments, the masses of all of the target compositions on the array are substantially the same. In other words, the amount of material that makes up each of the different target compositions on the array is substantially the same. In yet other embodiments, the target compositions on the array are normalized. By normalized is meant that the amount of each target composition on the array is selected to provide for normalization. By normalization is meant that the amount of targets in each target composition is selected to provide for a known relationship of expression of at least one housekeeping gene in two or more of the target compositions, including all or a substantial subpopulation of the target compositions. As such, the amount of each target composition on the array may be chosen to provide for an expression signal that is substantially equal in each spot for at least one housekeeping gene. Alternatively, the amount may be chosen to provide for known ratios of signals with respect to the at least one housekeeping gene. In certain embodiments, all of the different target compositions on the array may be normalized with respect to each other. In yet other embodiments, two or more subsets or populations of the target compositions may be normalized with respect to all of the members within the set, but not to other members in other set(s). For example, where the array includes a normal/disease pair which includes a plurality of normal target compositions and a plurality of disease target compositions, all of the normal target compositions may be normalized with respect to each other and all of the disease target compositions may be normalized with respect to each other. In another embodiment, if the array includes normal/disease pairs derived from different tissues, all the normal/disease pairs belonging to the same tissue may be normalized to each other and other normal/disease pairs will be normalized separately. The normalized amounts of each target composition may be chosen using any convenient protocol. Methods of identifying/determining normalized amounts of target are well known to those of skill in the art and any convenient protocol may be employed, where particular protocols of interest may be based on one or a plurality of different housekeeping genes, where when a plurality of housekeeping genes are employed, the number of different housekeeping genes may be at least 5, at least 10 or higher. Alternatively, a normalization protocol for reading the array in which the amount of each target composition is not normalized may be employed, where this normalization protocol is described in greater detail under the heading xe2x80x9cNormalizationxe2x80x9d infra.
As summarized above, the subject arrays comprise a plurality of different polymeric targets, where the number of targets is at least 5, usually at least 8, and may be much higher. In some embodiments, the arrays have at least 10 distinct spots, usually at least about 20 distinct spots, and more usually at least about 50 distinct spots, where the number of spots may be as high as 10,000 or higher. In yet other embodiments, the number of different spots is at least 100, where the number may range from about 100 to 2,000 or more. The density of the spots on the solid surface in certain embodiments is at least about 5/cm2 and usually at least about 50/cm2 but does not exceed about 1000/cm2, and usually does not exceed about 500/cm2, where in certain embodiments the density may range from about 300 to 400/cm2.
The arrays of the subject invention may be used directly in binding assays using well known technologies, e.g., contacting with probe in a suitable container, under a coverslip, etc, or may be incorporated into a structure that provides for ease of analysis, high throughput, or other advantages, such as in a biochip format, a multiwell format and the like. For example, the subject arrays could be incorporated into a biochip type device in which one has a substantially rectangular shaped cartridge comprising fluid entry and exit ports and a space bounded on the top and bottom by substantially planar rectangular surfaces, wherein the array is present on one of the top and bottom surfaces.
Alternatively, the subject arrays could be incorporated into a high throughput or multi-well device, wherein each array is bounded by raised walls in a manner sufficient to form a reaction container wherein the array is the bottom surface of the container. Representative high throughput devices that may be modified to incorporate arrays of the subject invention are described in U.S. patent application Ser. No. 5,545,531, the disclosure of which is herein incorporated by reference. Generally in such devices, the devices comprise a plurality of reaction chambers, each of which contains the array on the bottom surface of the reaction chamber. By plurality is meant at least 2, usually at least 4 and more usually at least 24, where the number of reaction chambers may be as high as 96 or higher, but will usually not exceed 100. The volume of each reaction chamber may be as small as 10 xcexcl but will usually not exceed 500 xcexcl. In a preferred embodiment, the volume is between about 50 and 200 xcexcl.
An important feature of the subject invention is that the arrays of the present invention are characterized by having at least one of a rigid solid support and/or at least one normal/disease pair of polymeric target compositions.
Array Preparation
The subject arrays may be prepared as follows. The substrate or support can be fabricated according to known procedures, where the particular means of fabricating the support will necessarily depend on the material from which it is made. For example, with polymeric materials, the support may be injection molded, while for metallic materials, micromachining may be the method of choice. Alternatively, rigid supports such as glass, plastic, or metal sheets can be purchased from a variety of commercial sources and used. The surface of the support may be modified to comprise one or more surface modification layers, as described above, using standard deposition techniques.
The next step in the preparation process is to prepare the target compositions and then stably associate or immobilize the target compositions onto the surface of the support. The complex source of target molecules that make up each target composition may be obtained from a naturally occurring physiological source using standard techniques. Protocols for isolating nucleic acids, proteins and their fractions from cells, tissues, organs and whole organisms are described in: Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press)(1989); Scope R., Protein Purification. Principle and Practice (Springer-Verlag)(1994); and Deutscher, Guide to Protein Purification (Academic Press)(1990). Such methods typically involve subjection/treatment of the original biological source to one or more of tissue/cell homogenization, nucleic acid/protein extraction, chromatography, centrifugation, affinity binding and the like. Target molecule composition preparation can further include one or more treatments designed to provide for one or more of the following: more stable association with the surface of the rigid support; better hybridization and detection; etc. Such treatments will necessarily depend on the nature of the target molecules being prepared, but may include one or more of: reverse transcription; nuclease treatment; protease digestion; in vitro transcription; in vitro translation; DNA amplification; enzymatic or chemical modification of RNA or proteins, such as the introduction of functional moieties, such as biotin, digoxigenin, fluorescent moieties, antigens, chelator groups, chemically active or photoactive groups, etc.; and the like.
Following preparation of the plurality of different complex mixtures of the target molecules, each mixture is stably associated with, i.e., immobilized on, the surface of the support. Any convenient protocol for immobilizing the complex mixtures on the support surface can be employed. Examples of non-covalent association include non-specific adsorption, binding based on electrostatic interactions (e.g. ion-pair interactions), hydrophobic interactions, hydrogen bonds, specific binding through a specific binding pair member covalently attached to the support surface, and the like. Examples of covalent binding include covalent bonds formed between the spotted nucleic acids and a functional group present on the surface of the rigid support, e.g. xe2x80x94OH, where the functional group can be naturally occurring or present as a member of an introduced linking group; etc. Following stable placement of the pattern of target molecules on the support surface, the resultant array may be used as is or incorporated into a biochip, multiwell or other device, as describe above, for use in a variety of binding applications.
The subject arrays or devices into which they are incorporated may conveniently be stored following fabrication for use at a later time. Under appropriate conditions, the subject arrays are capable of being stored for at least about 6 months and may be stored for up to one year or longer. The subject arrays are generally stored at temperatures between about xe2x88x9220xc2x0 C. to room temperature, where the arrays are preferably sealed in a plastic container, e.g. bag, and shielded from light.
