EP1248853A2 - Technologie des reseaux liquides - Google Patents

Technologie des reseaux liquides

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Publication number
EP1248853A2
EP1248853A2 EP00955728A EP00955728A EP1248853A2 EP 1248853 A2 EP1248853 A2 EP 1248853A2 EP 00955728 A EP00955728 A EP 00955728A EP 00955728 A EP00955728 A EP 00955728A EP 1248853 A2 EP1248853 A2 EP 1248853A2
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
array
sequence
probes
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00955728A
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German (de)
English (en)
Inventor
Mark B. Chandler
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Luminex Corp
Original Assignee
Luminex Corp
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Filing date
Publication date
Application filed by Luminex Corp filed Critical Luminex Corp
Publication of EP1248853A2 publication Critical patent/EP1248853A2/fr
Withdrawn legal-status Critical Current

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    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
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    • 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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
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    • 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
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00281—Individual reactor vessels
    • B01J2219/00283—Reactor vessels with top opening
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    • B01J2219/00277—Apparatus
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    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315—Microtiter plates
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    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/005—Beads
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    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00686—Automatic
    • B01J2219/00689—Automatic using computers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00729—Peptide nucleic acids [PNA]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/11—Compounds covalently bound to a solid support
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
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    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • This invention relates to compositions and methods of manipulating, classifying and obtaining information (including sequence infonnation) on large numbers of nucleic acid molecules.
  • the invention utilizes a plurality of oligonucleotide probes bound to addressable multicolored microparticles that are suspended in a fluid (e.g., a liquid array, suspension array, or gaseous array) and which thus provide at least a three-dimensional approach to carrying out the above-mentioned processes.
  • a fluid e.g., a liquid array, suspension array, or gaseous array
  • the present invention represents a radical departure from conventional approaches using "gene chip " ' technologies, which suffer from the inherent limitations imposed by their two- dimensional confinement.
  • oligonucleotide microarrays There are three basic types of DNA or gene chips (oligonucleotide microarrays). The first and oldest is the sequencing chip. This technology is based on sequencing by hybridization (SBH). A fairly straightforward methodology, it has gained increased utility through the power of computers. For example, an octamer probe constructed of the four basic nucleotides, A, C, T, or G, combined at random (4 8 ) produces 65,536 possible sequences. By having all possible 65,536 features or probe variants present on a chip and contacting a fragment of deoxyribonucleic acid (DNA) of unknown sequence to the chip, one should obtain useful sequence information about the DNA of interest.
  • DNA deoxyribonucleic acid
  • sequencing chips such as those initially introduced by Affymetrix and Hyseq, segments of DNA (usually 20 bases or nucleotides long) are placed in a microarray on the surface of a slide. Target samples are then introduced to the chip, and the particular segment(s) that the sample hybridizes with determine(s) the result.
  • SBH For additional discussion on the topic of SBH, see, for example, WO 98/31836 and WO 99/09217, the disclosures of which are incorporated by reference herein. Many other companies are now producing sequencing chips, most using an approach similar to SBH. But whatever their technique, such products are intended to determine the DNA sequence of the sample.
  • the second variety of DNA chips is known as the expression chip. These are designed to determine the degree of expression of a certain genetic sequence by measuring the rate or amount of messenger ribonucleic acid (mRNA) being produced by the target gene or by measuring complementary DNA (cDNA) corresponding to mRNA. This is done by creating chips with specific sets of base sequences (as opposed to sequencing chips, wherein every possible base sequence is arrayed). Results are then compared to a reference or control, and the degree of change is noted. These chips are useful in diagnosing and treating diseases linked to particular genetic expressions, such as some forms of cancer. Vysis and Synteni are examples of two companies engaged in marketing expression-chip-based products and services.
  • the third type of chip is based on comparative or quantitative genomic hybridization. It is designed to help diagnosticians determine the relative amount of a given genetic sequence in a particular sample. A certain amount of unusual genetic expression is normal, but for example in malignant cells the level of expression may be beyond a normal level. Many breast tumors, particularly at the end stages of the disease, are so aberrant genetically that they do not even exhibit the usual number of 46 chromosomes per human cell. (It is known that the 46 human chromosomes house 3 billion base pairs of DNA and encode about 60,000 to 100,000 proteins.) These coding regions on chromosomes make up only about 2% of the genome. The function of the remaining 98% is unknown, and some chromosomes have a higher density of genes than others.
  • This type of chip is designed to look at the level of aberration. This is usually done by using a healthy tissue sample as a reference and comparing it with a sample from the suspected tumor.
  • Prototype genotyping chips have been originally developed by Affymetrix (Santa Clara, CA, USA), which were used by Wang et al. (Wang DG, Fan JB, Siao CJ, Berno A, Young P, Sapolsky R, Ghandour G, Perkins N, Winchester E, Spencer J, Kruglyak L, Stein L, Hsie L, Topaloglou T, Hubbell E, Robinson E, Mittmann M, Morris MS, Shen N, Kilburn D, Rioux J, Nusbaum C, Rozen S, Hudson TJ, Lander ES, et al., Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome.
  • SNPs single nucleotide polymorphisms
  • Nanogen has developed a semiconductor microchip array that utilizes electrical currents to improve the speed and specificity of binding of nucleotide-specific probes to DNA samples and then assays the bound probes using a fluorescent imaging detector (see, U. S. Pat. No. 5,849,486 to Heller, et al., incorporated by reference herein). This technology allegedly can also perform multiple assays using a variety of different probes simultaneously on a single DNA sample.
  • Orchid Biocomputer and the University of Washington (Seattle) School of Medicine are establishing a new high-throughput SNP genotyping facility.
  • the center will utilize a proprietary automated genotyping system developed by Orchid, which will enable researchers to sort up to 30,000 SNP genotypes per day for the eventual pharmacogenetic analysis of clinical samples (see, PCT patent publications WO 99/27137 and WO 99/10538 assigned to Orchid, both incorporated by reference herein).
  • a microsphere based multiplexed DNA assay which allows simultaneous analysis of up to 64 unique DNA sequences, is known (see, U. S. Pat. No. 5,736,330 issued to Fulton on April 7, 1998).
  • oligonucleotide probes complementary to DNA or RNA molecules of interest are coupled to fluorescently labeled microspheres.
  • the probe is allowed to hybridize to analyte nucleic acid in the presence of a competitor molecule, and the amount of the analyte is determined, for example, from the known amount of competitor.
  • By combining just two dyes at eight different fluorescence levels (2 ) one obtains 64 distinct microspheres sets.
  • one unique microsphere set may contain orange and red dyes at a ratio of 85%: 15%, respectively, while another unique set may contain a 75%:25% ratio of the same orange and red dyes.
  • a separate and distinct variable the level of fluorescence intensity emitted by a given microsphere set, is provided.
  • an assay is now provided which allows the resolution of up to 1,000,000 or more unique sets of particles (e.g., microspheres), thus permitting the substantially simultaneous detection of a corresponding number of probes bound thereto.
  • This accomplishment is due in large part to the discovery of methods that allow for the introduction and use of additional dyes, which can be combined at ten or more distinguishable dye ratios giving rise to ten or more unique levels of fluorescence intensity.
  • the present invention provides for the use of at least six different dyes. The amount of each dye (either singly or in combination with one or more of the other dyes), which is used to stain a specific particle, can be manipulated to provide at least 10 levels of fluorescence intensity.
  • the number of unique sets of particles made possible by such combinations is about 6 10 or about 1,000,000.
  • the flexibility and versatility of the resulting "fluid array" system allows, in the same system, the pursuit of at least all three possible applications of conventional gene chip technology: That is, sequencing (SBH), detection of expression, and quantification of target nucleic acid.
  • the present invention provides superior, novel technology for multiplexed analyses of nucleic acid samples, which technology can be used by virtually any lab using an apparatus no more costly that standard laboratory equipment.
  • the present invention is able to provide an extremely large array of unique probes.
  • the instant invention is particularly well suited to a multiplexing analysis format and is easily adapted to automated procedures. In short, when using the present fluid arrays, the complexity, expense and limitations associated with prior gene chip manufacturing and/or testing procedures are avoided.
  • a collection comprising multiple subpopulations of particles.
  • the particles in each subpopulation have one or more characteristics that distinguish the particles of one subpopulation from those of another subpopulation, in which the collection is further characterized as having preferably about 10 3 or more distinct subpopulations of particles. In another preferred embodiment, 10 4 or more distinct subpopulations of particles are used. More preferably, the collection is further characterized as having about 10 5 or more distinct subpopulations of particles. An even more preferred collection is one which is further characterized as having about 10 6 or more distinct subpopulations of particles.
  • This collection further comprises bound nucleic acid.
  • this bound nucleic acid includes a predetermined polynucleotide sequence. Alternatively, this bound nucleic acid includes an ascertainable polynucleotide sequence.
  • This invention relates to a fluid array which preferably comprises a collection of subpopulations of particles.
  • the particles in each subpopulation preferably have one or more characteristics that distinguish the particles of one subpopulation from those of another subpopulation and are further characterized by a bound nucleic acid.
  • a collection is further characterized by having about 10 or more distinct subpopulations of particles and a fluid carrier.
  • the preferred fluid carrier is a liquid or gas in which the collection of the present invention is substantially co-mingled with one another, or, if desired, is substantially segregated from one another.
  • the preferred one or more characteristics include a distinctive fluorescence emission signature.
  • the preferred bound nucleic acid that is bound to the particles of one subpopulation differs from that bound to the particles of another subpopulation.
  • bound nucleic acid comprises DNA or RNA.
  • the preferred bound nucleic acid comprises 9 or more nucleotide residues. Preferably, 18 or more nucleotide residues are present. Even more preferably 36 or more nucleotide residues are present.
  • composition of the invention comprises a solid particle that has bound to it nucleic acid with a known polynucleotide sequence, a label comprising a dye that exhibits a distinctive fluorescent emission signature, and a substance that, in the absence of an analyte of interest comprising a polynucleotide sequence substantially complementary to said known polynucleotide sequence, can quench the fluorescence emission of the dye.
  • a device for identifying an analyte of interest, e.g., specific sequence of nucleic acid in a DNA or RNA fragment, among a plurality of unrelated sequences in a sample.
  • This device comprises a fluorescent microparticle having on its surface at least one bound probe of known sequence. Up to one million fluorescently distinct or addressable microparticle sets are contemplated as carrier solid phase substrates for at least the same number of different probes.
  • Non-limiting examples of material for microparticles suitable for use in the present invention include nitrocellulose, nylon, agar, glass, silica, plastic polymer, magnetic beadlets, and the like. Latex or polystyrene microspheres area preferred microparticle. The preferred diameter of microparticles is between about 1 nanometer and about 1,000 microns - a size that distinguishes them from larger size beads employed in the prior art for other applications.
  • the oligonucleotide probe is constructed to match the desired sequence of the target of interest.
  • a probe is built randomly by selecting any of four bases A, T, C, or G and stacking to these first bases any nucleotide, randomly selected from A, T, C, or G, and carrying the synthesis until the desired probe length is obtained.
