Classifications

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

Definitions

• Biochips or arrays of binding agents, such as ohgonucleotides and peptides, have become an increasingly important tool in the biotechnology industry and related fields
• binding agent arrays in which a plurality of binding agents are deposited onto a support surface, often a solid support surface, m the form of an array or pattern, find use in a variety of applications, including drug screening, nucleic acid sequencing, mutation analysis, 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 m the cells being compared
• arrays find use by serving as a substrate to which is bound polynucleotide "probe” fragments One then obtains "targets” from analogous cells, tissues or organs of a healthy and diseased organism The targets are then hybridized to the immobilized set of polynucleotide "probe” fragments Differences between the resultant hybridization patterns are then detected and related to differences in gene expression in the two sources
• the efficiency of hybridization of target nucleic acids to the array can be limited by experimental limitations, e g , different target nucleic acids can have different hybridization efficiencies to the probe nucleic acids of the array Differences in hybridization efficiency result in differences in the intensity of hybridization to different probe nucleic acids of the array, even though the targets are present in equivalent concentrations
• variation m the quality of array (reproducibi ty of array production) can preclude direct comparison of data obtained on the arrays, since conditions such as hybridization time, probe labeling, detection procedures may differ, and variations between the arrays may be present
• Patents and patent applications describing arrays of biopolyme ⁇ c compounds and methods for their fabrication include 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 applications describing methods of using arrays m various applications include 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, 5,800,992, WO 95/21265, WO 96/31622, WO 97/10365, WO 97/27317, EP 373 203, and EP 785 280
• nucleic acid means a polymer composed of nucleotides, e g deoxy ⁇ bonucleotides or ⁇ bonucleotides
• ⁇ bonucleic acid and RNA as used herein means a polymer composed of ⁇ bonucleotides
• deoxy ⁇ bonucleic acid and "DNA” as used herein means a polymer composed of deoxyribonucleotides
• o gonucleotide denotes single stranded nucleotide multimers of from about 10 to 100 nucleotides m length
• polynucleotide refers to single or double stranded polymer composed of nucleotide monomers of greater than about 120 nucleotides in length up to about 1000 nucleotides m length
• array refers to a plurality of nucleic acids stably associated with the surface of a solid support, where at least a portion of the stably associated nucleic acids are probe nucleic acids
• probe nucleic acid refers to nucleic acids stably associated with the surface of a solid support which correspond to a target gene of interest in a sample
• Probe nucleic acids are not random nucleic acids or nucleic acids that corresponding to genes that are not of interest in a sample, e g housekeeping genes, genes that are widely expressed among tissues, etc
• target nucleic acid refers to a nucleic acid isolated from a physiological sample which comprises a gene(s) of interest
• control target nucleic acid refers to a nucleic acid that is complementary to a probe nucleic acid on an array, where the control target nucleic acid is part of a set of target nucleic acids in which the amount of each nucleic acid in the set is known and in which each probe nucleic acid corresponding to a gene of interest present on an array is represented
• Control target nucleic acid is (in a preferred embodiment) synthetic RNA made artificially in vitro and not isolated from a biological source
• control target nucleic acid is distinguished from test target and test control, which are derived from a biological source
• Control target nucleic acids are not the same as test control nucleic acids, which are derived from the same physiological sample as the test target nucleic acids and are therefore unique and specific for that sample
• test control nucleic acids and the subject control target nucleic acids is that test control nucleic acids
• both a test set of target nucleic acids and a control set of target nucleic acids are employed, where the control set of target nucleic acids is characterized by including at least one nucleic acid that binds selectively to probe nucleic acids in at least a subset of the probe nucleic acids present on the array, I e at least a subset of, if not all of, the probe nucleic acids present on the array employed in the method is represented m the control set
• control and test sets of target nucleic acids may be labeled with the same label or differentially labeled, l e labeled such that they are simultaneously distinguishable from each other
• sets of control target nucleic acids for use m the subject methods, as well kits comprising the sets of control target nucleic acids for use in performing the subject methods
• the subject invention finds use in a variety of different applications, including gene expression analysis In further describing the subject invention, the methods will be described first, followed by a description of the subject kits and a discussion of representative applications in which the subject invention finds use
• the subject invention is directed to array-based hybridization assays in which a test set of labeled target nucleic acids is hybridized to an array of probe nucleic acids
• a critical feature of the subject invention is the use of a control set of target nucleic acids, where at least a subset of the probe nucleic acids, and m certain preferred embodiments all of the probe nucleic acids, present on the array are represented in the control set, l e the control set includes a nucleic acid capable of hybridizing to each different probe nucleic acid of at least a subset of all of the different probe nucleic acids of the array with which it is employed
• the subject methods include the following steps (1) procurement of the test and control target nucleic acids, (2) hybridization to an array(s), (3) washing, and (4) detection Each of these steps of the subject methods is described in greater detail below
• the first step in the subject methods is the procurement of the labeled control and test sets of target nucleic acids
• Both the control and test sets of nucleic acids are pools, mixtures or collections of a plurality of distinct nucleic acids that differ by sequence
• the number of distinct nucleic acids in the control set of target nucleic acids is at least about 50, usually at least about 100 and more usually at least about 200, where the number of distinct nucleic acids in a given set may be as high as 20,000 or higher, but will typically not exceed about 10,000 and usually will not exceed about 5,000
• the number of distinct nucleic acids in the test set is generally at least about 1000 and generally less than about 50,000, where the number generally ranges from about 5,000 to 20,000
• the control and test sets of target nucleic acids are now described separately in greater detail
• control sets of target nucleic acids are collections or pools of control target nucleic acids, as mentioned above
• a control target nucleic acid may be any nucleic acid capable of hybridizing selectively to its respective probe nucleic acid on an array
• the nucleotides that make up the control target nucleic acid molecule may be made up of nucleotides that are naturally occurring or nucleotides that are synthetically produced analogues that are capable of forming base-pair relationships with naturally occurring base pairs
• control target nucleic acids may include polymers ot ribonucleotides and deoxyribonucleotides, with the ribonucleotide and/or deoxyribonucleotides being connected together via 5 ' to 3 ' linkages.