Utility
Applications in which the subject arrays find particular use are expression analysis applications. Such applications generally involve the following steps: (a) preparation of probe; (b) contact of probe with the array under conditions sufficient for probe to bind with corresponding target, e.g. by hybridization or specific binding; (c) removal of unbound probe from the array; and (d) detection of bound probe. Each of these steps will be described in greater detail below.
How the probe is prepared will necessarily depend on the specific nature of the probe, e.g. whether the probe is nucleic or peptidic. For nucleic acid probes, the probes may be ribo- or deoxyribonucleotides, as well as hybridizing analogues or mimetics thereof, e.g. nucleic acids in which the phosphodiester linkage has been replaced with a substitute linkage, such as a phosphorothioate, methylimino, methylphosphonate, phosphoramidite, guanidine and the like; and nucleic acids in which the ribose subunit has been substituted, e.g. hexose phosphodiester, peptide nucleic acids; locked nucleic acids; and the like. The probe will have sufficient complementarity to its target to provide for the desired level of sequence specific hybridization. Where the probe is a nucleic acid, the length of the probe will generally range from about 10 to 2000 nt, where oligonucleotide probes will generally range in length from about 15 to 100 nt and polynucleotide probes will generally range in length from about 100 to 1000 nt, where such probes may be single or double stranded, but will usually be single stranded, e.g., in a sense or antisense orientation related to the corresponding sequence of the target on the array. The nucleic acid probes finding use in the subject methods may be synthesized using known chemical or enzymatic synthesis technologies, cloning procedures or obtained from a natural source. Where the assay protocol employs a single label, it is preferred that a single test probe that hybridizes to one gene of interest is employed. In multiplex approaches, several test probes may be employed so long as they are distinguishably labeled, where the number of different test probes may be at least 2 to 3, but generally does not exceed from about 10 to 20.
Peptidic probes that find use in the subject invention include: antibodies, e.g. polyclonal, monoclonal, and binding fragments thereof; peptides with high affinity to the target, as well as analogues and mimetics thereof; ligands, receptors, and the like. As with the nucleic probes, peptidic probes may be obtained from naturally occurring sources or synthesized using available technologies.
Generally, the probe molecule will be labeled to provide for detection in the detection step. By labeled is meant that the probe comprises a member of a signal producing system and is thus detectable, either directly or through combined action with one or more additional members of a signal producing system. Examples of directly detectable labels include isotopic and fluorescent moieties incorporated into, usually covalently bonded to, a moiety of the probe, such as a nucleotide monomeric unit, e.g. dNMP of the primer, or a photoactive or chemically active derivative of a detectable label which can be bound to a functional moiety of the probe molecule. Isotopic moieties or labels of interest include 32P, 33P, 35S, 125I, and the like. Fluorescent moieties or labels of interest include coumarin and its derivatives, e.g. 7-amino-4-methylcoumarin, aminocoumarin, bodipy dyes, such as Bodipy FL, cascade blue, fluorescein and its derivatives, e.g. fluorescein isothiocyanate, Oregon green, rhodamine dyes, e.g. Texas red, tetramethylrhodamine, eosins and erythrosins, cyanine dyes, e.g. Cy3 and Cy5, macrocyclic chelates of lanthanide ions, e.g. quantum dye(trademark), fluorescent energy transfer dyes, such as thiazole orange-ethidium heterodimer, TOTAB, etc. Also of interest are nanometer sized particle labels detectable by light scattering, e.g. xe2x80x9cquantum dots.xe2x80x9d Labels may also be members of a signal producing system that act in concert with one or more additional members of the same system to provide a detectable signal. Illustrative of such labels are members of a specific binding pair, such as ligands, e.g. biotin, fluorescein, digoxigenin, antigen, polyvalent cations, chelator groups and the like, where the members specifically bind to additional members of the signal producing system, where the additional members provide a detectable signal either directly or indirectly, e.g. antibody conjugated to a fluorescent moiety or an enzymatic moiety capable of converting a substrate to a chromogenic product, e.g. alkaline phosphatase conjugate antibody; and the like. Additional labels of interest include those that provide for signal only when the probe with which they are associated is specifically bound to a target molecule, where such labels include: xe2x80x9cmolecular beaconsxe2x80x9d as described in Tyagi and Kramer, Nature Biotechnology (1996) 14:303 and EP 0 070 685 B1. Other labels of interest include those described in U.S. Pat. No. 5,563,037; WO 97/17471 and WO 97/17076.
The next step in the subject method is to contact the probe with the array under conditions sufficient for binding between the probe and the target of the array. For example, where the probe and targets are nucleic acids, the probe will be contacted with the array under conditions sufficient for hybridization to occur between the probe and target, where the hybridization conditions will be selected in order to provide for the desired level of hybridization specificity. For peptidic probes, conditions will be selected to provide for specific binding between the probe and its target.
Contact of the array and probe involves contacting the array with an aqueous medium comprising the probe. Contact may be achieved in a variety of different ways depending on specific configuration of the array. For example, where the array simply comprises the pattern of size separated targets on the surface of a xe2x80x9cplate-likexe2x80x9d rigid substrate, contact may be accomplished by simply placing the array in a container comprising the probe solution, such as a polyethylene bag, small chamber, and the like. In other embodiments where the array is entrapped in a separation media bounded by two rigid plates, the opportunity exists to deliver the probe via electrophoretic means. Alternatively, where the array is incorporated into a biochip device having fluid entry and exit ports, the probe solution can be introduced into the chamber in which the pattern of target molecules is presented through the entry port, where fluid introduction could be performed manually or with an automated device. In multiwell embodiments, the probe solution will be introduced in the reaction chamber comprising the array, either manually, e.g. with a pipette, or with an automated liquid handling device.
In certain embodiments, the array is also contacted with one or more, usually no more than four and more usually no more than three, where the number is often 1 or at most 2, reference/control probes. The reference/control probes may be labeled that same as the test probe, or they may be distinguishably labeled, e.g., a different fluorescent color, where when they are distinguishably labeled, one has the opportunity to contact the array with the test and reference/control probe(s) at the same time. Use of reference/control probes provides for, among other things, normalization of the signal observed for the test probe, as described in greater detail infra.
Contact of the probe solution and the targets will be maintained for a sufficient period of time for binding between the probe and the target to occur. Although dependent on the nature of the probe and target, contact will generally be maintained for a period of time ranging from about 10 min to 24 hrs, usually from about 30 min to 12 hrs and more usually from about 1 hr to 6 hrs.
Following binding of probe and target, the resultant hybridization patterns of labeled probe may be visualized or detected in a variety of ways, with the particular manner of detection being chosen based on the particular label of the nucleic acid, where representative detection means include scintillation counting, autoradiography, fluorescence measurement, colorimetric measurement, light emission measurement and the like.