  • probes belong to DNA or RNA type molecules (RNA probes will contain as a base uracil or U instead of T), alternatively probes are made of so-called peptide or polyamide nucleic acid (PNA) probes.
  • the probe is synthesized according to standard oligonucleotide synthesis methods using as a solid phase substrate the surface of the microsphere.
  • a probe is synthesized separately and then coupled to the carrier microparticle of choice.
  • the oligonucleotide arrays are fabricated either by in situ combinatorial-type synthesis followed by automated sorting of fluorescent carrier particles or by conventional synthesis followed by immobilization of the probe on pre-selected particle sets having unique fluorescence signature.
  • a liquid array of up to one million nucleic acid probes is obtained wherein each of the probes is bound to a microparticle stained with at least two fluorescent dyes.
  • Preferably more than one pair of dyes is incorporated into a particle and dyes used in each pair are preferably admixed at variable ratios.
  • the preferred number of dyes is between two and six and preferred number of dyes ratio is up to eight or higher.
  • a probe is labeled with a fluorescent reporter dye, which upon binding of the analyte of interest undergoes through a change in fluorescence attributes indicating the presence of the analyte in a sample and said analyte is identified according to the fluorescent signature of the microparticle.
  • the operating principle of the fluorescent reporter dye is analogous to one used in TaqMan assay. Other principles of reporting systems are equally suitable and are selected from any of existing methods disclosed in published resources.
  • the target of interest is labeled with a fluorescent tag so that only those that hybridize to a probe are identified.
  • only hybrids are tagged by polymerase extension so that complementary duplexes but not single strand sequences are readily identified.
  • Another way to determine the specificity of hybridization is by providing a competitor molecule that has affinity to the probe.
  • probes used in this invention are not restricted by their length, but preferably they are anywhere between 3 and 120 nucleotides long. More preferably, they are between 6 and 50 bases long. Even more preferably they are between 8 and 25 bases long.
  • methods of the invention comprise hybridizing to a target nucleic acid a probe having desired length and sequence, wherein the probe hybridizes at various locations, e.g., upstream, within, or downstream of a target nucleic acid.
  • Another embodiment of the invention is to exploit hybridization process further whereby perfectly hybridized probe will serve as primer for enzymatic extension by polymerase-like enzymes and thus this process is useful for detecting primer-template mismatches, e.g., single nucleotide polymorphism (SNP).
  • SNP single nucleotide polymorphism
  • probes of this invention are used as mixed probes comprising various length oligomers between about 5 and about 120 nucleotides.
  • the mixture of probes takes advantage of the high selectivity of a short probe and the hybridization stability imparted by a longer probe.
  • N A/C/G/T.
  • the present invention allows up to one million probes to be present simultaneously in the reaction vessel.
  • An oligonucleotide probe of the invention is hybridized with one or more nucleic acid samples so that complementary complexes are formed. Accordingly, such complexes are analyzed to determine whether the sequence of interest in the complementary complexes match. Complementary sequences are identified by referring to the fluorescent signature associated with each set of microparticles carrying the probe of known sequence. This application is suitable for the SBH-type approach, especially when contiguous probes having predetermined overlapping sequences are used and degree of complementarily or mismatch among overlapping probes reveals the identity of the analyte.
  • a liquid array which comprises a mixture of sets of fluorescently addressable microspheres in a flowable liquid.
  • each set has a distinct fluorescent signature and each set is conjugated with a different oligonucleotide probe, whereby detection of a fluorescent signature identifies the oligonucleotide probe.
  • This array has no more than 1,000,000 probes of about 9 to 20 nucleotides in length.
  • This array also comprises at least four groups of microspheres, wherein a first group is exactly complementary to a reference sequence and comprises probes that completely span the reference sequence and, relative to the reference sequence, overlap one another in sequence, and wherein three additional groups of microspheres, each of which is identical to said first group but at least one different nucleotide, which different nucleotide is located in the same position in each of the three additional sets but which is a different nucleotide in each set.
  • this invention provides means of differential expression of a gene of interest by measuring the levels of mRNA or complementary DNA (or cDNA).
  • the preferred means of screening are those that are automated including but not limited to flow cytometry, laser scanning microscopy, fluorescence plate reader, and other standard methods known in the art.
  • a method of detecting at least one mutation in a gene or a set of related genes linked to a clinical condition is provided.
  • these . clinical conditions are associated with genetic aberrations.
  • These aberrations are selected from any mutation types or gene rearrangements, i.e., point mutation, substitution, insertion, deletion, inversion, repetition, amplification, translocation, and transposition.
  • Mutations are either of single nucleotide polymorphism (SNP) type or they involve multiple loci. Large-scale mutations involve chromosome aberrations; smaller mutations involve about
  • 10-50 kbp and even smaller involve point mutations.
  • these mutations are such that they are readily associated with or predisposed to hereditary diseases, neural diseases, muscle and bone diseases, malignant diseases, metabolic diseases, immune diseases, and infectious diseases.
  • liquid array assays which will replace conventional chemical, histological, microbiological, serological and antibody testing methods.
  • Gene based methods of the instant invention are better for: rapid diagnosis of infectious disease; testing latent viral infections; screening of hereditary genetic disease and risk assessment; identification of new therapeutic agents; detection of mutagenic changes to measure genotoxicity of biochemicals; detection of sexually transmitted diseases (STDs) such as gonorrhea, chlamydia, mycobacterium; industry application such as detection of food contaminants, water and sewage testing; identification of genetically transmitted disease; prenatal determination of fetal sex; risk assessment for developing genetically transmitted disease, such as fragile X syndrome, Alzheimer's, Huntington's disease, Gaucher's disease, Marfan syndrome, myotonic dystrophy, diabetes mellitus subtypes, and the like; DNA fingerprinting; forensic testing; paternity/familial testing; cancer diagnosis; agricultural applications; anthropological applications; among many others.
  • Genomic nucleic acid samples are isolated from a biological sample. Once isolated, the nucleic acids are employed in the present invention without further manipulation. Alternatively, one or more specific regions present in the nucleic acids are amplified.
  • Preferred target amplification techniques include but are not limited to PCR amplification; nucleic acid sequence-based amplification; (NASBA); strand-displacement amplification (SDA); transcription-mediated amplification (TMA); Q-beta replicase; and ligase chain reaction (LCR). Other means of amplification known in the art, e.g., cloning, are equally suitable.
  • An amplification step is not a prerequisite, although it provides the advantage of increasing the concentration of specific nucleic acid sequences within the target nucleic acid sequence population.
  • a preferred microparticle is any micron or submicron particle with size range between about 1 nm up to about 50 ⁇ m.
  • economics often dictate the limited use of reactants.
  • the nucleic acid probes are bound to discrete solid-phase supports, i.e., microparticles, suspended as an array in liquid-containing vessels or wells of microtiter plates.
  • an array of nucleic acid probes is formed wherein each distinct type of a probe is bound to a fluorescently addressable set of microparticles positioned in pre-determined wells of a microtiter plate.
  • This array comprises a plurality of fluorescently addressable microparticles, each stained with at least two fluorescent dyes and is spatially arrayed in a two-dimensional pattern over a plane of a microtiter plate. The identification by location allows the simultaneous processing and screening of a large number of samples.
  • the microtiter plate has a multiplicity of wells. Depending on the nature of inquiry the number of wells varies, typically 96 to 2,034 wells per plate are suitable. The use of such commercially available plates allows the simultaneous determination of a large number of samples and controls, and thus facilitates the analysis. Moreover, standard automated systems are available to dispense and manipulate reagents in such microtiter plates. "Plates" are also contemplated which are built specifically for this purpose and are not necessarily similar in their physical appearance to conventional multi-well plates. Considering the submicron scale of fluorescently addressable microparticles, such plates are preferably miniaturized and may fit a surface area that is orders of magnitude smaller than conventional 96- well plate.
  • the preferred minimum contemplated size for a well is one that is sufficient to accommodate a single microparticle, meaning that the total surface area sufficient to accommodate an array consisting of one million microparticles can be smaller than 1mm x 1mm or a surface area smaller than, for example, occupied by letter "o.”
  • Another object of the present invention is to provide a method to screen molecules that bind to nucleic acids representing targets for pharmaceutical intervention.
  • These molecules have various types of biological activities, including, but not limited to, hormonal, neurotransmitter, metabolic, genetic, pharmacologic, immunologic, pathologic, toxic, and anti-mitotic activities.
  • these molecules e.g., peptides, peptoids, other small organic and inorganic molecules, will be used to design compounds such as drugs, hormones, neurotransmitters, agonists and antagonists more efficiently and economically.
  • Fig. 1 illustrates segmental or combinatorial synthesis and fluorescence sorting of oligonucleotide probes immobilized on uniquely (preferably, fluorescently) addressable multicolored microparticles. 5. DETAILED DESCRIPTION OF THE INVENTION
  • the inventors have developed an improved process to perform hybridization, enzymatic manipulation, and classification of nucleic acid molecules.
  • the technique utilizes oligonucleotide probes bound to fluorescently addressable microspheres. The identity of the probe is determined by the fluorescent signature of the microparticle and by two-dimensional position of the well in a microtiter plate in which discrete sets of such microparticles are suspended in a hybridization liquid (liquid arrays).
  • the present invention provides arrays of nucleic acid probes immobilized on fluorescent microparticles, methods of making such arrays, and high-throughput screening methods for detecting, measuring, and identifying genetic characteristics of a biological sample.
  • the combination of the above-identified innovations obviates the need for so-called gene or DNA chips. As such, this new technique is a radical departure from conventional gene chip approaches.
  • Complementary refers to two nucleic acid strands that exhibit substantial normal base pairing characteristics. Complementary strands may contain one or more mismatches, however.
  • hybridization refers to the hydrogen bonding-mediated interaction that occurs between two complementary oligonucleotide strands.
  • multiplex analysis refers to the simultaneous assay of pooled DNA or RNA samples according to the disclosed methods.
  • mismatch means that a nucleotide in one strand of DNA or RNA does not or cannot pair through Watson-Crick base pairing and stacking interactions with a nucleotide in an opposing complementary DNA or RNA strand.
  • adenine in one strand of DNA or RNA would form a mismatch with adenine in an opposing complementary DNA or RNA strand.
  • Mismatches also occur where a first nucleotide cannot pair with a second nucleotide in an opposing complementary DNA or RNA strands because the second nucleotide is absent (i.e., deleted).
  • Flow cytometry also known as Fluorescence Activated Cell Sorting-FACS
  • Fluorescence Activated Cell Sorting-FACS Fluorescence Activated Cell Sorting-FACS
  • the target cells are tagged with fluorophores either directly or indirectly by coupling via fluorescently labeled antibodies or ligands.
  • the cells are then pumped in an aqueous stream where vibrational forces create a stream of droplets containing single cells.
  • lasers excite the fluorophores or fluorescent dyes and the resulting emission is measured in real-time.
  • Advanced flow cytometers also contain electrically coupled deflectors that deflect or gate cells with undesirable fluorescence parameters, thereby permitting the operator to physically separate cells within the population based on fluorescence spectrum and/or intensity.
  • Sets of fluorescently addressable microspheres are first mixed together.