• Control nucleic acids of the invention may be ribonucleic acids, for example sense or antisense ribonucleic acids, full-length or partial fragments of cRNA, full-length or partial fragments ot mRNA, and/or ribo-oligonucleotides
• control target nucleic acids of the invention may be deoxyribonucleic acids, preferably single-stranded full-length or fragments of sequences encoding the corresponding mRNAs.
• the form of the control and target nucleic acids should be chosen so that they are complimentary to and form appropriate Watson-Crick hydrogen bonds with probes present in an array. For example if probe sequences correspond in sequence to mRNA, then target sequences should be complementary, e.g. antisense or complementary RNA (cRNA)
• control target nucleic acids may be polymers of synthetic nucleotide analogs Such control target nucleic acids may be preferred in certain embodiments because of their superior stability under .assay conditions Modifications m the native structure, including alterations in the backbone, sugars or heterocychc bases, have been shown to increase intracellular stability and binding affinity Among useful changes in the backbone chemistry are phosphorothioates, phosphorodithioates, where both of the non-bridgmg oxygens are substituted with sulfur, phosphoroamidites, alkyl phosphot ⁇ esters and boranophosphates Achiral phosphate derivatives include 3'-O'-5'-S-phosphoroth ⁇ oate, 3'-S-5'-O- phosphorothioate,
• control target nucleic acids are structurally as similar as possible to the test target nucleic acids that are employed in the assay, e.g. , both sets of target and control nucleic acids are labeled cDNAs.
• control target nucleic acids should be similar to that of the test target nucleic acids in order to maximally imitate the hybridization of the test target nucleic acids
• Each target nucleic acid of the control set should bind to its corresponding probe nucleic acid with selectivity and sensitivity
• a nucleic acid that selectively binds with its corresponding probe nucleic acid is at least 10 times, preferably at least 100 times, and more preferably at least 1000 times more likely to bind with its designated probe nucleic acid than to a non-specific nucleic acid, and preferably any other sequence present on the array
• Non-specific nucleic acids include those of random sequence, coding sequences found in a particular array other than the designated probe nucleic acid, and coding sequences of non-probe sequences specific to the organism from which the probe nucleic acids are derived
• Control target nucleic acids of the invention also display sufficient sensitivity upon bindmg with their designated probe nucleic acids
• sufficient sensitivity is meant that binding of the probe nucleic acid is significantly greater than the binding of background nucleic acids of random sequence, where the strength of binding is at least 10 times, preferably at least 100 times, and more preferably 500 times greater than the recognition of background nucleic acids of random sequence
• the nucleotide sequences of the subject control target nucleic acids are chosen with algorithms, where such algorithms are described in detail m PCT publication WO 97/10365 and PCT/US96/14839, the disclosures of which are herein incorporated by reference
• a critical feature of the control sets of target nucleic acids is that they include at least one target nucleic acid complementary to each probe nucleic acid present in at least a subset of the probe nucleic acids present on the array with which they are used
• at least a subset of the probe nucleic acids present on a given array are represented in the control set intended for use with the given array, where the probe members of the subset preferably correspond to genes for which quantitative analysis is desired
• at least a subset is meant that at least 20, usually at least 30 and more usually at least 50 of the probe nucleic acids present on the array are represented in the control set
• at least 20%, usually at least 30% and more usually at least 50%) of the probe nucleic acids present on the array are represented in the array
• all of the probe nucleic acids present on the array are represented in the control set
• a given array includes 500 distinct probe nucleic acids which are distinct from each other based on sequence
• a control set for use with this particular array includes at least 500 different
• Non-probe sequences on the array may not have a target nucleic acid in the control set, e g array sequences such as orientation sequences, negative and positive control sequences, etc that may be present on an array
• control target nucleic acids are not necessary for sequences on an array which do not require quantification, where a particular protocol is intended to provide qualification data only
• the number of unique control target nucleic acids in the set or pool of control target nucleic acids will, in most embodiments, be at least about 20, usually at least about 50, more usually at least about 100, where the number may be as high as 1000 or higher, but m many embodiments will not exceed about 800, and usually will not exceed about 750
• control sets that include a representative or representational number of target nucleic acids
• the total number of different target nucleic acids in any given set will be only a fraction of the total number of different or distinct RNAs in the sample from which the test set of target nucleic acids (described infra) is derived, where the total number of target nucleic acids in the control set will generally not exceed 80%, usually will not exceed 50% and more usually will not 20% of the total number of distinct RNAs in the original sample from which the test set of target nucleic acids is derived, e g the total number of distinct messenger RNAs (rnRNAs) m the original sample Any two given RNAs m a sample will be considered distinct or different if they comprise a stretch of at least 100 nucleotides m length in which the sequence similarity is less then 98%, as determined using the FASTA program (default settings)