The method may or may not further comprise a non-bound label removal step prior to the detection step, depending on the particular label employed on the probe. For example, in homogenous assay formats a detectable signal is only generated upon specific binding of probe to target. As such, in homogenous assay formats, the hybridization pattern may be detected without a non-bound label removal step. In other embodiments, the label employed will generate a signal whether or not the probe is specifically bound to its target. In such embodiments, the non-bound labeled probe is removed from the support surface. One means of removing the non-bound labeled probe is to perform the well known technique of washing, where a variety of wash solutions and protocols for their use in removing non-bound label are known to those of skill in the art and may be used. Alternatively, in those situations where the targets are entrapped in a separation medium in a format suitable for application of an electric field to the medium, the opportunity arise to remove non-bound labeled probe from the target by electrophoretic means.
The above methods can be varied to provide for multiplex analysis. For example, one can employ a plurality of different probe molecules that are each distinguishably labeled, e.g. with different fluorophores.
The above assays can be used to determine both the expression level of the target bound by the probe. The target expression level in the particular tissue being analyzed can be derived from the intensity of the detected signal. To ensure that an accurate level of expression is derived, a housekeeping gene of known expression level can also be detected, e.g. using a multiplex approach as described above, to provide for a control signal level in order to calibrate the detected probe signal.
As such, the subject arrays find use in a variety of different gene expression analysis applications, including differential expression analysis of diseased and normal tissue, e.g. neoplastic and normal tissue; different tissues or subtypes; tissues and cells under different condition states, like predisposition to disease, age, exposure to pathogens or toxic agents, etc.; and the like.
Normalization
In certain embodiments, the above described methods further include use of a normalization protocol. This particular normalization protocol provides a method for normalizing relative amounts of target compositions, e.g., samples (such as nucleic acids derived from an RNA sample, as described above), by employing an integral factor that is determined based on a set of internal reference ribonucleic acids. The reference ribonucleic acids, e.g., housekeeping genes, used to generate the integral factor are preferably expressed in all cells with medium to high relative abundance and possess low or moderate tissue to tissue variability. Once determined, the integral factor allows a relative determination of integral or total transcript levels in a sample based on the levels of one or more of the reference ribonucleic acids in the sample. Based on the measured expression level of test mRNA, the level of any reference mRNA, and the integral factor, the procedure allows one to normalize the expression level of test mRNAs between multiple samples. This procedure allows the determination of the relative level of a test transcript between samples without being dependent of the actual levels of total RNA assayed, thus obviating the requirement for normalization of total mRNA amount prior to loading multiple samples onto an array. Preferably, the selected reference nucleic acids are housekeeping gene products (or transcripts), and more preferably the reference nucleic acids of a selected set belong to different functional classes of genes.
This particular normalization protocol provides a systematic, reliable and reproducible method for normalizing test mRNA levels between different total RNA samples without regard to the actual amount of RNA sample used for detection (RNA sample is equivalent to an RNA target composition of the subject arrays). The present invention employs an integral factor for mathematical xe2x80x9cnormalizationxe2x80x9d between RNA samples, which is determined based on a set of reference RNAs. The reference RNAs are sequences expressed in all cells with medium to high relative abundance, and which possess low or moderate sample to sample variability in case they are derived from the same biological species. The integral factor is determined based on the levels of the reference RNAs in a sample, and is used to quantitate levels of other transcripts on the array based on detected levels of one or more of the reference RNAs.
This procedure allows the determination of the relative level of a test mRNA in each RNA sample without the requirement for normalization of total RNA sample amount prior to loading multiple RNA samples on an array using the level of reference RNA and integral factor as a guideline. Preferably, the selected reference RNAs correspond to housekeeping genes, and more preferably the reference RNAs of a set belong to different functional classes of housekeeping genes.
The integral factor for any RNA sample used for normalizing the amount of total RNA sample is determined by comparison of levels of a set of reference RNAs from that sample. In the method of the invention, at least six reference RNAs are used in each set for normalizing levels of total RNA between samples. Multiple reference RNAs are used to determine the integral factor to minimize the effects of expression level variation of each reference RNA between different cell/tissue types or between different healthy and diseased individuals. By using a variety of reference RNAs instead of a single mRNA to normalize levels of total RNAs between samples, it is also possible to minimize non-specific variations in expression that can be observed in different hybridization experiments, different sample preparations, etc.
For example, expression of test mRNA isolated from the same tissue sample, but isolated in separate preparations, can be normalized by determining an integral factor that will account for the differences in methodology. Integral factors can also be determined for identifying equivalent amounts of RNA from size-selected fractions of different RNA samples from a single preparation. An integral factor can be determined to normalize total RNA or test mRNA in two or more different tissue types in different physiological states, e.g. liver tissue and smooth muscle, different tissues in development, aging, etc. An integral factor can be determined to normalize differences in expression in the same underlying tissue type at different disease/pathological stages, e.g. an adenomatous polyp and a colorectal carcinoma in comparison with normal control tissue. In another example, an integral factor can be determined to normalize expression levels in the same tissue type from different individuals, e.g. pathologically xe2x80x9cnormalxe2x80x9d pancreas tissue from individual A and individual B. The integral factor can be used to normalize RNA samples isolated from treated or exposed cells, tissues, organisms, i.e. hypoxia, heat shock, drug treatment, physical activity, diet, etc.
The set of reference RNAs all have certain characteristics that make them desirable for determination of the integral factor: 1) they are expressed in substantially all cells with a relatively constant abundance and possess low or moderate tissue to tissue variability; 2) the different reference RNAs used within a single set preferably encode proteins belonging to at least three different functional classes; 3) the reference RNAs usually have transcript sizes between 1 to 3 kilobases, as ribonucleic acids of this size provide a sharp hybridization signal in Northern blot analysis. These reference RNAs, when used as a set, help to eliminate specific variation of expression between tissue/cell types that may be present using only one or a few reference RNAs.
To determine an integral factor for a sample, different total RNA samples or nucleic acids derived from RNA, e.g., SMART(trademark) amplified cDNA target (in preferred embodiment poly A+ RNAs) are spotted on a series of flexible supports (i.e. membranes) or solid supports (i.e. glass) in a known gram amount. Each flexible or solid support of the series preferably contains the same amount and spot pattern of poly A+ RNAs. Each spotted array is then hybridized with one or more gene-specific control/reference probes (in preferred embodimentxe2x80x94one) for a potential reference RNA target. Generally, the probe will be labeled either directly or through combined action with one or more additional members of a signal producing system. Examples of directly detectable labels include isotopic and fluorescent moieties incorporated into, usually covalently bonded to, a moiety of the probe, such as a nucleotide monomeric unit, or a photoactive or chemically active derivative of a detectable label, which can be bound to a functional moiety of the probe molecule. Isotopic moieties or labels of interest include 32P, 33P, and the like. Fluorescent moieties or labels of interest include coumarin and its derivatives, fluorescein and its derivatives, rhodamine dyes, cyanine dyes, Alexa dyes, Bodipy dyes and the like, etc. For multiplex analysis one can employ a plurality of different probe molecules that are each distinguishable labeled, e.g. with different fluorescent fluorophores.