  • a computer file assigning a unique oligonucleotide sequence to each encoded microsphere is created to control flow sorting.
  • the starting mixture of microparticles is then sorted to 4 outputs of the flow cytometer depending upon which base (A, C, G, or T) is attached first to the surface of the microparticle (various methods of attaching nucleic acid to solid base support are known, e.g., carbodiimide coupling, and details for alternative methods can be found for example in U. S. Pat. Nos. 5,919,626, 5,610,287, and 5,837,860, which are incorporated herein by way of reference).
  • microspheres that require deoxyadenosine (A) are sorted to output 1 and all those requiring C are sorted to output 2, while G and T are sorted to outputs 3 and 4 respectively.
  • the microspheres are then transferred to standard nucleic acid synthesis machine or automated DNA synthesizer, such as an ABI 392, and appropriate coupling of A, C, G, or T is performed on each of microsphere mixtures.
  • standard nucleic acid synthesis machine or automated DNA synthesizer such as an ABI 392
  • the methods of synthesis are standard methods well known in the art, e.g., U. S. Pat. No. 5,869,644; Southern EM, Case-Green SC, Elder JK, Johnson M, Mir KU, Wang L, Williams JC.
  • Arrays of complementary oligonucleotides for analyzing the hybridization behavior of nucleic acids are standard methods well known in the art, e.g., U. S. Pat. No. 5,869,644; Southern EM, Case
  • Another means of obtaining an array is to synthesize in advance the oligonucleotides on an automated DNA synthesizer and then attach the oligonucleotides onto the solid phase (Lamture JB, Beattie KL, Burke BE, Eggers MD, Ehrlich DJ, Fowler R, Hollis MA, Kosicki BB, Reich RK, Smith SR, et al. Direct detection of nucleic acid hybridization on the surface of a charge coupled device. Nucleic Acids Res 1994 Jun 11;22(1 1):2121-5 and Guo Z, Guilfoyle RA, Thiel AJ, Wang R, Smith LM.
  • the method allows massive parallel synthesis to efficiently produce high diversity of oligomers, i.e., oligonucleotide library.
  • oligonucleotide library i.e., oligonucleotide library.
  • only four parallel nucleotide reactions they are run simultaneously in the same DNA synthesizer
  • each addition or stacking step one for each of A, C, G, and T
  • the total for an array or a library consisting of 65,536 discrete sets of probes associated with an equal number of sets of microparticles are produced in just 8 reactions.
  • each probe is made individually, a total of 4 separate addition steps would be required.
  • this invention overcomes the complexity of the prior art methods for constructing an oligonucleotide library.
  • Libraries resulting from above-described synthesis approach are characterized by the phrase "one microsphere, one probe.”
  • Each microparticle in the library holds at least one and preferably multiple copies of a single library member.
  • Particle-based approach greatly simplifies the isolation and identification of analyte molecules because beads are large enough to be observed by automated means and sorted mechanically.
  • Genomic regions suspected to contain one or more mutations are identified by reference to a nucleotide database, such as GENBANK, EMBL, or any other appropriate database or publication disclosing such mutation. If the genomic code is unknown and unavailable in these databases then it is deduced by standard sequencing methods such as disclosed in U. S. Pat. No. 4,962,020, which incorporated herein by way of reference.
  • GENBANK is a computerized database of nucleotide and amino acid sequences that are constantly revised and updated on a daily basis.
  • GENBANK itself originated as genetic sequence database from the National Institutes of Health (NIH), it is not a single entity. Indeed, it is an annotated collection of all publicly available DNA sequences.
  • GENBANK comprises, for example, the International Nucleotide Sequence Database Collaboration, which in turn comprises the DNA DataBank of Japan (DDBJ), the European Molecular Biology Laboratory (EMBL), and GenBank at NCBI.
  • DDBJ DNA DataBank of Japan
  • EMBL European Molecular Biology Laboratory
  • GenBank GenBank at NCBI.
  • Protein sequences in the GENBANK are from Swiss-Prot, PIR, PRF, PDB, as well as translated protein sequences from the DNA sequence databases. Additional information is provided by National Library of Medicine through MEDLINE and by the American Type Culture Collection (www.atcc.org). In the preferred embodiment of this invention the content of these databases and updates thereof are incorporated by reference in their entirety.
  • this data is downloaded from Internet to a computer attached to a flow cytometer and desired probe sequences are constructed by using specially designed program according to the combinatorial synthesis strategy described above.
  • a complete set of 4 N nucleotides of length N is immobilized as an ordered array on a solid support and an unknown DNA sequence is hybridized to this array.
  • the resulting hybridization pattern provides all n-tuple "words" in the sequence.
  • this method has been applied successfully to determination of short sequences, but tends to lead to sequence ambiguities for longer sequences, e.g., 100 Kb or larger.
  • One reason for the sequence ambiguity is duplicated short sequences in the analyte, which can lead to unresolved sequence determination.
  • this problem can be solved by generating longer-sequence oligo libraries, this solution requires a much larger and more complex permutation libraries. It also increases the likelihood of single-mismatch hybridization, since longer stretches of duplex will accommodate more base pair mismatching, even under high stringency conditions, especially in duplex regions with high GC content.
  • a first step is to create a plurality of sets of microspheres with probes or fragments of DNA, wherein fragments are known in advance.
  • the sequence of one fragment or probe may overlap with the fragment of another set of probes.
  • the overlapping of hybridized probe and analyte in a contiguous manner enables one to reconstitute the DNA sequence of interest. Therefore, invented method also provides an efficient means of sequencing of DNA by providing contiguous overlapping probes.
  • the process of sequentially manipulating molecules involves the use of miniaturized reaction vessels arranged as microtiter plates. Fragments having an average length of about 250 bases are generated by cutting target DNA, with specific restriction endonucleases. These fragments are directly sequenced by the use of contiguous stacking hybridization on a sequencing array.
  • element of an array must contain oligonucleotide strings that are unique for specific fragments.
  • the longer the oligonucleotide string and the shorter the fragmented DNA the higher the probability that a sequence complementary to the oligonucleotide string will be unique for only one of the fragments. Concurrently, the probability that the oligonucleotide string will hybridize at all with any fragment present in the mixture, will be lowered.
  • the shorter the length of immobilized oligonucleotide strings the higher the hybridization sensitivity to single-base-pair mismatches; however, the stability of the formed duplexes decrease.
  • single-stranded nucleic acids form relatively stable hairpins and tertiary structures that interfere with their hybridization with shorter oligonucleotide immobilized fractions.
  • the invented method is also appropriate for drug screening or to construct a protein assay.
  • an array of monoclonal antibodies, heavy and light chains from a spleen library or from polyvalent sera is a suitable source. Then each of these antibodies is immobilized in separate elements of an array. The array is then subjected to an antigen, which is tagged. Those elements that "light up” would serve as starting points for building antibodies specific for that antigen. Further, depending on the size of the array, such arrays of microspheres will serve as a universal antibody diagnostic device allowing for thousands of assays to occur simultaneously via protein affinity processes. Drug screening approach is based, for example, on affinity binding of tested molecules to an array of nucleic acid targets representing a sequence of a gene responsible for a given disease.
  • DNA-binding molecules are for example estrogen receptor, androgen receptor, thyroid hormone receptor, glucocorticoid receptor, vitamin D receptor, human vascular endothelial growth factor (VEGF), human chorionic gonadotropin (ACG) and human thyroid stimulating hormone (hTSH).
  • VEGF vascular endothelial growth factor
  • ACG human chorionic gonadotropin
  • hTSH human thyroid stimulating hormone
  • DNA binding molecules are also contemplated including but not limited to NF-kB binding unit, an SP1 binding unit, a TATA binding unit, a human papillomavirus (HPV) E2 binding unit, an HPV LTR binding unit, and human immunodeficiency virus (HTV) LTR binding unit.
  • HPV human papillomavirus
  • HPV LTR HPV LTR binding unit
  • HTV human immunodeficiency virus
  • the likelihood of each identification associated with a set of hybridization values is computed by comparing an unknown set of probes to a set of example cases for which the correct base identification was known.
  • Methods of the invention permit the detection of a mutation at a locus in which there is more than one nucleotide to be interrogated. Moreover, methods of the invention allow one to screen a locus in which more than one single base mutation is possible. Once regions of interest are identified, at least one probe is prepared to detect the presence of a suspected mutation. This probe is then arrayed with other probes having affinity to other identified mutations so that an array specific to a disease or set of diseases becomes available. Appropriate control probes are included as well, e.g., wild type sequence corresponding to non-mutated normal sequence. The ability to detect mutations in coding and non- coding DNA (exon/intron), as well as mRNA, is important for the diagnosis of inherited diseases.
  • a gene mutation can be a single nucleotide change (SNP) or multiple nucleotide changes in a DNA sequence encoding an essential protein.
  • SNP single nucleotide change
  • a single nucleotide change or multiple nucleotide changes can result in frame shift mutations, stop codons, or non-conservative amino acid substitutions in a gene, each of which can independently render the encoded protein inactive.
  • a gene mutation can be harmless, resulting in a protein product with no detectable change in function (i.e., a harmless gene polymorphism). Mutations in repetitive DNA can also lead to diseases as is the case, for example, in human fragile-X syndrome, spinal and bulbar muscular dystrophy, and myotonic dystrophy.
  • a mutant nucleic acid that includes a single nucleotide change or multiple nucleotide changes will form one or more base pair mismatches after denaturation and subsequent annealing with the corresponding wild type and complementary nucleic acid.
  • G:A, C:T, C:C, G:G, A:A, T:T, C:A, and G:T represent the eight possible single base pair mismatches which can be found in a nucleic acid heteroduplex, where U is substituted for T when the nucleic acid strand is RNA.
  • Nucleic acid mismatches can form when the two complementary strands of a heteroduplex are derived from DNA or RNA molecules that differ in sequence such that one contains deletions, substitutions, insertions, transpositions, or inversions of sequences compared to the other.
  • Detection of such mutations provides an important diagnostic tool in areas including cancer diagnosis and prognosis, prenatal screening for inherited diseases, differential diagnosis of diseases not readily detectable by conventional tests (for example, Marfan's syndrome and the fragile X syndrome), and the analysis of genetic polymorphisms or DNA fingerprinting (for example, for genetic mapping or identification purposes in legal and forensic matters).
  • Methods disclosed herein allow one skilled in the art to detect mutations such as insertions, deletions, and substitutions.
  • Nucleic acid samples to be screened with the methods of the present invention comprise human, animal, plant, and microbial nucleic acid samples. Methods disclosed herein are useful to detect mutations associated with diseases such as cancer.
  • the specific nucleic acid sequence comprises a portion of a particular gene or genetic locus in the patient's genomic nucleic acid known to be involved in a pathological condition or syndrome.
  • Non-limiting examples include cystic fibrosis, Tay-Sachs disease, sickle-cell anemia, thalassemia, and Gaucher's disease.
  • Single base mutations can be detected by differential hybridization techniques using allele- specific oligonucleotide probes as disclosed in incorporated reference by Saiki RK, Walsh PS, Levenson CH, Erlich HA.