• Control target nucleic acids can be the same length, shorter or longer than their corresponding probe sequences on the array or test nucleic acid in the solution (if present) However, each control target nucleic acid should have a least partial complementarity to its corresponding probe nucleic acid and at least partial sequence identity with its corresponding test target nucleic acids (if present) In addition, the control target nucleic should have structural and hybridization characteristics very similar to its corresponding test target nucleic acid, l e , it should have similar hybridization efficiencies, similar kinetics with complementary probe sequences, similar background hybridization with other sequences, etc
• the control set of target nucleic acids comprises labeled cDNAs reverse transcribed from a control set of a representative pool of synthetic RNAs
• the test target nucleic acids will also generally be labeled cDNAs reverse transcribed from rnRNAs, e g synthetic mRNAs
• Each target nucleic acid in the control set may be the same length as its corresponding probe nucleic acid, longer than its corresponding probe nucleic acid or shorter than its corresponding probe nucleic acid
• the length of each target nucleic acid m a given control set is at least about 25 nucleotides, usually at least about 50 nucleotides, and more usually at least about 100 nucleotides, where the length could be as a long as 2 kb or longer, but will generally not exceed about 1 kb and more usually will not exceed about 800 nucleotides
• a critical feature of control sets of target nucleic acids is that the concentration of each control target nucleic acid present in the set be known
• the control sets of target nucleic acids are further characterized m that at least two different gene functional classes are represented in a given control set, where the number of different functional classes of genes represented in the a given control set will generally be at least 3, and will usually be at least 5
• the sets of control target nucleic acids comprise nucleotide sequences complementary to RNA transcripts of at least 2 gene functional classes, usually at least 3 gene functional classes, and more usually at least 5 gene functional classes
• Gene functional classes of interest include oncogenes, genes encoding tumor suppressors, genes encoding cell cycle regulators, stress response genes, genes encoding ion channel proteins, genes encodmg transport proteins, genes encoding intracellular signal transduction modulator and effector factors, apoptosis related genes, DNA synthesis/recombination/repair genes, genes encoding transcription factors, genes encoding DNA-binding proteins, genes encoding receptors, including receptors for growth factors, chemokines, mterleukms, interferon
• control target nucleic acids are synthetic nucleic acids and not isolated from a biological source
• the control target nucleic acids may be generated using any convenient protocol, including reverse transcription protocols (e g using AMV or MoMLV reverse transcriptase), bacteriophage RNA polymerase (T7 RNA polymerase, T3 RNA polymerase, etc ) mediated transcription, PCR protocols, ohgonucleotide synthesis protocols (I e nucleotide chemistry), and the like
• the control target nucleic acid sequences are generated using cDNA fragments cloned into appropriate expression vectors using a set of a representative number of gene specific primers, as described U S Application Serial nos 08/859,998, 08/974,298,09/225,998, the disclosures of which are incorporated herein by reference
• These cloned cDNAs are then used to produce RNA control targets using techniques such as PCR and/or bacteriophage RNA polymerase mediated transcription
• each control target nucleic acid is quantitated using procedures such as spectrophotometry, fluorescence me ⁇ asurement, etc Known quantitative amounts of each control target nucleic acid are then mixed together for use in the hybridization assays, described in greater detail infra
• the control target nucleic acids are mixed together in equal molar amounts, at predetermined ratios, at equal weight amounts, etc, where m many embodiments they will be mixed together in equal weight amounts, such that the amount of each individual target nucleic acid m the control set is the same as any every other individual target nucleic acid m the set
• the test target nucleic acids are generally isolated trom a biological sample (cells, tissues, organs, etc ) preparation, and then converted to other nucleic acids using known in the art technology, such as PCR, reverse transcription, etc , e g mRNA, cDNA, PCR products, cRNA, and the like
• the target nucleic acids may be isolated from a tissue or cell of interest using any method known in the art Total RNA or its trans c ⁇ ptionally active fraction mRNA can be isolated from a tissue and labeled and used directly as a target nucleic acid, or it may be converted to a labeled cDNA, cRNA, etc via methods such as reverse transcription, transcription and/or PCR Generally, such methods will employ the use of ohgonucleotide primers, and the primers can be anchored by bacteriophage RNA polymerase promoter
• the primers may be designed to copy a large spectrum of RNA species, e g
• test and control sets of target nucleic acids may be labeled with the same label, such that the test and control sets cannot be distinguished from one another, or the test and control sets of target nucleic acids may be differentially labeled, such that the two sets are readily distinguishable from each other
• the test and control sets of target nucleic acids are differentially labeled
• “differentially labeled” is meant that the test and control sets of target nucleic acids are labeled differently from each other such that they can be simultaneously distinguished from each other