Following binding of labeled control/reference probe to target ribonucleic acid, the resultant hybridization patterns of labeled targets can be visualized or detected in a variety of ways, with the particular manner of detection being chosen based on the particular label of the nucleic acid, where representative detection means include autoradiography, fluorescence measurement, or light emission measurement and the like. Usually, the hybridization signal is quantitated using phosphorimaging technology. At this stage of the procedure it should be already clear which reference RNAs give minimal signal variation between different cells/tissues, and which should be considered as standards for the best determination of the integral factor.
An average signal is calculated for each tissue/cell, pooling the data from those potential reference RNAs that are considered to be useful for determination of the integral factor. In the next step the quantities for each tissue/cell sample necessary to produce equal average signal is calculated. For example, if some RNA samples show 2-fold less average signal intensity than others, the calculated (adjusted) amount of RNA for these samples will be 2-fold more than for others. The original RNA sample amounts are adjusted to reflect the fluctuations in the relative average signal intensity for the next round of spotting.
The subsequent round of determining the integral factor will be carried out with generally between 20 and 6, but not less than 3, selected reference ribonucleic acids (e.g. the ribonucleic acids that display the best signal and consistency between tissues). The process is repeated as described above, with different adjusted amounts of total RNA spotted on supports and each array hybridized with a probe for a specific reference RNA from the set. These finally chosen reference RNAs are used for the mathematical calculation of the integral factor.
The integral factor for mathematically normalizing a set of samples is calculated using the following procedure:
First, a set of arrays with known gram quantities of each RNA sample, and more preferably identical array with each RNA sample in a specific position on the support, are assayed for levels of the reference RNAs in preferred embodiment. Each support is probed with a single labeled control/reference probe, and the amount of signal of this reference RNA is determined for each RNA sample on the support. Thus, for each RNA sample (R1, R2, etc.) a quantified signal is measured (SR1, SRR2, etc.). The signal is preferably measured using a phosphor imager, but can also be measured using other techniques known in the art.
Second, the percentage (P) of signal that each RNA sample contributes to the sum of all signals on the array is calculated for each hybridization (or for each reference RNA). For example, this can be determined by the equation:
P=(S/xcexa3)xc3x97100
where P is the percentage of the signal, S is the measured intensity of the single signal (e.g., SR1), and xcexa3 is the level of all measured signals of the different RNA samples on the support (SR1+SR2+SR3+ . . . ). This percentage (P) is determined for each RNA sample on each array, resulting in a single sample (e.g., R1) having a P value for each array assayed, i.e. a P value for each reference ribonucleic acid.
Third, the sum (H) of all of the P values for a particular RNA sample on all of the assayed arrays is calculated. For example, H can be calculated for sample R1 by adding the P values for R1 from each array assayed. Some P values, which show obvious tissue or cell-specific variation for some tissues/cells, could be excluded from sum calculation. Usually, the P value could be rejected if it shows more than 3-fold, more commonly 5-fold, and most commonly 10-fold variation from the average P value for a particular tissue across all reference RNAs. For example, xcex2-actin reference RNA usually shows about 5-fold higher signals in skeletal muscle and heart than in other tissues, which reflects high level of muscle-specific expression of actin isoform.
An integral factor can then be determined for each RNA sample by a calculation in which the integral factor (IF) is arbitrarily set to 1 for a single RNA sample. The integral factor can be determined for the other RNA samples to allow a relative determination of mRNA levels between the samples. For example, if the level of sample R1 was set to 1, the integral factor for each of the other samples (e.g. R2 and R3) can be determined using the following equation:
IFR1=HR1/HR1=1
IFR2=HR1/HR2
IFR3=HR1/HR3
The whole calibration process described above can be repeated to ensure accuracy of the calculated integral factor, and the numbers compared for accuracy and/or averaged for more consistent results. Preferably, at least two rounds are performed to determine the IF for each sample. In another embodiment the integral factor can be calculated by using other well known in art algorithms, for example developed for calculation of NASDAQ or DOW JONES indexes. In such approach, for any particular tissue the P values calculated for each reference RNA are used directly to calculate sum. An additional coefficient k reflects accuracy and significance of P value measurement. Accuracy and significance for each reference RNA depends on tissue to tissue variation and relative signal intensities, reproducibility from experiment to experiment, etc. The formula for sum calculation for RNA sample 1 looks like:
H1=P1xc2x7k1+P2xc2x7k2+P3xc2x7k3+ . . . Pnxc2x7kn
In most cases, k is equal or less than 1, usually between 1 and 0.1, more usually between 1 and 0.3, and most commonly between 1 and 0.5.
Once the integral factor has been calculated for all RNA samples used, it can be used to normalize the amount of spotted RNA samples in order to achieve equal average intensity of hybridization signals from all tested reference RNAs. Such produced array based on normalized amount of spotted RNA samples can be used directly for comparing normalized expression levels of any test mRNA. In this approach, a labeled gene-specific cDNA fragment or oligonucleotide specific for the test mRNA is hybridized with the normalized RNA array. The intensity of the hybridization signal can be directly used to compare the expression level of the test mRNA in different tissues/cells.
If the RNA amounts in an equivalent gram amount of each sample are to be compared, a signal obtained for test mRNA in each RNA sample is multiplied by the IF to determine the normalized levels of the test mRNA in each sample. Thus, if an array contains RNA samples R1, R2 and R3, and each sample is present in a 1 gram quantity, the normalized expression levels of a test RNA can be determined by multiplying the signal intensity of each sample with the integral factor for that sample.
In another example, if test mRNA expression levels are to be compared in different amounts of each RNA sample, the signal generated from a labeled test probe is first adjusted to reflect the difference in the gram amounts of each RNA sample, and then the relative levels of test mRNA present in each sample are determined using the integral factor. Thus, if an array contains RNA samples R1, R2 and R3, and the samples are present in a 1, 2 and 6 grams quantity, respectively, the detected signals for test mRNA are first adjusted to reflect the actual levels: the signal of R1 is multiplied by 6, the signal of R2 is multiplied by 3, and signal of R3 is left as is. Once the test mRNA signals have been altered to reflect for amounts of sample, the relative levels of signal generated by the test probe in each sample can be determined by multiplying the signals with its respective integral factor.
For instance, if there is relatively little of sample Ri available, a smaller gram quantity of R1 can be spotted on an array with sample R2 and the integral factor determined for these samples can be used to provide a meaningful comparison of test mRNA levels between R1 and R2. In another example, if a RNA sample has a very high or very low amount of transcription, various levels of a single RNA sample can be compared to another sample, and the relative levels determined using the calculated integral factor.