  • a mutation is interrogated by two oligonucleotides capable of annealing immediately adjacent to each other on a target DNA or RNA molecule, one of the oligonucleotides having its 3' end complementary to the point mutation. Adjacent oligonucleotide sequences are only covalently attached when both oligonucleotides are correctly base-paired. Thus, the presence of a point mutation is indicated by the ligation of the two adjacent oligonucleotides as disclosed in incorporated reference by Grossman PD, Bloch W, Brinson E, Chang CC, Eggerding FA, Fung S, Iovannisci DM, Woo S, Winn-Deen ES.
  • primers which span the region to be interrogated for the mutation
  • primers which hybridizes proximally and upstream of the region to be interrogated for the mutation.
  • primer connotes essentially same meaning as a probe except that after initial binding step as in probe-target binding, the primer serves to direct the synthesis of new strands substantially identical to the template sequence in the amplified product.
  • primer-dependent DNA polymerases have, in general, a low replication error rate. This feature is essential for the prevention of genetic mistakes, which would have detrimental effects on progeny.
  • Methods in a second category exploit the high fidelity inherent in this enzymological reaction. Detection of mutations is based on primer extension and incorporation of detectable, chain-terminating nucleoside triphosphates. The high fidelity of DNA polymerases ensures specific incorporation of the correct base labeled with a reporter molecule.
  • Such single nucleotide primer-guided extension assays have been used to detect aspartylglucosaminuria, hemophilia B, and cystic fibrosis; and for quantifying point mutations associated with Leber Hereditary Optic Neuropathy (LHON). See. e.g., Kuppuswamy MN, Hoffmann JW, Kasper CK, Spitzer SG, Groce SL, Bajaj SP.
  • Single nucleotide primer extension to detect genetic diseases experimental application to hemophilia B (factor IX) and cystic fibrosis genes.
  • the selectivity and stability of the oligonucleotide primer extension assay is determined by the length of the oligonucleotide primer.
  • short primers i.e., less than about a 15-mer
  • short primers may exhibit non-specific binding to a wide variety of perfectly-matched complementary sequences.
  • detection methods based on primer extension assays use oligonucleotide primers ranging in length from 15-mer to 25-mer.
  • genomic DNA is isolated from the biological sample and/or amplified with PCR using primers, which flank the region to be interrogated.
  • the primer extension analysis is then conducted on the purified PCR products. See PCT Patent Publications WO 91/13075; WO 92/15712; and WO 96/30545.
  • multiplexing can be achieved by using primers of different lengths and by monitoring the wild-type and mutant nucleotide at each mutation site in two separate single nucleotide incorporation reactions. Such methods are provided herein.
  • Factors affecting hybridization are well known in the art and include temperature, ion concentration, pH, probe length, and probe GC content.
  • a probe can hybridize at numerous places in an average genome. For example, any given 8-mer occurs about 65,000 times in the human genome. However, an octamer has a low melting temperature (Tm) and a single base mismatch will greatly exaggerate this instability.
  • Tm melting temperature
  • a 25-mer probe typically hybridizes with more stability than an 8-mer.
  • a longer probe will form a stable hybrid but will have a lower selectivity because it will tolerate nucleotide mismatches.
  • the short probe hybridizes with high selectivity (i.e., hybridizes poorly to sequence with even a single mismatch), but forms unstable hybrids.
  • Longer probe will form a stable hybrid but will have a lower selectivity because of its tolerance of mismatches.
  • a mixture of probes is made of overlapping contiguous sequences of varying length.
  • the first and second probes hybridize to substantially contiguous portions of the target.
  • substantially contiguous portions are those that are close enough together to allow hybridized first and second probes to function as a single probe.
  • Substantially contiguous portions are preferably between zero (i.e., exactly contiguous so there is no space between the portions) nucleotides and about one nucleotide apart. It has now been realized that the adjacent probes bind cooperatively so that the longer, second probe imparts stability on the shorter, first probe. However, the stability imparted by the second probe does not overcome the selectivity (i.e., intolerance of mismatches) of the first probe. Therefore, methods of the invention take advantage of the high selectivity of the short first probe and the hybridization stability imparted by the longer second probe.
  • DNA molecules contain internucleotide phosphodiester linkages, which are degraded by exonucleases present in cells, culture media and human serum.
  • exonucleases degradation by exonucleases in tissue culture media of DNA may be observed within about 30 minutes to about six hours.
  • Various 3' exonucleases are known, such as phosphodiesterase from snake venom, exonuclease VII from E. coli, Bal 31 exonuclease, exonuclease III, 5' lambda exonuclease, and the 3'-5' exonuclease activity of some DNA polymerases exerted in the absence of dNTPs, as for example T4
  • PNA oligomers are synthesized like DNA oligomers in that the synthesis begins with the four bases (adenine, guanine, thymine, or cytosine) and linked together to form the oligomer of desired sequence.
  • the monomers for making PNA each contain one of the four bases attached to 2-aminoethyl glycine.
  • PNA monomers have amino and carboxyl termini, which are similar to amino acids.
  • PNA monomers are thus linked by peptide bonds to form an oligomer and the synthesis protocols required to link the monomers are the same as those used for standard peptide synthesis.
  • Non-limiting examples and details are found in U. S. Pat. No. 5,821,060 to Arlinghaus, et al., as incorporated herein by way of reference.
  • a variety of alternate methods have been developed which exploit sequence variation in DNA using enzymatic and chemical cleavage techniques.
  • a commonly used screening method for DNA polymorphisms consists of digesting DNA with restriction endonucleases and analyzing the resulting fragments by means of Southern blots, as reported by Botstein D, White RL, Skolnick M, Davis RW. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet 1980 May;32(3):314-31 and incorporated herein by reference. RFLP analysis suffers from low sensitivity and requires a large amount of sample.
  • RFLP analysis When RFLP analysis is used for the detection of point mutations, it is, by its nature, limited to the detection of only those single base changes which fall within a restriction sequence of a known restriction endonuclease. Mutations that do not reside at the cleavage site of restriction endonuclease are not detected.
  • RFLP analysis Jeffreys AJ.DNA sequence variants in the G gamma-, A gamma-, delta- and beta-globin genes of man. Cell 1979 Sep; 18(1): 1-10).
  • It is an object of the present invention to provide a method for conducting affinity fractionation and sequencing of DNA comprising cleaving DNA into predetermined lengths; labeling the cleaved DNA; contacting the labeled DNA to an array of isolated oligonucleotide sequences, wherein said sequences are complementary to portions of the DNA; allowing hybridization to occur between said cleaved DNA and the sequences; determining the sequence of hybridized DNA from the sequences; contacting said extracted hybridized DNA.
  • the technical details of this approach are well known to those skilled in art and are found, for example, in U. S. Pat. No. 5,905,024, as incorporated herein by way of reference.
  • the majority of the available enzymes have 4 to 6 base-pair recognition sequences, and cleave too frequently for many large-scale DNA manipulations (Eckstein and Lilley (eds.), Nucleic Acids and
  • Nla III catg/, Nla IV, ggn/ncc, Not I, gc/ggccgc, Nru I, tcg/cga, Nsi I, atgca/t, Nsp I, rcatg/y, Pac I, ttaat/taa, Pae R7I, c/tcgag, Pci I, a/catgt, Pfl FI, gacn/nngtc, Pfl MI, ccannnn/ntgg, #Ple I, gagtc, Pme I, gttt/aac, Pml I, cac/gtg, Ppu 101, a/tgcat, Ppu MI, rg/gwccy, Psh Al, gacnn/nngtc, Psi I, tta/ta, Psp
  • Cytoplasmic RNA is extracted from cultured cells by the method of Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987 Apr; 162(1): 156-9, treated with DNAse I to remove DNA contamination, then extracted with phenol/chloroform and ethanol precipitated. Reverse transcriptions and PCR are performed to obtain cDNA as described in the exemplary differential display protocol disclosed by Nishio Y, Aiello LP, King GL. Glucose induced genes in bovine aortic smooth muscle cells identified by mRNA differential display. FASEB J 1994 Jan;8(l): 103-6, as incorporated by way of reference.
  • PCR products Prior to hybridization, PCR products are fluorescently labeled by random priming and unincorporated label is removed by Centricon filtration. Prior to hybridization, microparticles are rinsed with a solution of 1% "Blotto" or 50 mM tripolyphosphate and then washed with hybridization solution (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1M NaCl). Labeled PCR fragments representing the 3'-end of expressed genes are recovered from the Centricon filtration units in hybridization buffer and beads representing probes complementary to expressed genes of interest are flooded with appropriately diluted cDNA solution. The plate is placed at 65° C for 30-60 minutes and then washed three times with hybridization buffer.
  • microtiter plates To facilitate washing and operating procedures the individual wells of microtiter plates have microporous filter bottoms to allow fluid passage but to retain microparticles with probes. Following hybridization and washing, the plate is placed into fluorescence reading plate reader and the intensity of signal is read.
  • Other labeling means in addition to fluorescence detection are known, e.g., horse radish peroxidase, alkaline phosphatase, an isotope such as 32 P, chemiluminiscent reporter, and are disclosed for example in Eggers M, Hogan M, Reich RK, Lamture J, Ehrlich D, Hollis M, Kosicki B, Powdrill T, Beattie K, Smith S, et al.
  • Reference material is usually nucleic acid or molecule, which is identical or substantially homologous to the analyte of interest.
  • the known quantity of reference material is diluted serially and standard curves are obtained accordingly, from which the quantity of the analyte is extrapolated.
  • TaqMan indicates the probe or Fluorescence Energy Transfer (FET) probe used to detect specific sequences in PCR products by employing the 5'>3' exonuclease activity of Taq DNA polymerase.
  • FET Fluorescence Energy Transfer
  • the TaqMan probe hybridizes to its complementary single strand DNA sequence within the PCR target. When amplification occurs the
  • TaqMan probe is degraded due to the 5'— >3' exonuclease activity of Taq DNA polymerase, thereby separating the quencher from the reporter during extension. Due to the release of the quenching effect on the reporter, the fluorescence intensity of the reporter dye increases. This allows the real time
  • the TaqMan assay offers a sensitive method to determine the presence or absence of specific sequences. Therefore, this technique is particularly useful in diagnostic applications, such as the screening of samples for the presence or incorporation of favorable traits and the detection of pathogens and diseases.
  • the TaqMan assay allows high sample throughput because no gel-electrophoresis is required for detection.
  • TaqMan behaves as a codominant marker.
  • the technical details regarding TaqMan assay are found in Holland PM, Abramson RD, Watson R, Gelfand DH. Detection of specific polymerase chain reaction product by utilizing the 5' — 3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A 1991 Aug 15;88(16):7276-80; Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996 Oct;6(10):986-94; and also at the web site by Molecular Beacons (http://molecular-beacons.org), all these references are incorporated herein by reference.
  • PNAs are polyamide analogs of DNA and RNA. See, e.g., U. S. Pat. No. 5,539,082 to Nielsen et al. Nielsen et al. discloses that PNAs simulate natural polynucleotides by binding complementary single stranded (ss) DNA and RNA strands. The method is conducted without separating unhybridized probes from the hybridization complex prior to signal detecting, without providing a signal quenching agent on the probe, or on the nucleotide sequence of interest, and without the use of enzymes. The details of this method are provided in U. S. Pat. No. 5,846,729 issued to Wu, et al. December 8, 1998 and incorporated herein by way of reference.