• each target nucleic acid in the test set will be labeled with the same first label and each target nucleic acid in the control set will be labeled with the same second label that is different and distinguishable from the first label
• each target nucleic acid in the second control set will be labeled with a third label different and distinguishable from both the first and second label
• test and control sets of target nucleic acids are labeled with the same label, so as to be indistinguishable from each other
• each target nucleic acid of each set is labeled with the same label
• labeled primers can be employed to generate the labeled target nucleic acid during first strand synthesis or subsequent synthesis, labeling or amplification steps m order to produce labeled target Label can also be incorporated directly to mRNA using chemical modification of RNA with reactive label derivatives or enzymatic modification using labeled substrates Representative methods of producing labeled target are disclosed m U S Application Serial nos 08/859,998, 08/974,298, 09/225,998, the disclosures of which are incorporated herein by reference.
• labels include fluorescent labels, lsotopic labels, enzymatic labels, particulate labels, etc
• suitable labels include fluorochromes, e g fluorescem lsothiocyanate (FITC), rhodamine, Texas Red, phycoerythrm, allophycocyanin, 6-carboxyfluoresce ⁇ n (6-FAM), 2',7'-d ⁇ methoxy-4',5'- d ⁇ chloro-6-carboxyfluorescem (JOE), 6-carboxy-X-rhodamme (ROX), 6-carboxy-2',4',7',4,7- hexachlorofluorescein (HEX), 5-carboxyfluoresce ⁇ n (5-FAM) or N,N,N',N'-tetramethyl-6- carboxyrhodamine (TAMRA), cyanme dyes, e g Cy5, Cy3, BODIPY dyes, e
• FITC fluorescem ls
• any combination of labels e g first and second labels, first, second and third labels, etc , may be employed for the test and control target sets, provided the labels are distinguishable from one another
• distinguishable labels include two or more different emission wavelength fluorescent dyes, like Cy3 and Cy5, or Alexa 542 and Bodipy 630/650, two or more isotopes with different energy of emission, like 32 P and 33 P, labels which generate signals under different treatment conditions, like temperature, pH, treatment by additional chemical agents, etc , and labels which generate signals at different time points after treatment
• Using one or more enzymes for signal generation allows for the use of an even greater variety of distinguishable labels based on different substrate specificity of enzymes (e g alkaline phosphatas e/p eroxidas e)
• the next step in the subject methods is the hybridization of the test and control sets of target nucleic acids to a nucleic acid array(s)
• the nucleic acid arrays employed in the subject hybridization assays have a plurality of nucleic acid spots, and preferably in many embodiments polynucleotide spots, stably associated with a surface of a solid support, where the solid support may be rigid, e g glass, or flexible, e g nylon membrane At least a portion of the nucleic acid spots on the array are made up of probe nucleic acids
• Arrays with which the subject methods find use include nucleic acid biochips, e g cDNA biochips, RNA biochips, polynucleotide biochips, ohgonucleotide biochips, and the like Of particular interest are the arrays described in U S Patent Application Serial No 08/859,998, U S Patent Application Serial No 08/974,298, U S Patent Application Serial No 08/859,998, U S
• test and control sets of target nucleic acids are hybridized to an array, where the sets of target nucleic acids may be hybridized to the same array or different arrays, where when the sets of target nucleic acids are hybridized to different arrays, all of the different arrays will at least share common arrays of probe nucleic acids, l e they will be identical with respect to their probe nucleic acids
• the test and control sets of target nucleic acids are hybridized to the same array
• the array is hybridized with a test set of labeled target nucleic acids and at least one control set of labeled target nucleic acids
• the number of different control sets may range from 2 to 6, usually 2 to 4 and more usually 2 to 3
• 1 or 2 different control sets of target nucleic acids are employed
• the probe and control sets of target nucleic acids may be hybridized to the array and/or detected simultaneously or sequentially
• the two sets of target nucleic acids may be combined prior to hybridization and the array hybridized to both simultaneously to minimize potential variability in hybridization conditions
• a known amount of labeled sets of test target and control target nucleic acids can be added to the same hybridization buffer, and then contacted with one or more arrays simultaneously under hybridization conditions
• a known amount of labeled sets of test target and control target nucleic acids can be added to the same hybridization buffer, and then contacted with one or more arrays simultaneously under hybridization conditions
• test and control target nucleic acids are premixed or pooled prior to contact with the array
• mixtures of test and control target nucleic acids have amounts of control and target nucleic acids which are sufficient to generate signals that are at least 10 fold, usually at least 20 fold and more usually at least 50 fold higher than background signals observed with the array
• the relative amounts of control and test target nucleic acids in the mixture are selected to be sufficient to allow reliable detection of the test sequences complimentary to the probe nucleic acid while at the same time allowing complete binding of the test target nucleic acids with a nofold excess of unbound probe nucleic acid on the array
• the amount of test nucleic acid present m the mixture is usually determined by available amount of sample and sensitivity of technology employed in a particular protocol
• the amount of test nucleic acid present in the mixture ranges from about 0 01 -100 ⁇ g of nucleic acid.