In preferred embodiment instead of using the integral factor for normalizing the amount of signal for test mRNA the invention disclosed to use the absolute level of any reference RNA used in order to calculate the integral factor. Once the integral factor for each RNA is calculated based on a set of selected reference RNAs, each signal intensity (S) described above for each reference RNA is proportional (and described by formulas above) to the integral factor of all reference RNA set. It means, that if you know the absolute intensity of hybridization signals generated by any reference RNA (used in a set), signal generated by test mRNA and integral factor for each RNA sample on array is enough in order to normalize expression level of test mRNA between all RNA samples. The invention discloses that the first step in preferred protocol will be to perform the hybridization assay with two hybridization probes (sequentially or mixed together in hybridization assay) designed for reference RNA and test mRNA. Then based on intensities of hybridization signals for test mRNA, reference RNA, and value of integral factor the normalized expression level of test mRNA will be calculated using the integral factor. This protocol does not require performing multiple hybridizations with several reference RNAs for each array type and each RNA sample. As soon as the integral factor is calculated for each RNA sample, the normalization of test mRNA expression level between RNA samples requires only one single hybridization with any reference RNA. In preferred embodiment there can be one reference RNA, in some cases it can be two or three, but not more than 6 and in most cases less than 4. Using a single reference RNA signal as internal standard for calculating normalized expression level of test RNA allow to adjust (compensate) variation generated by inaccuracy in spotting technology (i.e. differences in amount of spotted RNA), differences in quality (total mRNA amount in RNA sample preparation) of RNA sample, variation in hybridization, washing, detection conditions, etc.
A number of different ribonucleic acids can be used to determine the integral factor. For example, a number of housekeeping genes were tested for use as reference ribonucleic acids. Ubiquitin (2.3 kilobases), phospolipase A2 (2.8 kilobases), and 23 kDa highly basic protein (0.7 kilobases) belong to multigene families and generate multiple bands on a Northern Blot. The intensity of the major mRNA sizes in brackets may therefore vary in different tissues and cell types. xcex2-tubulin (1.6 kilobases), hypoxanthine guanine phosphoribosyl transferase (HPRT) (1.3 kilobases), ribosomal protein S9 (RPS9) (0.7 kilobases), and ornithine decarboxylase (2.5 kilobases) on the other hand are only moderately abundant. Other reference ribonucleic acids can also be used, as will be apparent to one skilled in the art upon reading the present disclosure. Alternatively, 18S and 28S rRNAs can be used as internal standards. The expression level of these rRNAs is less likely to fluctuate under conditions that affect the expression of mRNAs, because they are transcribed by a distinct polymerase.
Kits
Also provided are kits for performing binding assays using the subject arrays, where kits for carrying out differential gene expression analysis assays are preferred. Such kits according to the subject invention will at least comprise an array according to the subject invention, where the array may simply comprise a pattern of target compositions on a planar support or be incorporated into a multiwell configuration, biochip configuration, or other configuration. The kits may further comprise one or more additional reagents for use in the assay to be performed with the array, where such reagents include: probe generation reagents, e.g. buffers, primers, enzymes, labels and the like; reagents used in the binding step, e.g. hybridization buffers; signal producing system members, e.g. substrates, fluorescent-antibody conjugates, etc.; control probes, e.g., pre-labeled control probes; washing and/or hybridization containers; and the like.
Finally, systems which incorporate the subject arrays, particularly the biochip and multiwell configurations of the subject arrays, are provided, where the systems find use in high throughput gene expression analysis in which information regarding the expression level a gene in a tissue is desired. By the term xe2x80x9csystemxe2x80x9d is meant the working combination of the enumerated components thereof, which components include those components listed below. Systems of the subject invention will generally comprise the array, a fluid handling device capable of contacting the probe fluid and all reagents with the pattern of target molecules on the array and delivery and removing wash fluid from the array surface; a reader which is capable of providing identification of the location of positive probe target binding events and the intensity of the signal generated by such binding events; and preferably a computer means which capable of controlling the actions of the various elements of the system, i.e. when the reader is activated, when fluid is introduced and the like.

Polymeric arrays and methods for their use in binding assays


An alignment fixture includes a rotor fixture, a stator fixture and a sensor system which measures a rotational displacement therebetween. The fixture precisely measures rotation of a generator stator assembly away from a NULL position referenced by a unique reference spline on the rotor shaft. By providing an adjustable location of the stator assembly within the housing, the magnetic axes within each generator shall be aligned to a predetermined and controlled tolerance between the generator interface mounting pin and the reference spline on the rotor shaft. Once magnetically aligned, each generator is essentially a line replaceable unit which may be readily mounted to any input of a multi-generator gearbox assembly with the assurance that the magnetic alignment will be within a predetermined tolerance.

This invention was made with government support under Contract No.: F19628-03-0014. The government therefore has certain rights in this invention.
The present invention relates to a generator system, and more particularly to an alignment fixture therefor.
Vehicles, such as aircraft, utilize an electric generator system to provide electric power. The generators convert mechanical energy from rotation of the engine into electrical energy for the vehicle.
Oftentimes, vehicles such as aircraft require more power than a single generator can provide and utilize a multiple of generators on a multi-headed gearbox all driven by a common drivetrain. To allow for optimum parallel operation, each generator that feeds a common electrical bus is required to be synchronized with every other generator to which it is hard connected through the geartrain so that the voltage of each generator is in phase.
The mechanical alignment of the each coupled rotor to the common drive train defines the angular position of the rotor magnetic axis in its respective generator and thus the phasing of its alternating voltage. The voltage of two or more generators must be in phase with each other within a predetermined angular separation to permit connecting them in parallel. Assuming all the generators to be paralleled are identical and the magnetic axes aligned with each other, conventional mechanical alignment requires the housing assembly of each generator to be individually rotationally adjusted or "timed" relative the gearbox assembly to accommodate the magnetic variance and align the output phasing of each generator to each other generator. Although effective, such individualistic mounting may be relatively time consuming from a maintenance perspective. Adjustment to a multiple of generators is also often required whenever a single generator requires replacement which may still further increase maintenance down time.
Accordingly, it is desirable to provide an electric generator assembly system that accommodates electrical and mechanical variance and produces multiple generators all demonstrating similar angular relationship between individual output phasing and mounting interface position which results in apparently identical generators that minimizes individualized mounting alignment and maintenance concerns.


The invention relates to a device for injecting liquid, comprising a piece of tubing on which there is situated at least a first liquid occlusion system. According to the invention, a regulation system (4) is situated upstream from the first occlusion system, the two systems defining an intermediate segment in which the pressure, in the absence of injection, is greater than the pressure that exists downstream from the first occlusion system so as to direct any leak of liquid from the first occlusion system downstream. The invention is applicable to a pump used for injecting contrast liquid for medical imaging.

The present invention relates to a medical device for injecting liquid.
An application for such a device can lie, for example, with a pump used for injecting contrast liquid for medical imaging.
A major problem that results from injecting liquid into patients lies in the risk of the injection device being contaminated by the patient. When a liquid is administered to a patient, there is a risk of the injected liquid flowing back after it has already come into contact with the patient. Also, in the absence of injection, there still exists a risk of contamination by contaminating agents, such as germs, migrating from the patient to the injection device. As a general rule, this situation makes it essential, when treating a new patient, to change all of the portions of the device that have already been used.