  • the samples of nucleic acid are labeled with fluorescein and biotin, respectively. Reactions are carried out at 1.25 mM of ATP, CTP, GTP, and UTP and 0.5 mM fluorescein- 12-UTP or 0.25 mM biotin- 16-UTP (Boehringer Mannheim). Labeled samples are denatured at 95 degrees C for 5 min, chilled on ice for 5 min, and equilibrated to 37 degrees C. See for further details in Melchior WB Jr, Von Hippel PH. Alteration of the relative stability of dA-dT and dG-dC base pairs in DNA. Proc Natl Acad Sci U S A 1973 Feb;70(2):298-302; Lipshutz RJ, Morris D,
  • Triton X-100 Triton X-100
  • microparticles are washed and scanned at a resolution of 74 pixels in a fluorescence plate reader.
  • a fluorescein scan is obtained with a 515- to 545-nm band-pass fitter and a phycoerythrin scan with a 560-nm long-pass filter.
  • PCR Polymerase Chain Reaction
  • LCR Ligase Chain Reaction
  • SR/NASBA Self-Sustained Synthetic Reaction
  • Q-beta Replicase Q-beta Replicase
  • PCR polymerase chain reaction
  • U.S. Pat. Nos. 4, 683, 195 and 4, 683, 202 to Mullis and Mullis et al. is a method for increasing the concentration of a segment of target sequence in a mixture of genomic DNA without cloning or purification.
  • This technology provides one approach to the problems of low target sequence concentration.
  • PCR is usually used to directly increase the concentration of the target to an easily detectable level.
  • This process for amplifying the target sequence involves introducing an excess of two oligonucleotide primers, which are complementary to their respective strands of the double-stranded target sequence in the DNA mixture containing the desired target sequence. The mixture is denatured and then allowed to hybridize.
  • the primers are extended with polymerase so as to form complementary strands.
  • the steps of the denaturation, hybridization, and polymerase extension are repeated as often as needed, in order to obtain high concentrations of a segment of the desired target sequence.
  • the length of the segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and this length is a controllable parameter. Because the desired segments of the target sequence become the dominant sequences (in terms of concentration) in the mixture, they are said to be "PCR-amplified.”
  • LCR ligase chain reaction
  • LCR Ligase chain reaction
  • ligase will covalently link each set of hybridized molecules.
  • two probes are ligated together only when they base-pair with sequences in the target sample, without gaps or mismatches. Repeated cycles of denaturation, hybridization and ligation amplify a short segment of DNA.
  • LCR has also been used in combination with PCR to achieve enhanced detection of single-base changes. Segev, PCT Publication No. W09001069 Al (1990).
  • the four oligonucleotides used in this assay can pair to form two short ligatable fragments, there is the potential for the generation of target-independent background signal.
  • the use of LCR for mutant screening is limited to the examination of specific nucleic acid positions.
  • the self-sustained sequence replication reaction (SR/NASBA) (Guatelli JC, Whitfield KM, Kwoh DY, Barringer KJ, Richman DD, Gingeras TR. Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. Proc Natl Acad Sci U S A 1990 Mar;87(5): 1874-8) is a transcription-based in vitro amplification system that can exponentially amplify RNA sequences at a uniform temperature. The amplified RNA is then utilized for mutation detection (Fahy E, Kwoh DY, Gingeras TR. Self-sustained sequence replication (3SR): an isothermal transcription-based amplification system alternative to PCR.
  • 3SR Self-sustained sequence replication
  • an oligonucleotide primer is used to add a phage RNA polymerase promoter to the 5' end of the sequence of interest.
  • a cocktail of enzymes and substrates that includes a second primer, reverse transcriptase, RNase H, RNA polymerase and ribo-and deoxyribonucleoside triphosphates, the target sequence undergoes repeated rounds of transcription, cDNA synthesis and second-strand synthesis to amplify the area of interest.
  • the use of 3SR to detect mutations is kinetically limited to screening small segments of DNA (e.g., 200-300 base pairs).
  • RNA template for Q-beta replicase e.g., see incorporated by reference an article by Abramson RD, Myers TW. Nucleic acid amplification technologies. Curr Opin Biotechnol 1993 Feb;4(l):41-7.
  • a previously identified major problem with false positives resulting from the replication of unhybridized probes has been addressed through use of a sequence- specific ligation step.
  • available thermostable DNA ligases are not effective on this RNA substrate, so the ligation must be performed by T4 DNA ligase at low temperatures. This prevents the use of high temperature as a means of achieving specificity as in the LCR, the ligation event can be used to detect a mutation at the junction site, but not elsewhere.
  • the cycling probe reaction uses a long chimeric oligonucleotide in which a central portion is made of RNA while the two termini are made of DNA. Hybridization of the probe to a target
  • RNA and exposure to a thermostable RNase H causes the RNA portion to be digested. This destabilizes the remaining DNA portions of the duplex, releasing the remainder of the probe from the target DNA and allowing another probe molecule to repeat the process.
  • the signal in the form of cleaved probe molecules, accumulates at a linear rate. While the repeating process increases the signal, the RNA portion of the oligonucleotide is vulnerable to RNases that may carried through sample preparation.
  • Branched DNA described by Urdea et al., (Urdea MS, Running JA, Horn T, Clyne J, Ku LL, Warner BD.
  • Urdea MS Running JA, Horn T, Clyne J, Ku LL, Warner BD.
  • oligonucleotides with branched structures that allow each individual oligonucleotide to carry 35 to 40 labels (e.g., alkaline phosphatase enzymes).
  • alkaline phosphatase enzymes e.g., alkaline phosphatase enzymes
  • Single nucleotide polymorphisms are DNA point mutations and is estimated that SNPs occur once every 100-300 base pair (bp). SNPs are useful for gene mapping, defining population structure, and performing functional studies. SNPs are expected to greatly facilitate large-scale genetic studies concerned with determining linkage between sequence variations and heritable phenotypes. SNPs are also an efficient tool for genetic identification for legal and forensic applications. SNPs are particularly interesting as markers because many known genetic diseases, such as sickle cell anemia, are caused by single base mutations. Therefore, an assay for an SNP marker is useful for identification of the disease-causing mutation. Some genetic diseases, such as cystic fibrosis, are caused by any of a large number of different mutations in a single gene.
  • SNPs are used in a multiplex assay for all known alleles in a large gene.
  • SNPs have been proposed as an ideal tool for the emerging discipline of pharmacogenomics for fine-tuning of patients' diagnosis and treatment.
  • Large-scale analysis of the associations between the effects of drugs and genetic markers allows physicians to match drugs to the genetic makeup of individual patients to better predict beneficial and harmful effects.
  • Databases of gene expression profiles can be predictive of different classes of drug toxicity.
  • SNPs over other types of genetic markers such as isozymes, restriction fragment length polymorphisms (RFLP), variable number tandem repeats and simple sequence repeats include: very large numbers of polymorphic loci; loci distributed throughout the genome; markers present within coding regions or exons, introns and regions that flank exons; simple and unambiguous assay techniques; high levels of polymorphism in the population; stable Mendelian inheritance; and low levels of spontaneous mutation.
  • Kwok et. al Kwok, P.Y., Q. Deng, H. Zakeri, S.L. Taylor and D.A. Nickerson.
  • NIHPDR National Cancer Institute
  • GAI Annotation Initiative
  • Genome Anatomy project which collects STS data from a broad set of cancerous and noncancerous tissues.
  • the Whitehead Institute/MIT Center for Genome Research has a Human SNP Mapping
  • SNPs Human mitochondrial sequence is found at web site of Emory University: http://infinity.gen.emory.edu/mitomap.html.
  • EST expressed sequence tag
  • SNPs are found by scanning for regions of overlapping sequence in bacterial artificial chromosome (BAC) and Pl-derived artificial chromosome (PAC) clones that come from different individuals or from homologous chromosomes in a single individual. It is also possible to discover SNPs by scanning for variation among homologous EST sequences.
  • the EST approach is particularly attractive since these sequences are by definition derived from expressed genes, and in fact, some SNPs may represent mutations with detectable phenotypes.
  • BRCA1 on chromosome 17 and BRCA2 on chromosome 13 Two breast cancer susceptibility genes were identified: BRCA1 on chromosome 17 and BRCA2 on chromosome 13.
  • BRCA 1 or BRCA2 Two breast cancer susceptibility genes were identified: BRCA1 on chromosome 17 and BRCA2 on chromosome 13.
  • Oncogene myc mutation is associated with Burkitt lymphoma (Bhatia K, Huppi K, Spangler G, Siwarski D, Iyer R, Magrath I. Point mutations in the c-Myc transactivation domain are common in Burkitt's lymphoma and mouse plasmacytomas. Nat Genet 1993 Sep;5(l):56-61). Mutations in MSH2 and MLH1 are associated with colon cancer (Bronner CE, Baker SM, Morrison PT, Warren G, Smith LG, Lescoe MK, Kane M, Earabino C, Lipford J, Lindblom A, et al. Mutation in the DNA mismatch repair gene homologue hMLHl is associated with hereditary non-polyposis colon cancer.
  • Lung cancer is also associated with genetic mutations (Braun MM, Caporaso NE, Page WF, Hoover RN. Genetic component of lung cancer: cohort study of twins. Lancet 1994 Aug 13;344(8920):440-3; Sjalander A, Birgander R, Rannug A, Alexandrie AK, Tornling G, Beckman G. Association between the p21 codon 31 Al (arg) allele and lung cancer. Hum Hered 1996 Jul- Aug;46(4):221-5; and Weston A, Willey JC, Modali R, Sugimura H, McDowell EM, Resau J, Light B, Haugen A, Mann DL, Trump BF, et al. Differential DNA sequence deletions from chromosomes 3, 1 1,
  • MEN Multiple endocrine neoplasia
  • hyperplasia abnormal multiplication or increase in the number of normal cells in normal arrangement in a tissue
  • hyperfunction excessive functioning of 2 or more components of the endocrine system.
  • NF-2 Neurofibramatosis type 2
  • NF2 Neurofibramatosis type 2
  • NF2 is a rare inherited disorder characterized by the development of benign tumors on both auditory nerves (acoustic neuromas). The disease is also characterized by the development of malignant central nervous system tumors as well.
  • the NF2 gene has been mapped to chromosome 22 and is thought to be a so-called 'tumor- suppressor gene'.
  • tumor suppressor genes such as p53 and Rb
  • the normal function of NF2 is to act as a brake on cell growth and division, ensuring that cells do not divide uncontrollably, as they do in tumors.
  • a mutation in NF2 impairs its function, and accounts for the clinical symptoms observed in neurofibromatosis sufferers (Rouleau GA, Merel P, Lutchman M, Sanson M, Zucman J, Marineau C, Hoang-Xuan K, Demczuk S, Desmaze C, Plougastel B, et al. Alteration in a new gene encoding a putative membrane-organizing protein causes neuro-fibromatosis type 2. Nature 1993 Jun 10;363(6429):515-21).