• the amount of control target nucleic acid employed in the hybridization protocol is about the same or less than the amount of test target nucleic acid that is employed, where less than typically means 10 fold less, usually 100 fold less and more usually 1000 fold less Of interest are mixtures of labeled nucleic acids that provide for an intensity of signal from each probe nucleic acid in the control detection channel that ranges from about 0 001 -0 1%, usually from about 0 001 to 0 01%) abundance level
• one or more arrays may be hybridized with the control and test sets of target nucleic acids sequentially
• arrays may be hybridized with a hybridization mix containing the labeled test target nucleic acids to allow these molecules uninhibited access to the probe sequences of the array Following this hybridization, control target nucleic acids could then be exposed to the array for use as an internal control
• the hybridization of the control target nucleic acids may be completely separate from the hybridization of the test target nucleic acids, e g using different hybridization mixes at different times, or the control target sequences may be added to the hybridization buffer containing the test target nucleic acids following an incubation period with the test target nucleic acids The latter is especially appropriate when the test target nucleic acids require a longer hybridization incubation period than the control test nucleic acids
• the control and test target nucleic acids may be differentially labeled or labeled with the same label, since detection occurs separately
• test and control sets of target nucleic acids are hybridized to different arrays, where each of the different arrays has an identical population of probe sequences, l e the different arrays do not vary with respect to their probe sequences
• control and test target nucleic acids may be labeled with the same label so as to be indistinguishable from one another, and discussed above
• the test and control target nucleic acids are hybridized to high throuhput array devices, as described in 5,545,531 and PCT/US99/00248, the disclosures of which are herein incorporate by reference
• hybridization conditions conditions sufficient to promote Watson-Crick hydrogen bonding to occur between the target and probe nucleic acids
• hybridization conditions such as hybridization tme, temperature, wash buffers used, etc can be altered to optimize the efficient and specific binding of the target sequences
• Test target nucleic acids having sequence similarity to the probes may be detected by hybridization under low stringency conditions, for example, at 50°C and 6 ⁇ SSC (0 9 M sodium chlor ⁇ de/0 09 M sodium citrate, 1% SDS) and remain bound when subjected to washing at 55°C m 1 xSSC (0 15 M sodium chlor ⁇ de/0 015 M sodium citrate, 1% SDS)
• Test target sequences with sequence identity may be determined by hybridization under stringent conditions, for example, at 60°C or higher and 6 ⁇ SSC (15 mM sodium chloride/01 5 mM sodium cit
• Levels of hybridization of test target RNA to the probe compositions can be standardized by comparing the hybridization signal of the test with control target sequences on each array Differences in hybridization of the control target sequences allows a comparison of relative hybridization levels between arrays
• non-hybridized labeled nucleic acid is removed from the support surface, conveniently by washing, generating a pattern of hybridized nucleic acid on the substrate surface
• wash solutions and protocols are known to those of skill m the art and may be used See Sambrook, F ⁇ tsch & Mamatis, Molecular Cloning A Laboratory Manual (Cold Spring Harbor Press)(l 989) Detection
• the resultant hybridization patterns of labeled nucleic acids 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 target nucleic acid, where representative detection means include scintillation counting, autoradiography, fluorescence measurement, colorimet ⁇ c measurement, light emission measurement, light scattering and the like
• the hybridization patterns generated by control and test target nucleic acids may be compared to identify differences between the signals Where arrays in which each of the different probes corresponds to a known gene are employed, differences in signal intensity can be related to a different target concentration of a particular gene
• the comparison of the intensity of binding of a test target nucleic acid to a probe sequence can be compared to the intensity of the binding of the corresponding control target sequence, and the measurement converted to a quantitative RNA concentration for that target sample
• the quantitative RNA levels of the test target can be compared between arrays to identify or confirm differential expression of genes in particular samples
• the subject methods find use in, among other applications, standardization of differential gene expression assays
• one may use the subject methods in the differential expression analysis of (a) diseased and normal tissue, e g neoplastic and normal tissue, (b) different tissue or tissue types, (c) developmental stage, (d) response to external or internal stimulus, (e) response to treatment, and the like
• the methods of the subject invention therefore find use in broad scale expression screening for drug discovery, diagnostic and research, such as the effect of a particular active agent on the expression pattern of genes in a particular cell, where such information can be used to reveal drug toxicity, carcmogenicity, etc , environmental monitoring, disease research and the like