There exist several devices in the prior art for reducing the risk of such backflow: EP-A-648513 in the name of MEDEX S. A. describes an injection unit that includes a non-return valve, for example. EP-A-279028 in the names of KABI PHARMACIA GmbH and PFRIMMER-VIGGO GmbH claims a liquid injection device having a non-return valve characterized by the fact that the opening pressure can be set to either of two positions.
Although prior art devices do indeed reduce the risk of backflow, there still remains the risk of the non-return valve leaking. This risk is particularly high in the absence of injection.
The present invention thus seeks totally to eliminate any risk of backflow. In addition, the present invention also seeks to make it possible to detect any leak through the safety means that are installed to avoid such backflow, assuming such backflow might indeed take place.
To this end, use is made of a device for injecting liquid, the device comprising a piece of tubing on which there is situated at least a first liquid occlusion system such as a non-return valve. In addition, a regulation system is situated upstream from the first occlusion system, the two systems defining an intermediate segment in which the pressure, in the absence of injection, is greater than the pressure that exists downstream from the first occlusion system. In this way, any leak of liquid at the first occlusion system is directed downstream and any leak from the intermediate segment can be detected by measuring the associated pressure drop.
The terms xe2x80x9cdownstreamxe2x80x9d and xe2x80x9cupstreamxe2x80x9d mean respectively towards the patient and in the opposite direction.
By way of example, the regulation system can be a second non-return valve or a system for flattening the tubing, such as the wheels of the peristaltic cassette corresponding to patent FR 89/03234 in the name of MALBEC S. A.
It should be observed that the regulation system cannot be considered merely as constituting a safety system in addition to and independent of the first occlusion system.
On the contrary, the two systems together constitute an interactive assembly since the presence of the regulation system makes it possible to set up, and optionally to maintain, a positive pressure difference between the two systems to prevent any leakage of liquid located downstream from the first occlusion system from flowing upstream towards the intermediate segment.
It should also be observed that the pressure difference between the two systems is constant in the absence of a leak since the space between the two systems is filled with liquid which, like any other liquid, is incompressible.
In order to increase the safety of the device, it is also desirable to have systems of different kinds. As a result, if one system should fail, the other can nevertheless continue to act, in a manner similar to that described in the prior art.
Furthermore, when using systems of different kinds, and more precisely systems characterized by the fact that each of them has a different opening pressure, it is possible to direct the flow direction of the liquid in the event of a leak or high pressure in the intermediate segment.
In particular, if the opening pressure of the first occlusion system is less than the opening pressure of the regulation system, any liquid movement during an interruption of injection will take place downstream, the regulation system closing before the first occlusion system, thereby avoiding any contamination of the portions of the device situated upstream from the first occlusion system.
Similarly, in the event of high pressure in the intermediate segment, the first occlusion system will be the first to open in the event of a leak, thereby causing the liquid to go downstream from the first occlusion system and thus preventing any backflow towards the intermediate segment.
It is also possible to envisage that the two opening pressures of the regulation systems are identical, however that can only be envisaged if the opening pressure is greater than the pressure that exists downstream from the first occlusion system when there is no injection.
In addition, it is desirable to have an opening pressure for the first occlusion system which is greater than the maximum pressure that can be set up downstream therefore because of the patient in the absence of injection. Also, in the absence of injection, it is necessary for the opening pressure of the first occlusion system to be greater than the pressure of the intermediate segment.
In most cases, in the absence of injection, the pressure that exists downstream from the first occlusion system corresponds to the venous pressure of the patient.
In another preferred embodiment, it is desirable to provide the tubing with a disconnection system situated between the two regulation systems. The disconnection system marks the boundary between a downstream tubing for single use only and an upstream tubing for multiple use.
Thus, when the tubing is contaminated only downstream from the disconnection system, it is possible to change only that portion of the device while reusing the remainder thereof (pumping system, peristaltic cassette, reservoir, etc.) with other patients.
The disconnection system is preferably situated as close as possible to the second regulation device or, which comes to the same thing, as far as possible from the non-return valve. Thus, in the highly improbable event of contamination propagating upstream, the risk of contaminating the zone situated upstream from the disconnection system is minimized since the path to be followed to reach the disconnection system is as long as possible.
In another preferred embodiment, the disconnection system is provided with occlusion means for the tubing which are activated prior to disconnection, thereby avoiding any risk of liquid being ejected from the tubing which is under pressure. Such an automatic closure system is described, for example, in U.S. Pat. Nos. 5,549,566 and 5,533,996 in the names of ABBOTT LAB and BAXTER INT. INC., respectively.
The above-mentioned occlusion means can also be designed so as to open during connection.
By way of example, such disconnection systems can be of the xe2x80x9cluer-lockxe2x80x9d type coupled to a rotary cock system on each of the two connected segments. In addition, the connection system between the two pieces can be of the key and lock type, thus allowing interconnection to take place only between two elements that are properly encoded relative to each other. Such a rotary system makes it possible to open and close the two cocks placed on either side of the connection when the key is turned in the lock, while preventing the two pieces from being disconnected when the key is engaged in the position corresponding to the open position of the occlusion systems.
The occlusion system situated in the downstream segment can also be a non-return valve, and the system in the upstream segment can be a check valve that is open when the two pieces are engaged or, alternatively, the two occlusion systems can be check valves that are open only when the two pieces are engaged.
In another preferred embodiment, a pressure sensor is situated on the intermediate segment. As a result, it is possible to detect any pressure variation in this zone.
When the pressure sensor comes back into contact with the tubing, it is advantageous to place it between the disconnection system and the regulation system; this configuration has the merit of making it possible to reuse the same pressure sensor configuration for a plurality of patients.
In the absence of injection, any pressure drop between the two systems implies that a leak is present which, very likely, might have taken place in the first occlusion system or in the regulation system.
If the leak has taken place in the first occlusion system, then contamination can propagate into the tubing for multiple use.
If the leak has taken place in the regulation system, then contamination has not necessarily propagated into the tubing for multiple use, but the risk of that happening has nevertheless been increased since any leak at the first occlusion system gives rise to a drop in pressure in the intermediate segment, thus eliminating the effect of the means installed in the context of the present invention.
In order to mitigate the two above-described situations, it is advantageous to associate an alarm 102 with the pressure sensor placed on the intermediate segment. Furthermore, in even more advantageous manner, liquid injection can be triggered as to reestablish the initial pressure when the alarm is activated.
It should also be observed that maintaining a high pressure in the intermediate segment, close to the pressure for opening the first occlusion system, can serve to favor leakage at said first occlusion system.
To this end, means are provided to reduce the pressure in the intermediate segment. These means can be constituted by an intermediate chamber of adjustable volume. By way of example, the volume of the intermediate chamber can be adjusted by a piston. Thus, the pressure which exists in the intermediate segment can be selected so as to be greater than the pressure downstream from the first occlusion system and far enough away from the opening pressure of said first occlusion system to make full use of the sealing characteristics of said first occlusion system. Another means enabling said pressure to be lowered below the opening threshold of the first occlusion system consists in selecting a regulation system whose opening can be adjusted in such a manner as to lower the pressure of the intermediate segment to a predetermined value.