  • the p53 gene like the Rb gene, is a tumor suppressor gene, i.e., its activity stops the formation of tumors. If a person inherits only one functional copy of the p53 gene from their parents, they are predisposed to cancer and usually develop several independent tumors in a variety of tissues in early adulthood. This condition is rare, and is known as Li-Fraumeni syndrome. However, mutations in p53 are found in most tumor types, and so contribute to the complex network of molecular events leading to tumor formation.
  • the p53 gene has been mapped to chromosome 17. In the cell, p53 protein binds DNA, which in turn stimulates another gene to produce a protein called p21 that interacts with a cell division-stimulating protein (cdk2).
  • oncogenes or tumor suppressor genes of a eukaryotic (for example, mammalian) cell are contemplated as useful markers for malignancies; preferable mammalian oncogenes include, without limitation, abl, akt, crk, erb-A, erb-B, ets, fes/fps, fgr, frns, fos, jun, kit, mil/raf, mos, myb, myc, H-ras, K-ras, rel, ros, sea, sis, ski, src, and yes; preferable tumor suppressor genes in addition to p53 include, retinoblastoma (preferably RBI), adenomatous polyposis coli, NF-1,
  • NF-2, MLH-1 , MTS-1, MSH-2, and human non-polyposis genes are also derived from any cell cycle control gene, preferably p21 , p27, or pl6. (U. S. Pat. No. 5,876,941 to Landegren, et al., on March 2, 1999).
  • DPC4 tumor suppressor gene
  • TGF-beta transforming growth factor-beta
  • Other tumor suppressor genes include p53 and Rb, which, if mutated or absent from the genome can contribute to cancerous growth in a variety of tissues (Hahn SA, Schutte M, Hoque AT, Moskaluk CA, da Costa LT, Rozenblum E, Weinstein CL, Fischer A, Yeo CJ, Hruban RH, Kern SE.
  • DPC4 a candidate tumor suppressor gene at human chromosome 18q21.1. Science 1996 Jan 19;271(5247):350-3).
  • prostate cancer Despite the high prevalence of prostate cancer, little is known about the genetic predisposition of some men to the disease. Numerous studies point to a family history being a major risk factor and thought to be responsible for an estimated 5-10% of all prostate cancers.
  • a susceptibility locus for prostate cancer on chromosome 1 accounts for about 1 in 500 cases of prostate cancer (Smith JR, Freije D, Carpten JD, Gronberg H, Xu J, Isaacs SD, Brownstein MJ, Bova GS, Guo H, Bujnovszky P, Nusskern DR, Damber JE, Bergh A, Emanuelsson M, Kallioniemi OP, Walker-Daniels J, Bailey- Wilson JE, Beaty TH, Meyers DA, Walsh PC, Collins FS, Trent JM, Isaacs WB.
  • Major susceptibility locus for prostate cancer on chromosome 1 suggested by a genome- wide search. Science 1996 Nov 22;274(5291):1371-4).
  • Ras is one such oncogene product that is found on chromosome 11. It is found in normal cells, where it helps to relay signals by acting as a switch. When receptors on the cell surface are stimulated (by a hormone, for example), Ras is switched on and transduces signals that tell the cell to grow. If the cell-surface receptor is not stimulated, Ras is not activated and so the pathway that results in cell growth is not initiated. In about 30% of human cancers, Ras is mutated (Lowy DR, Willumsen BM. Function and regulation of ras. Annu Rev Biochem 1993;62:851-91; Russell MW, Munroe DJ, Brie E, Housman
  • Retinoblastoma occurs in early childhood and affects about 1 child in 20,000.
  • hereditary and non-hereditary forms of retinoblastoma In the hereditary form, multiple tumors are found in both eyes, while in the non-hereditary form only one eye is effected and by only one tumor.
  • a gene called Rb is lost from chromosome 13. Since the absence of Rb seemed to be linked to retinoblastoma, it has been suggested that the role of Rb in normal cells is to suppress tumor formation. Rb is found in all cells of the body, where under normal conditions it acts as a brake on the cell division cycle by preventing certain regulatory proteins from triggering DNA replication.
  • Rb is missing, a cell can replicate itself over and over in an uncontrolled manner, resulting in tumor formation. Untreated, retinoblastoma is almost uniformly fatal, but with early diagnosis and modern methods of treatment the survival rate is over 90%. Since the Rb gene is found in all cell types, studying the molecular mechanism of tumor suppression by Rb will give insight into the progression of many types of cancer, not just retinoblastoma (Friend SH, Bernards R, Rogelj S, Weinberg RA, Rapaport JM, Albert DM, Dryja TP. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma.
  • Von Hippel-Lindau Syndrome is an inherited multi-system disorder characterized by abnormal growth of blood vessels. While blood vessels normally grow like trees, in people with VHL little knots of blood capillaries sometimes occur. These knots are called angiomas or hemangioblastomas. Growths may develop in the retina, certain areas of the brain, the spinal cord, the adrenal glands and other parts of the body.
  • the gene for Von-Hippel Lindau disease (VHL) is found on chromosome 3, and is inherited in a dominant fashion. If one parent has a dominant gene, each child has a 50-50 chance of inheriting that gene.
  • the VHL gene is a tumor suppressor gene. This means that its role in a normal cell is to stop uncontrolled growth and proliferation.
  • Asthma is a what is known as a complex heritable disease. This means that there are a number of genes that contribute towards a person's susceptibility to a disease, and in the case of asthma, chromosomes 5, 6, 1 1, 14, and 12 have all been implicated. The relative roles of these genes in asthma predisposition are not clear, but one of the most promising sites for investigation is on chromosome 5.
  • asthma Although a gene for asthma from this site has not yet been specifically identified, it is known that this region is rich in genes coding for key molecules in the inflammatory response seen in asthma, including cytokines, growth factors, and growth factor receptors.
  • the search for specific asthma genes can be assisted by the present invention (A genome-wide search for asthma susceptibility loci in ethnically diverse populations. The Collaborative Study on the Genetics of Asthma (CSGA). Nat Genet 1997 Apr;15(4):389-92).
  • Autoimmune polyglandular syndrome type I (APS1, also called APECED) is a rare autosomal recessive disorder that maps to human chromosome 21.
  • AIRE autoimmune regulator
  • IBD Inflammatory bowel diseases
  • Candidate genes found in this region include several involved in the inflamatory response, including: CD 19, involved in B-lymphocyte function; sialophorin, involved in leukocyte adhesion; the CD 1 1 integrin cluster, involved in microbacterial cell adhesion; and the interleukin-4 receptor, which is interesting, as IL-4-mediated functions are altered in IBDs (Hugot JP, Laurent-Puig P, Gower-Rousseau C, Olson JM, Lee JC, Beaugerie L, Naom I, Nors JL, Van Gossum A, Orholm M, Bonaiti-Pellie C, Weissenbach J, Mathew CG, Lennard- Jones JE, Cortot A, Colombel JF, Thomas G.
  • IBDs Human JP, Laurent-Puig P, Gower-Rousseau C, Olson JM, Lee JC, Beaugerie L, Naom I, Nors JL, Van Gossum A, Orholm M,
  • DiGeorge syndrome is a rare congenital (i.e. present at birth) disease whose symptoms vary greatly between individuals, but commonly include a history of recurrent infection, heart defects and characteristic facial features. DiGeorge syndrome is caused by a large deletion from chromosome 22, produced by an error in recombination at meiosis (the process that creates germ cells and ensures genetic variation in the offspring). This deletion means that several genes from this region are not present in DiGeorge syndrome patients.
  • Familial Mediterranean fewer is an inherited disorder usually characterized by recurrent episodes of fever and peritonitis (inflammation of the abdominal membrane).
  • FMF Familial Mediterranean fewer
  • the gene, found on chromosome 16 codes for a protein that is found almost exclusively in granulocytes.
  • the protein, pyrin is a member of a family of nuclear factors homologous to the Ro52 autoantigen and is likely to normally assist in keeping inflammation under control. Discovery of the gene mutations will allow the development of a simple diagnostic blood test for FMF (Ancient missense mutations in a new member of the RoRet gene family are likely to cause familial Mediterranean fever.
  • SCID Severe combined immunodeficiency
  • IL2RG interleukin 2 receptor gamma
  • ADA adenosine deaminase
  • Immature lymphoid cells of the immune system are particularly sensitive to the toxic effects of these unused substrates, so fail to reach maturity (Valerio D, Duyvesteyn MG, Dekker BM, Weeda G, Berkvens TM, van der Voorn L, van Ormondt H, van der Eb AJ. Adenosine deaminase: characterization and expression of a gene with a remarkable promoter. EMBO J 1985 Feb;4(2):437-43; Noguchi M, Yi H, Rosenblatt HM, Filipovich AH, Adelstein S, Modi WS, McBride OW, Leonard WJ. Interleukin-2 receptor gamma chain mutation results in X-linked severe combined immunodeficiency in humans. Cell 1993 Apr 9;73(1): 147-57).
  • IKBL gene HLA class II genes
  • MHC major histocompatibility complex
  • DMD Duchenne muscular dystrophy
  • X-linked The gene for DMD, found on the X chromosome, encodes a large protein - dystrophin.
  • Dystrophin is required inside muscle cells for structural support: it is thought to strengthen muscle cells by anchoring elements of the internal cytoskeleton to the surface membrane.
  • Ellis-van Creveld syndrome also known as 'chondroectodermal dysplasia', is a rare genetic disorder characterized by short-limb dwarfism, polydactyly (additional fingers or toes), malformation of the bones of the wrist, dystrophy of the fingernails, partial hare-lip, cardiac malformation and often prenatal eruption of the teeth.
  • the gene causing Ellis-van Creveld syndrome, EVC has been mapped to the short arm of chromosome 4.
  • the function of a healthy EVC gene is not known (Polymeropoulos MH, Ide SE, Wright M, Goodship J, Weissenbach J, Pyeritz RE, Da Silva EO, Ortiz De Luna RI, Francomano CA.
  • Marfan syndrome is a connective tissue disorder, so affects many structures, including the skeleton, lungs, eyes, heart and blood vessels. The disease is characterized by unusually long limbs. Marfan syndrome is an autosomal dominant disorder that has been linked to the FBN1 gene on chromosome 15.
  • FBN1 encodes a protein called fibrillin, which is essential for the formation of elastic fibres found in connective tissue (Dietz HC, Cutting GR, Pyeritz RE, Maslen CL, Sakai LY, Corson GM, Puffenberger EG, Hamosh A, Nanthakumar EJ, Curristin SM, et al. Marfan syndrome caused by a recurrent de novo missense mutation in the fibrillin gene. Nature 1991 Jul 25;352(6333):337-9).
  • Myotonic dystrophy is an inherited disorder in which the muscles contract but have decreasing power to relax. With this condition, the muscles also become weak and waste away. Myotonic dystrophy can cause mental deficiency, hair loss and cataracts.
  • the myotonic dystrophy gene found on chromosome 19, codes for a protein kinase that is found in skeletal muscle, where it likely plays a regulatory role.