• a number of different tasks can be accomplished with the subject invention, which tasks mclude, but are not limited to detecting relative hybridization of target sequences, calibrating a hybridization assay, harmonizing data between hybridization assays, and testing reagents used in a hybridization assay
• the methods of the present invention are useful in detecting relative levels of hybridization of different genes in a sample by providing a set of internal hybridization controls Since the control set of nucleic acids are of a known sequence, m a known quantity, and of a known specific activity (where in a preferred embodiment the control and test target are labeled with the same specific activity), the level of hybridization of the control nucleic acids can be used to determine the level of expression of each gene m a test sample based on its level of binding to a probe sequence The fact that each probe sequence has its own internal control also allows for the detection of potential expression differences between samples and differences in binding affinities between probe sequences, both on a single array and between arrays Thus, the intensity level of hybridization of a control sequence can be used to calculate the expression level of a gene in a sample based upon the intensity of the test target hybridization to the corresponding probe sequence
• the methods of the subject invention also find use in the calibration of hybridization assays Using known concentrations of probe nucleic acid, test target nucleic acids, and control target nucleic acids allows one to optimize the hybridization conditions for a particular use, such as increasing stringency to allow better detection of nucleic acids with some level of sequence homology (e g differential expression between genes from a single family or alternative splice forms for the same gene)
• the use of the internal standards of the method of the subject invention allows hybridization, labeling procedures, and the like to be optimized for a particular use, which is especially valuable for standardization of large scale of hybridization assays, such as high- throughput screening of biological samples Optimization thus means that one can change hybridization conditions in order to achieve maximal intensity of specific hybridization signals with complimentary probe sequences and minimal level of non-specific hybridization with non- complementary probe sequences
• the methods of the subject invention also find use m the harmonization of data between hybridization assays, thus allowing for a direct comparison of expression levels despite potential differences due to variables such as differences in hybridization conditions, differences m sample preparation and even between different types of arrays, differences in quality and performance withm and between different arrays, differences in specific activity of the labeled target sequences, and the like Because each hybridization assay has its internal control for at least a subset of the probe sequences on the array, the data can be compared using ratios of the intensity of the control target nucleic acids and the intensity of the test target nucleic acids Thus, the use of simple mathematical formulations to correct for differences between assays allows the levels of gene expression in these different assays to be adjusted to the same level and then compared in a biologically relevant fashion
• the methods of the present invention are also useful in determining the efficacy of hybridization reagents
• Such reagents may be, for example, new reagents, e g different buffer solutions for prehyb ⁇ dization and hybridization, or established reagents, e g a new batch of a known, commercially available reagent
• the internal control of the methods of the subject invention provide for two levels of quality assurance upon testing the reagents, basically providing an extra control for determining the efficacy of a reagent in a single hybridization Efficiency means maximum specific signal with minimal level of non-specific signal and background binding to solid surface Other parameters such as temperature, buffer composition, length of hybridization and/ washing times, etc , may be optimized using calibration controls
• the same calibration target nucleic acids can be used routinely to test and calibrate detection equipment to expected level intensity of signals, thus limiting variability due to functionality of the equipment, and may be used to test and calibrate the quality of arrays for control procedures
• the subject methods in which control and test target nucleic acids are employed can be used to determine real RNA concentrations in sample This information can m turn be used to compile a gene expression database
• Use of subject methods in building a database has advantages over databases derived from images or signal intensities Such advantages include the generation of more compact information (number/versus image file), the identification of expression levels that are not dependent on type of array, hybridization conditions, lot of array, etc. These advantages are significant m that expression data obtained with the subject methods does not need annotation to be meaningful, and the database generated from the data can be universal, l e it can be generated using data generated in different labs, or at different times, or even using different types of arrays
• the present invention also provides kits for performing the subject array-based hybridization assays
• the subject kits at least include a control set of target nucleic acids, as defined above, or a precursor thereof