Although adjusting the pressure in the intermediate segment considerably reduces any risk of contamination, it is still possible after an alarm is triggered to change the portions that might have been contaminated, for example portions such as the peristaltic cassette. Under such circumstances, it is advantageous for this purpose to provide a second disconnection system upstream from the regulation system.
Optionally, a second pressure sensor can be placed on the segment of tubing which is to be found downstream from the first occlusion system, thus making it possible, for example, to detect any excess pressure compared with the pressure in the intermediate segment.
It is also possible to provide the device with a system for measuring the pressure difference as measured by the two sensors, thus having the advantage of determining the risk of backflow more directly. An alarm can be triggered when the pressures situated upstream and downstream of the non-return valve are tending to become equal.
The medical device of the invention can also be designed in such a manner as to deactivate the alarm when the single-use tubing is disconnected.
The medical device of the invention may advantageously include a syringe driver 100. A pressure sensor 108 can be placed on the distal end of the piston 106 of the syringe driver.
Assuming that syringe driver 100 is used, it is possible to consider the syringe 104 itself as being an integral portion of the intermediate segment, and the syringe itself can form the regulation system. In addition, under these circumstances, the syringe 104 can also act as a system for regulating the intermediate chamber so as to enable the pressure in the intermediate chamber to be adjusted.
The medical device of the invention can be used with any type of liquid, and in particular it can be used for injecting contrast liquids.


A fitment has walls with a spout aperture therebetween. Reduced wall thickness results in a reduced amount of plastic in the fitment. An alternative embodiment uses radially extending meltable fins. During installation in asealing apparatus, a heated mandrel engages the inner diameter of the spout aperture thereby pre-heating and physically supporting the fitment during the installation process. The heated mandrel typically engages the fitment at least one cycle prior to be installed. The fitment may be canoe-shaped.

1. Field of the Invention
This invention pertains to a fitment for a spouted pouch, particularly one with a canoe-type base or meltable fin wherein the plastic is reduced in transition areas and in the wall thickness of the base. To accelerate and maintain the integrity of the process for installing a fitment, a heated mandrel shaped and sized to the inner diameter of the fitment passageway is used.
2. Description of the Prior Art
In the prior art, it is known to use a fitment/spout with a canoe shaped base with extended meltable fins oriented 180xc2x0 apart, and multiple horizontal ribs spaced evenly across the height of the canoe. The canoe shape and the meltable fins assure gradual transition from the two plies of film (one on each side of the fitment) to the maximum width of the canoe in the diametrical center of the fitment.
In order to seal or weld the fitments to the film at the fastest possible rate, and to reduce potential leaking at the base, the base can be made with horizontal.ribs to increase the sealing pressure and to reduce the dwell time. However, the sealing pressure must not damage or misshape the inner diameter of the fitment. To avoid such problems, the canoe base is typically made more massive than would otherwise be required.
Because the maximum temperature of the jaws is limited by the characteristics of the film, the two driving variables of the sealing equation become the dwell time and the pressure. That is, a canoe base which is enlarged for the above reasons requires increased dwell time and pressure to bring it to the sealing temperature, which is counterproductive to the production rates of the fitment attachment process.
The attachment of this kind of fitment is disclosed in U.S. patent application Ser. No. 09/452,714 entitled xe2x80x9cMethod for Attaching Fitment at Longitudinal Fin Seal and Package Resulting Therefromxe2x80x9d filed on Dec. 2, 1999.
Other prior art includes U.S. Pat. No. 5,911,340 entitled xe2x80x9cSpout Assembly, Spout Assembly Manufacturing Apparatus and Package with Spout Asssemblyxe2x80x9d issued on Jun. 15, 1999 to Uematsu; U.S. Pat. No. 5,716,471 entitled xe2x80x9cMethod for Securing Articles to Laminatesxe2x80x9d issued on Feb. 10, 1998 to Pape; U.S. Pat. No. 4,909,434 entitled xe2x80x9cMoisture Impervious Carton Having One-Piece Pouring Spout Sealed to Innermost and Outermost Surfacesxe2x80x9d issued on Mar. 20, 1990 to Jones et al.; and U.S. Pat. No. 3,894,381 entitled xe2x80x9cMethod and Means for Attaching Fitments to a Bag or Pouch on a Packaging Machinexe2x80x9d issued on Jul. 15, 1975 to Christine et al.
It is therefore an object of the present invention to provide a fitment with canoe-shaped walls which can be installed between two plies of plastic film with reduced dwell time and/or reduced jaw pressure.
It is therefore a further object of the present invention to provide a fitment with canoe-shaped walls which can be installed at high speeds.
It is therefore a still further object of the present invention to provide a fitment with canoe-shaped walls which can reliably maintain its shape during installation.
These and other objects are attained by providing an injection molded fitment with reduced-plastic in the canoe base, to retain the shape and length of the canoe, but to reduce the amount of plastic in transition areas and in the wall thickness of the base. This reduces the mass of the canoe and the required preheat/sealing dwell time. An alternative embodiment employs radially extending meltable fins in place of the canoe base. In order to compensate for the reduction in the rigidity of the base and the resulting reduction in ability to withstand the pressure of the sealing bars, the fitment is installed with a heated mandrel inserted through the inner diameter of the fitment. More specifically, as the fitment travels along the track from the vibratory bowl to the sealing station, a heated mandrel shaped and sized to the inner diameter of the fitment is inserted into the inner diameter of the fitment. In order to avoid unnecessary softening of the fitment, the temperature of the mandrel is typically 20-30xc2x0 F. below the softening temperature of the resin from which the fitment is molded. In order to heat the fitment thoroughly from the inside prior to installation without slowing down the installation process, the mandrel is typically inserted at least one cycle away from the sealing position. The insertion of the heated mandrel into the inside diameter of the fitment will further prevent the collapse or misshaping of the base. Indeed, the sealing dwell time is thereby reduced by both the reduction of plastic in the base and the heating of the fitment by the mandrel.


A method and system for a search based on diversity is described. The system calculates a diversity index for a result set comprising results for a query. The diversity index is a measure of diversity among the results of the query. The diversity index relates to differences among the results. The system compares the diversity index to a threshold value. Finally, the system expands the query with one or more query terms based on the comparison.

Information retrieval relates to the searching of information stored on computing systems, such as searches for web pages, items for sale, documents, and images. To locate particular information, a user specifies a query about the information of interest and a search engine, for example, uses this query in the search. For example, online shopping and auction websites provide a number of publishing, listing, and price-setting mechanisms whereby a seller may list (or publish information concerning) items for sale. A buyer can express interest in or indicate a desire to purchase such items by, for example, submitting a query to the website for use in a search of the requested items.