  • An unusual feature of this illness is that its symptoms usually become more severe with each successive generation. This is because mistakes in the faithful copying of the gene from one generation to the next result in the amplification of a 'AGC triplet repeat', similar to that found in Huntington disease.
  • AD Alzheimer disease
  • PS1 or AD3 on chromosome 14
  • PS2 or AD4 on chromosome 1.
  • the formation of lesions made of fragmented brain cells surrounded by amyloid-family proteins are characteristic of the disease.
  • ALS Amyotrophic lateral sclerosis
  • Lou Gehrig disease is a neurological disorder characterized by progressive degeneration of motor neuron cells in the spinal cord and brain, which ultimately results in paralysis and death.
  • a team of researchers linked familial ALS to chromosome 21. Two years later, the SOD 1 gene was identified as being associated with many cases of familial ALS.
  • the enzyme coded for by SOD1 carries out a very important function in cells: it removes dangerous superoxide radicals by converting them into non-harmful substances (Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P, Hentati A, Donaldson D, Goto J, O'Regan JP, Deng HX, et al. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 1993 Mar 4;362(6415):59-62).
  • CMT Charcot-Marie-Tooth disease
  • PMP22 protein
  • CMT CMT
  • Type IB autosomal-recessive and X-linked.
  • the same proteins involved in the Type IA and Type IB CMT are also involved in a disease called Dejerine-Sottas syndrome
  • DSS human myelin protein zero
  • Epilepsy affects approximately 1% of the population making it one of the most common neurological diseases. There are many forms of epilepsy - most are rare. To date, twelve forms of epilepsy have been demonstrated to possess some genetic basis. For example, LaFora Disease (progressive myclonic, type 2) is thought to result from a mutation in the EPM2A gene, which is located on chromosome 6. This gene is thought to produce laforin, a protein similar to a group of protein-tyrosine phosphatases that help maintain a balance of sugars in the blood stream. Too much laforin destroys brain cells, which may then lead to the development of LaFora Disease. Much progress has been made in narrowing down regions of chromosomes associated with different forms of epilepsy.
  • This invention will be particularly useful in helping to further the research and diagnostics of this disease (Minassian BA, Lee JR, Herbrick JA, Huizenga J, Soder S, Mungall AJ, Dunham I, Gardner R, Fong CY, Carpenter S, Jardim L, Satishchandra P, Andermann E, Snead OC 3rd, Lopes-Cendes I, Tsui LC, Delgado-Escueta AV, Rouleau GA, Scherer SW. Mutations in a gene encoding a novel protein tyrosine phosphatase cause progressive myoclonus epilepsy. Nat Genet 1998 Oct;20(2): 171-4).
  • Tremor or uncontrollable shaking, is a common symptom of neurological disorders such as Parkinson disease, head trauma and stroke.
  • many people with tremor have what is called idiopathic or essential tremor.
  • the tremor itself is the only symptom of the disorder.
  • essential tremor is inherited as an autosomal dominant trait, which means that children of an affected individual will have a 50 percent chance of also developing the disorder.
  • ETM1 gene also called FET1 was mapped to chromosome 3 in a study of Icelandic families, while another gene, called ETM2, was mapped to chromosome 2.
  • Fragile X syndrome is the most common inherited form of mental retardation currently known. Fragile X syndrome is a defect in the X chromosome and its effects are seen more frequently, and with greater severity, in males than females. In normal individuals, the FMR1 gene is transmitted stably from parent to child. However, in Fragile X individuals, there is a mutation in one end of the gene (the
  • FMR1 gene is not expressed, so no FMR1 protein is made. Although the exact function of FMR1 protein in the cell is unclear, it is known that it binds RNA. A similar nucleotide repeat expansion is seen in other diseases, such as Huntington disease (Siomi H, Siomi MC, Nussbaum RL, Dreyfuss G.
  • the protein product of the fragile X gene, FMR1 has characteristics of an RNA-binding protein.
  • FRDA Friedreich's ataxia
  • FRDA is a rare inherited disease characterized by the progressive loss of voluntary muscular coordination (ataxia) and heart enlargement.
  • FRDA is an autosomal recessive disease caused by a mutation of a gene called frataxin, which is located on chromosome 9. This mutation means that there are many extra copies of a DNA segment, the trinucleotide GAA.
  • a normal individual has 8 to 30 copies of this trinucleotide, while FRDA patients have as many as 1,000 (Campuzano V, Montermini L, Molto MD, Pianese L, Cossee M, Cavalcanti F, Monros E, Rodius F, Duclos F, Monticelli A, et al. Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion. Science 1996 Mar 8;271(5254): 1423-7).
  • Huntington disease is an inherited, degenerative neurological disease that leads to dementia. About 30,000 Americans have HD and about 150,000 more are at risk of inheriting the disease from a parent.
  • the HD gene whose mutation results in Huntington disease, was mapped to chromosome 4 in 1983 and cloned in 1993.
  • the mutation is a characteristic expansion of a nucleotide triplet repeat in the DNA that codes for the protein huntingtin.
  • the number of repeated triplets - CAG (cytosine, adenine, guanine) - increases with the age of the patient. Since people who have those repeats always suffer from Huntington disease, it suggests that the mutation causes a gain-of-function, in which the mRNA or protein takes on a new property or is expressed inappropriately.
  • Type A is the acute infantile form
  • Type B is a less common, chronic, non-neurological form
  • Type C is a biochemically and genetically distinct form of the disease.
  • NP-C Niemann-Pick type C
  • NPC1 was found to have known sterol-sensing regions similar to those in other proteins, which suggests it plays a role in regulating cholesterol traffic (Carstea ED, Morris JA, Coleman KG, Loftus SK, Zhang
  • Parkinson disease is a neuodegenerative disease that manifests as a tremor, muscular stiffness and difficulty with balance and walking.
  • a candidate gene for some cases of Parkinson disease was mapped to chromosome 4. Mutations in this gene have now been linked to several Parkinson disease families.
  • the product of this gene, a protein called alpha-synuclein, is a familiar culprit: a fragment of it is a known constituent of Alzheimer disease plaques.
  • alpha-synuclein fragments are implicated in both Parkinson and Alzheimer diseases (Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, Dutra A, Pike B, Root H, Rubenstein J, Boyer R, Stenroos ES, Chandrasekharappa S, Athanassiadou A, Papapetropoulos T, Johnson WG, Lazzarini AM, Duvoisin RC, Di Iorio G, Golbe LI, Nussbaum RL. Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. Science 1997 Jun 27;276(5321):2045-7).
  • Spinocerebellar atrophy of which there are several types, is a degeneration of the spinal cord and the cerebellum, the small fissured mass at the base of the brain, behind the brain stem.
  • the cerebellum is concerned with coordination of movements, so atrophy or "wasting away" of this critical control center results in a loss of muscle coordination.
  • the basic defect in all types of spinocerebellar atrophy is a an expansion of a CAG triplet repeat. In this way, it is similar to fragile-X syndrome, Huntington disease and myotonic dystrophy, all of which exhibit a triplet repeat expansion of a gene.
  • the gene is SCA1, found on chromosome 6 (Banfi S, Servadio A, Chung MY, Kwiatkowski TJ Jr, McCall AE, Duvick LA, Shen Y, Roth EJ, Orr HT, Zoghbi HY. Identification and characterization of the gene causing type 1 spinocerebellar ataxia. Nat Genet 1994 Aug;7(4):513-20). Williams syndrome is a rare congenital disorder characterized by physical and development problems. In Williams syndrome individuals, both the gene for elastin and an enzyme called LIM kinase are deleted. Both genes map to the same small area on chromosome 7.
  • elastin is a key component of connective tissue, conferring its elastic properties. Mutation or deletion of elastin lead to the vascular disease observed in Williams syndrome.
  • LIM kinase is strongly expressed in the brain, and deletion of LIM kinase is thought to account for the impaired visuospatial constructive cognition in Williams syndrome.
  • Williams syndrome is a contigious disease, meaning that the deletion of this section of chromosome 7 involves several more genes (Lenhoff HM, Wang PP,
  • Adrenoleukodystrophy is a rare, inherited metabolic disorder wherein myelin sheath on nerve fibers in the brain is lost, and the adrenal gland degenerates, leading to progressive neurological disability and death.
  • ALD gene was discovered in 1993 and corresponding protein is ember of a family of transporter proteins (Mosser J, Douar AM, Sarde CO, Kioschis P, Feil R, Moser H, Poustka AM, Mandel JL, Aubourg P. Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters. Nature 1993 Feb 25;361(6414):726-30).
  • Atherosclerosis is a disease that can affect people at any age, although it usually doesn't pose a threat until people reach their forties or fifties. It is characterized by a narrowing of the arteries caused by cholesterol-rich plaques of immune-system cells. Key risk factors for atherosclerosis, which can be genetic and/or environmental.
  • a protein called apolipoprotein E which can exist in several different forms, is coded for by a gene found on chromosome 19. Detection of defects in apolipoprotein E gene is useful for diagnosis and treatment atherosclerosis (Wenham PR, Newton CR, Price WH. Analysis of apolipoprotein E genotypes by the Amplification Refractory Mutation System. Clin Chem 1991 Feb;37(2):241-4).
  • Gaucher disease is characterized by the accumulation of glucocerebroside, leading to enlargement of the liver and spleen and lesions in the bones. It is caused by an inherited deficiency of the enzyme glucocerebrosidase. Many mutations exist, but four of these account for over 97% of the mutations in Ashkenazi Jews, the population group in which Gaucher disease is the most common. Detection of genetic mutations is useful for genetic counseling and gene therapy (Beutler E. Gaucher disease: new molecular approaches to diagnosis and treatment. Science 1992 May 8;256(5058):794-9). Glanzmann's thrombasthenia (GT) arises from a qualitative or quantitative defect in the GPIIb-
  • Illa complex (integrin alphallbeta3a), the mediator of platelet aggregation (Ruan J, Schmugge M, Clemetson KJ, Cazes E, Combrie R, Bourre F, Nurden AT. Homozygous Cys542— >Arg substitution in GPIIIa in a swiss patient with type I Glanzmann's thrombasthenia. Br J Haematol 1999 May;105(2):523-31). Gyrate atrophy of the choroid and retina leads to a progressive loss of vision, with total blindness usually occurring between the ages of 40 and 60. The disease is an inborn error of metabolism.
  • the gene whose mutation causes gyrate atrophy is found on chromosome 10, and encodes ornithine ketoacid aminotransferase (OAT) enzyme.
  • OAT ornithine ketoacid aminotransferase
  • Different inherited mutations in OAT cause differences in the severity of symptoms of the disease (Akaki Y, Hotta Y, Mashima Y, Murakami A, Kennaway NG, Weleber RG, Inana G.
  • Gyrate atrophy of the choroid and retina assignment of the ornithine aminotransferase structural gene to human chromosome 10 and mouse chromosome 7. Am J Hum Genet 1988 Dec;43(6):922-8).