• nucleic acid precursor any nucleic acid from which with the control set may be prepared, e g a set of RNAs encoding the nucleic acids of the control set, plasmids containing nucleic acids for generation of the control set, and the like
• Labeled cDNA can be derived from these precursors by enzymatic synthesis, or ohgonucleotides chemically synthesized based on sequence information of these precursors
• the set may contain RNAs that recognizes each probe composition on an array, and such RNAs may be pre-labeled, may be labeled for use with the test target nucleic acids, or may be converted to labeled cDNA for hybridization
• Kits of the present invention may also contain cDNA or o gonucletodies that selectively binds to the
• kits for carrying out differential gene expression analysis assays are preferred Such kits according to the subject invention will at least comprise the subject sets of nucleic acids
• the kits may further comprise one or more arrays corresponding to the set of control target nucleic acids Of particular interest are the arrays disclosed in U S Patent Application Serial No 08/859,998, U S Patent Application Serial No 08/974,298, U S Patent Application Serial No 08/859,998, U S Patent Application Serial No 08/974,298, U S Patent Application Serial No 09/225,998, U S Application Serial No 09/221,480, U S Application Serial No 09/222,432, U S Application Serial No 09/222,436, U S Application Serial No 09/222,437, U S Application Serial No
• kits may further comprise one or more additional reagents employed m the various methods, such as primers for generating target nucleic acids, dNTPs and/or rNTPs, which may be either premixed or separate, one or more uniquely labeled dNTPs and/or rNTPs, such as biotinylated or Cy3 or Cy5 tagged dNTPs, or other post synthesis labeling reagents, such as chemically active derivatives of fluorescent dyes, enzymes such as reverse transcriptases, DNA polymerases, RNA polymerases and the like, various buffer mediums, e g hybridization and washing buffers, prefabricated probe arrays, labeled probe pur
• 236 cDNA fragments corresponding to 236 different human genes were amplified from quick-clone cDNA (CLONTECH) m 236 separate test tubes using a combination of sense and antisense human stress gene-specific primers as described in U S Patent Application Serial No 09/222,256, the disclosure of which is herein incorporated by reference
• Amplification was conducted in a 100- ⁇ l volume containing 2 ⁇ l of mixture of 10 Quick-clone cDNAs from placenta, brain, liver, lung, leukocytes, spleen, skeletal muscle, testis, kidney and ovary (CLONTECH), 40 mM T ⁇ cine-KOH (pH 9 2 at 22 °C), 3 5 mM Mg(OAc) 2 , 10 mM KOAc, 75 ⁇ g/ml BSA, 200 ⁇ M of each dATP, dGTP, dCTP and dTTP, 0 2 ⁇ M of each sense and antisense gene-specific primers and 2
• the above described human array is used to screen a human tissue sample to determine gene expression of each of the 236 genes in a human sample
• Total RNA is isolated from homogenized human tissue using a ATLASPURETM RNA isolation kit and mRNA is isolated from the total RNA using an ohgo-(dT)-cellulose spin column (both from CLONTECH, Palo Alto CA) according to the manufacturer's protocols.
• the mRNA of the tissue sample is used to generate target cDNAs for hybridization to the probe array C Preparation of control target ribonucleic acids
• a control set of ribonucleic acids was synthesized by T7 transcription from cDNA fragments corresponding to the 236 genes on the Human Stress array prepared as described above, where each of the cDNA fragments included a T7 promoter
• the fragments were amplified by PCR in 236 separate tubes as described in section A using a combination of sense and antisense gene specific primers
• Antisense primers carry a T7 promoter for following transcription resulting in RNA fragments mimicking the corresponding mRNA fragment T7 transcription was accomplished m 236 separate tubes in a 200 ⁇ l volume containing 200 ng of the individual cDNA fragment, 80 mM HEPES-KOH (pH 7 5 at 22 °C), 15 mM MgCl 2 , 2 mM spermid e, 10 mM DTT, 3 mM each ATP, GTP, CTP, UTP, and 200 units of T7 RNA polymerase (Epicentre Tech , Madison, WI) Incubation of reaction mixture was
• RNA fragment sample was determined on a spectrophotometer by UV absorption with 1 optical density unit corresponding to 40 ⁇ g of RNA
• the adjusted equal molar weight concentration for each fragment was then calculated A length of 2200 nucleotides (which is equal to the average length of mRNA) was used as a standard For example, if the real concentration for a particular fragment with a size of 200 nucleotides was lng/ ⁇ l, its adjusted equal molar weight concentration was 11 ng/ ⁇ l
• all 236 synthetic RNAs were mixed in equal molar ratio and this mixture of synthetic RNAs was used as a standard, as described below
• RNA dissolved in water at 0 5-1 ⁇ g/ ⁇ l was mixed to 1 ⁇ l of l OxCDS primer mix (0 2 mM each, comprising mixture of antisense primers complementary to 236 rnRNAs related to the Stress/toxicology array)
• the sample m the tube was then mixed by vortexmg and spun down by microcentrifuge
• the tubes were then incubated in a prewarmed PCR thermal cycler at 70 °C for 2 mm
• the temperature was then reduced to 50 °C and incubation was continued for another 2 mm
• the remaining reagents were then added and incubation was continued for 20 mm
• the final reaction conditions were 50 mM T ⁇ s-HCl pH 8 3, 75 mM KC1, 3 mM MgCl 2 , 0 5 mM each of dGTP, dCTP, dATP, 0 1 mM of dTTP and 0 1 mM of allylamine-
• control target nucleic acids and the test target nucleic acids are labeled using differentially detectable fluorescent labels