The accurate matching of a query to relevant items is currently a major technical challenge in the field of information retrieval. An example of such a challenge is that item descriptions (e.g., title) tend to be short and such descriptions may be uniquely defined by the sellers. The buyers seeking to purchase the items might use a different vocabulary from the vocabulary used by the sellers to describe the items. As an example, an item identified in the title as a "garnet" does not match a query "January birthstone" submitted by a buyer, although garnet s known as the birthstone for January. In another example, an item identified in the title as "Martin guitar" does not match the query "acoustic guitar" submitted by a buyer, although the Martin guitar is a type of an acoustic guitar. As a result, for example, online shopping and auction websites that use a conventional search engine to locate items may not effectively connect the buyers to the sellers.

This patent is subject to a terminal disclaimer.


A centrifugal chiller includes both a unit-mounted full-voltage starter and a unit-mounted reduced-voltage starter. The chiller is rather large with a rated cooling capacity of at least 300 tons and is driven by a motor that is powered by at least 2,000 volts. The chiller includes an evaporator shell and a condenser shell in a staggered arrangement to provide a combined height of between 6 and 12 feet and a combined width of between 5 and 8.5 feet. The staggered arrangement of the shells plus mounting the two starters at opposite sides of the motor provide a chiller whose outer dimensions permit the chiller to be shipped as a single package without exceeding certain shipping limits.

1. Field of the Invention
The subject invention generally pertains to refrigerant systems and more specifically to unit-mounted starters for centrifugal chillers.
2. Description of Related Art
Typical centrifugal chillers are refrigerant systems that includes a centrifugal compressor driven by an electric motor. The cooling capacity of the chiller may range from a few tons to a thousand tons or more. The smaller units often operate at 480 volts or less and can be turned on and off by a full-voltage starter, which selectively applies or removes the full supply voltage to the motor.
Larger chillers that can provide at least 300 tons of cooling are often powered by higher voltages of 2,000 volts or more. The higher voltage means less current is needed for a given size motor, and less current means lighter gauge power supply lines and/or less power losses in the wires. However, suddenly applying or removing a full 2,000+ volts across the motor leads can place a significant load on a facility""s electrical supply system and can be quite a shock to the motor, compressor, and the contactors that switch the power on and off. Consequently, such units often employ some type of reduced-voltage starter, whereby reduced voltage is applied at startup, and full voltage is applied shortly thereafter.
Perhaps the three most common types of reducedvoltage starters are the primary reactor, autotransformer, and solid-state starter. A primary reactor includes a resistor in series with each power line leading to the motor. A set of contacts serves as a shunt across each resistor to effectively add or remove the resistor from its respective line. When the contacts open, the resistors reduce the line voltage to the motor for reduced-voltage starting. When the contacts close, the current bypasses the resistors to apply full voltage to the motor during normal operation. An autotransformer includes a transformer with multiple leads that a set of contacts selectively taps to apply full-voltage or reduced-voltage across the motor leads. A solid-state starter includes at least one solid-state electrical xe2x80x9cswitchingxe2x80x9d device (e.g., SCR, triac, diac, power transistor, etc.) that interrupts or changes the waveform of the power leading to the motor to deliver less power to the motor at startup, and full power afterwards.
For typical higher voltage chillers, a full-voltage starter and/or a reduced-voltage starter are often contained within a single enclosure that is installed off the unit in an electrical room or in another area within the building served by the chiller. The starters are often installed alongside other electrical equipment of the building. Main electrical supply lines of the building lead to the full-voltage starter, which in turn feeds the reduced-voltage starter. Wires from the reduced-voltage starter convey the electrical power to the motor. The reduced-voltage starter provides reduced voltage to the motor at startup and full voltage afterwards.
Installing a higher voltage chiller separate from its full-voltage and reduced-voltage starters adds cost and time to the initial installation of the chiller because of the extra electrical work that is required in the field to install the two starters. Unit-mounted starters are commonly found on relatively small chillers; however, larger chillers are different.
Larger chillers generally have larger starters, yet they have less room for them because of shipping rules that limit a chiller""s overall physical size. Although some full-voltage starters have been mounted to larger chillers (i.e., at least 300 tons and over 2,000 volts), such large chillers have not been known to include both a unit-mounted full-voltage starter and a unit-mounted reduced-voltage starter. The physical size of such chillers, even without any unit-mounted starters, is often already pushing the allowable envelope for normal shipping. And starters for larger chillers are quite heavy and bulky, which can make them difficult to mount to a chiller.
Moreover, certain areas on a chiller should be left clear of obstructions to provide room for maintenance, lifting, and to meet applicable codes and regulations. Other areas of the chiller may be taken up by chilled water piping, condenser water piping, a purge unit, a control panel, an economizer, and various sensors. In the end, there is often insufficient room left on the chiller for installing bulky unit-mounted starters.
Consequently, there is a need for providing a strategic location and method for mounting both a full-voltage starter and a reduced-voltage starter to a relatively large centrifugal chiller while still being able to use standard modes of shipping.
To provide a large centrifugal chiller with both a full-voltage starter and a reduced-voltage starter, it is an object of some embodiments of the invention to install the two starters in their own separate enclosure rather than in the same one. For larger chillers, it can be easier to find room to mount two smaller enclosures than to find room for a single, much larger enclosure.
Another object of some embodiments is to reduce the time and cost of installing a large centrifugal chiller.
Another object of some embodiments is to mount both a full-voltage starter and a reduced-voltage starter to a large centrifugal chiller without exceeding maximum dimensions of standard shipping methods.
Another object of some embodiments is to create a convenient user interface with a large centrifugal chiller by mounting a control panel and a full-voltage motor starter on one side of the chiller""s motor and mounting a reduced-voltage starter on the opposite side of the motor. This arrangement can make the control panel and full-voltage starter more accessible than the reduced-voltage starter, which can be a benefit in cases where the reduced-voltage starter requires less attention by operators and service personnel.
Another object of some embodiments is to mount a relatively heavy starter enclosure atop a condenser or evaporator shell and to mount a lighter starter enclosure in a more off centered position to one of the shells, whereby the heavier enclosure receives substantial vertical support, and the lighter enclosure is readily accessible.
Another object of some embodiments is to place an evaporator shell and condenser shell in a staggered arrangement that provides room for two separately mounted motor starters.
Another object of some embodiments is to route a chiller""s electrical power first into a full-voltage starter enclosure, then into a reduced-voltage starter enclosure, and then back to the full-voltage starter enclosure before delivering the power to the motor. This allows the full-voltage starter enclosure to be placed in a location that is accessible to a user, adjacent to the motor leads, and convenient for feeding the main power supply lines to the chiller. Also, the reduced-voltage starter enclosure can be placed in a more out-of-the-way location that does not interfere with operation or servicing of the chiller.
One or more of these and/or other objects of the invention are provided by a rather large centrifugal chiller that includes both a unit-mounted full-voltage starter and a unit-mounted reduced-voltage starter. The chiller has a rated cooling capacity of at least 300 tons and is driven by a motor that is powered by at least 2,000 volts. The chiller includes an evaporator shell and a condenser shell in a staggered arrangement to provide a combined height of between 6 and 12 feet and a combined width of between 5 and 8.5 feet.

Centrifugal chiller with high voltage unit-mounted starters