• For labeling of the test target nucleic acids following conversion of the target ribonucleic acids to ammo-modified cDNAs, 1 mg of Cy3 succimmide ester is dissolved in 10 ⁇ l of dimethyl sulfoxide, and 10 ⁇ l of the test target cDNAs are added to it
• For labeling of the control target set 1 mg of Cy5 succimmide ester is dissolved in 10 l of dimethyl sulfoxide and 10 ⁇ l of control target cDNAs are added to it Both mixtures are incubated at room temperature overnight
• Each labeled set of target nucleic acids is purified separately by column chromatography using a CHROMA SPLN-200 column (CLONTECH, Palo Alto.CA) according to the manufacturer's instructions See also U S Patent Application Serial No 08/859,998, the disclosure of which is herein incorporated by reference
• control target and test target cDNAs are then pooled for hybridization to the array
• 1 ⁇ g of the Cy3 labeled test target cDNA and 1 ⁇ g of the Cy5 labeled control target prepared above are combined
• a solution of ExpressHybTM (CLONTECH) and sheared salmon testes DNA (Sigma) is prepared as follows 5 ml of ExpressHybTM is prewarmed at 50-60°C 0 5 mg of the sheared salmon testes DNA is heated at 95-100 °C for 5 mm, and then chilled quickly on ice Heat- denatured sheared salmon testes DNA is mixed with prewarmed ExpressHybTM
• the human cDNA array is placed in a hybridization container, and 1 ml of the ExpressHybTM/salmon DNA solution is added Prehyb ⁇ dization is conducted for 5 min with continuous agitation at 60 °C
• Labeled cDNA test and control target nucleic acids, as prepared above, (about 200 ⁇ l) are mixed with 2 ⁇ l ( 1 ⁇ g/ ⁇ l) of human Cot- 1 DNA , and denaturated at 99 °C for 2 min
• the mixture is added to the hybridization solution and mixed together thoroughly
• the container is sealed by
• a method to determine differentially expressed genes in normal and cancerous cells, or treated v untreated cells is conducted as follows
• test target nucleic acids Two separate sets of test target nucleic acids are produced, one set isolated from a cancerous human tissue, and the second set isolated from the corresponding physiologically normal human tissue Each tissue sample is tested for differential expression by isolating mRNA from each sample and subsequently transcribing the mRNA into labeled cDNA Each labeled pool of cDNA is then used as test target nucleic acids for hybridization against the probe sequences of one of the two arrays, and the levels of expression between the two arrays is compared to determine relative differences in expression between the normal and the cancerous tissues Following preparation, both the set of control target nucleic acids and the two sets of test target nucleic acids, corresponding to the normal and the cancerous tissue, are labeled using detectable fluorescent labels Each set of test target nucleic acids is labeled in a separate reaction with Cy3 For each labeling procedure, an aliquot of test cDNAs is added to a 0 5 ml Eppendorf tubes containing 1 mg of Cy3 succimmide ester
• Each hybridization mixture is prepared by mixing about 200 ⁇ l of one set of test target cDNAs synthesized from 1 ⁇ g of polyA RNA, 100 ⁇ l of the set of control target cDNAs synthesized from l ⁇ g of control RNA mix containing 0 01%) of each synthetic control RNA for each probe on the array (the size of each synthetic RNA is adjusted to average size of mRNA, 1 e , 2 2 kb mixed together and then equal molar weight % was adjusted to 0 01%), and 2 ⁇ l ( 1 ⁇ g/ ⁇ l) of human Cot-I DNA
• the mixture is denatured at 99 °C for 2 min prior to hybridization
• the hybridization mixture containing the test nucleic acids from the cancerous tissue is added to the prehybndization solution of the first array, and the hybridization mixture containing the test nucleic acids from the normal tissue is added to the prehybndization solution of the second array
• Each array is also contacted with the labeled control set of target
• RNA corresponding to the Human Stress array was synthesized for 236 fragments arrayed on membrane or glass RNA fragments were mixed in equal molar ratio and this mixture was used for further experiments cDNA from a control mixture of RNA was labeled with Cy5 fluorescent dye as described earlier cDNA from placenta poly(A)+ mRNA or liver poly(A)+ mRNA was labeled with Cy3 dye Hybridization was performed on the glass slides The first probe contained labeled control cDNA Hybridization of this probe to a glass Human Stress array was done at different concentrations relative to amount of poly(A)+RNA Hybridization was to 5 different glass slides at concentrations of 0 01%, 0 04%, 0 1%, 0 4%, and 1%, corresponding to 0 1 ng, 0 4 ng, 1 ng, 4 ng, 10 ng of adjusted weight amount of individual RNA species in the mixture At the same time, 0 1 ng of each individual control Cy5 labeled cDNA (cor
• the intensity of the signal in the spot increased proportionally (and linearly) with an increasing amount of RNA
• the linear change in intensity of the signal from synthetic RNA in each spot allowed the direct comparison of the abundance of an mRNA of interest on two arrays with liver and placenta poly(A)+ RNA samples using the intensity of the Cy5 channel as a normalization value
• control RNA mix may be used alone or added to poly(A)+ RNA before labeling
• Design of this experiment was similar to the glass experiment descnbed above Five different concentrations of control RNA were used (from 0 01% to 1%) Labeling of these probes was done separately and after the mixing to 1 ⁇ g poly(A)+ RNA

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