  • Type I diabetes is a chronic metabolic disorder that adversely affects the body's ability to manufacture and use insulin, a hormone necessary for the conversion of food into energy. The disease greatly increases the risk of blindness, heart disease, kidney failure, neurological disease and other conditions for the pproximately 16 million Americans who are affected by it.
  • Type I diabetes is what is known as a 'complex trait', which means that mutations in several genes likely contribute to the disease. About 10 loci in the human genome have now been found that seem to confer susceptibility to Type I diabetes.
  • a gene at the locus IDDM2 on chromosome 1 1 and (2) the gene for glucokinase (GCK), an enzyme that is key to glucose metabolism which helps modulate insulin secretion, on chromosome 7 (Davies JL, Kawaguchi Y, Bennett ST, Copeman JB, Cordell HJ, Pritchard LE, Reed PW, Gough SC, Jenkins SC, Palmer SM, et al. A genome-wide search for human type 1 diabetes susceptibility genes. Nature 1994 Sep 8;371(6493): 130-6). Genetic mutations in the coding or exon-intron regions of the uncoupling protein 2 (UCP2) gene have been associated with non-insulin-dependent diabetes mellitus (NIDDM).
  • NIDDM non-insulin-dependent diabetes mellitus
  • IDDM7 gene has previously been mapped, involved in type 1 but not type 2 diabetes (Iwata I, Nagafuchi S, Nakashima H, Kondo S, Koga T, Yokogawa Y, Akashi T, Shibuya T, Umeno Y, Okeda T, Shibata S, Kono S, Yasunami M, Ohkubo H, Niho Y. Association of polymorphism in the NeuroD/BETA2 gene with type 1 diabetes in the Japanese. Diabetes 1999 Feb;48(2):416-9).
  • She adaptor proteins's gene useful as a marker for identifying impaired insulin secretion, insulin resistance, and type 2 diabetes mellitus (Almind K, Ahlgren MG, Hansen T, Urhammer SA, Clausen JO, Pedersen O. Discovery of a Met300Val variant in She and studies of its relationship to birth weight and length, impaired insulin secretion, insulin resistance, and type 2 diabetes mellitus. J Clin Endocrinol Metab 1999 Jun;84(6):2241-4). Obesity is an excess of body fat that frequently results in a significant impairment of health.
  • Paroxysmal nocturnal hemoglobinuria is associated with a high risk of major thrombotic events, most commonly thrombosis of large intra-abdominal veins. Most patients who die of their disease die of thrombosis. PNH blood cells are deficient in an enzyme known as PIG-A, which is required for the biosynthesis of cellular anchors. The PIG-A gene is found on the X chromosome.
  • PNH is a genetic disorder, known as an acquired or somatic genetic disorder (Bessler M, Mason PJ, Hillmen P, Miyata T, Yamada N, Takeda J, Luzzatto L, Kinoshita T. Paroxysmal nocturnal haemoglobinuria (PNH) is caused by somatic mutations in the PIG-A gene. EMBO J 1994 Jan 1; 13(1): 1 10-7).
  • Phenylketonuria is an inherited error of metabolism caused by a deficiency in the enzyme phenylalanine hydroxylase. Loss of this enzyme results in mental retardation, organ damage, unusual posture and can, in cases of maternal PKU, severely compromise pregnancy.
  • Classical PKU is an autosomal recessive disorder, caused by mutations in both alleles of the gene for phenylalanine hydroxylase (PAH), found on chromosome 12. In some cases, mutations in PAH will result in a phenotypically mild form of PKU called hyperphenylalanemia. Both diseases are the result of a variety of mutations in the PAH locus (DiLella AG, Marvit J, Brayton K, Woo SL. An amino-acid substitution involved in phenylketonuria is in linkage disequilibrium with DNA haplotype 2. Nature 1987 May 28- Jun 3;327(6120):333-6).
  • Refsum disease is a rare disorder of lipid metabolism that is inherited as a recessive trait. Symptoms may include a degenerative nerve disease (peripheral neuropathy), failure of muscle coordination (ataxia), retinitis pigmentosa (a progressive vision disorder), and bone and skin changes. Refsum disease is characterized by an accumulation of phytanic acid in the plasma and tissues. In 1997 the gene for Refsum disease was identified and mapped to chromosome 10.
  • the protein product of the gene, PAHX is an enzyme that is required for the metabolism of phytanic acid (Jansen GA, Ofman R, Gustavusse S, Ijlst L, Muijsers AO, Skjeldal OH, Stokke O, Jakobs C, Besley GT, Wraith JE, Wanders RJ. Refsum disease is caused by mutations in the phytanoyl-CoA hydroxylase gene. Nat Genet 1997 Oct;17(2): 190-3).
  • X-linked liver glycogenosis is probably the most frequent glycogen-storage disease.
  • XLG can be divided into two subtypes: XLG I, with a deficiency in phosphorylase kinase (PHK) activity in peripheral blood cells and liver; and XLG II, with normal in vitro PHK activity in peripheral blood cells and with variable activity in liver.
  • Both types of XLG are caused by mutations in the same gene, PHKA2, that encodes the regulatory alpha subunit of PHK (Hendrickx J, Lee P, Keating JP, Carton D, Sardharwalla IB, Tuchman M, Baussan C, Willems PJ.
  • PHKA2 phosphorylase kinase
  • Cystic fibrosis is the most common fatal genetic disease in the US today. It causes the body to produce a thick, sticky mucus that clogs the lungs, leading to infection, and blocks the pancreas, stopping digestive enzymes from reaching the intestines where they are required to digest food. CF is caused by a defective gene, which codes for a sodium and chloride transporter found on the surface of the epithelial cells. Several hundred mutations have been found in this gene, all of which result in defective transport of salt ions. CF research has accelerated sharply since the discovery of
  • Diastrophic dysplasia is a rare growth disorder in which patients are usually short, have club feet and have malformed hands and joints.
  • the gene whose mutation results in DTD maps to chromosome 5 and encodes a novel sulfate transporter. This ties in with the observation of unusual concentrations of sulfate in various tissues of DTD patients (Hastbacka J, de la Chapelle A, Mahtani MM, Clines G, Reeve-Daly MP, Daly M, Hamilton BA, Kusumi K, Trivedi B, Weaver A, et al.
  • LQTS Long-QT syndrome
  • Menkes' syndrome is an inborn error of metabolism that markedly decreases the cells' ability to absorb copper. The disorder causes severe cerebral degeneration and arterial changes, resulting in death in infancy. Menkes' disease is transmitted as an X-linked recessive trait.
  • a number of other diseases including type IX Ehlers-Danlos syndrome, are the result of allelic mutations (i.e. mutations in the same gene, but having slightly different symptoms) and research into these genes is useful in finding cure (Chelly J, Tumer Z, Tonnesen T, Petterson A, Ishikawa-Brush Y, Tommerup N, Horn N, Monaco AP. Isolation of a candidate gene for Menkes disease that encodes a potential heavy metal binding protein. Nat Genet 1993 Jan;3(l): 14-9).
  • Pendred syndrome (osteochondrodysplasia) is an inherited disorder that accounts for as much as 10% of hereditary deafness. Patients usually also suffer from thyroid goiter.
  • the normal gene makes a protein, called pendrin, that is found at significant levels only in the thyroid and is closely related to a number of sulfate transporters. When the gene for this protein is mutated, the person carrying it will exhibit the symptoms of Pendred syndrome.
  • the discovery of pendrin should also stimulate new angles of research into thyroid physiology and the role of altered sulfur transport in human disease (Kopp P. Pendred's syndrome: identification of the genetic defect a century after its recognition. Thyroid 1999 Jan;9(l):65-9).
  • APKD Advanced polycystic kidney disease
  • APCD Advanced polycystic kidney disease
  • PKDl a gene from chromosome 16 that was disrupted in a family with APCD.
  • the protein encoded by the PKDl gene is an integral membrane protein involved in cell-cell interactions and cell-matrix interactions.
  • APKD The International Polycystic Kidney Disease Consortium. Polycystic kidney disease: the complete structure of the PKDl gene and its protein. Cell 1995 Apr 21;81(2):289-98).
  • Wilson's disease is a rare autosomal recessive disorder of copper transport, resulting in copper accumulation and toxicity to the liver and brain.
  • the cornea of the eye can also be affected: the 'Kayser-Fleischer ring' is a deep copper-colored ring at the periphery of the cornea.
  • the gene for Wilson's disease (ATP7B) was mapped to chromosome 13. The sequence of the gene was found to be similar to sections of the gene defective in Menkes disease. These genes will be useful for studying copper transport and liver pathophysiology, and are useful in the development of a therapy for Wilson disease (Bull PC, Thomas GR, Rommens JM, Forbes JR, Cox DW.
  • Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene. Nat Genet 1993 Dec;5(4):327- 37).
  • Zellweger syndrome (ZS) is a hereditary disorder affecting infants, and usually results in death.
  • the PXR1 gene has been mapped to chromosome 12; mutations in this gene cause ZS.
  • the PXR1 gene product is a receptor found on the surface of peroxisomes - microbodies that carry out a number of metabolically important reactions such as cellular lipid metabolism and metabolic oxidations (Marynen P, Fransen M, Raeymaekers P, Mannaerts GP, Van Veldhoven PP.
  • the gene for the peroxisomal targeting signal import receptor (PXR1 ) is located on human chromosome 12pl3, flanked by TPI1 and D12S1089.
  • PPARgamma protein associated with bone mineral density (BMD) and osteoporosis in postmenopausal women
  • BMD bone mineral density
  • osteoporosis in postmenopausal women
  • Peroxisome Proliferator-Activated Receptor gamma Gene PPARgamma Expression in Osteoblasts.
  • Paramyotonia congenita is a temperature-sensitive skeletal muscle disorder caused by missense mutations that occur in the adult skeletal muscle voltage-gated sodium channel genes (Bendahhou S, Cummins TR, Kwiecinski H, Waxman SG, Ptacek LJ. Characterization of a new sodium channel mutation at arginine 1448 associated with moderate paramyotonia congenita in humans. Nat Genet
  • A-T telangiectasia
  • cerebellar degeneration characterized by cerebellar degeneration, immunodeficiency, radiosensitivity (sensitivity to radiant energy, such as x- ray) and a predisposition to cancer.
  • radiosensitivity sensitivity to radiant energy, such as x- ray
  • predisposition to cancer.
  • the gene responsible for A-T was mapped to chromosome 11.
  • the diverse symptoms seen in A-T reflect the main role of ATM, which is to induce several cellular responses to DNA damage.
  • 5-alpha reductase is an enzyme that was first discovered in the prostate. Here, it catalyzes the conversion of testosterone to dihydrotestosterone, which in turn binds to the androgen receptor and initiates development of the external genitalia and prostate.
  • the gene for 5-alpha reductase has been mapped to chromosome 5. More recently, 5-alpha reductase was found in human scalp and elsewhere in the skin, where it carries out the same reaction as in the prostate.
  • Cockayne syndrome is a rare inherited disorder in which people are sensitive to sunlight, have short stature and have the appearance of premature aging. In the classical form of Cockayne syndrome
  • Cockayne syndrome the symptoms are progressive and typically become apparent after the age of one year. An early onset or congenital form of Cockayne syndrome (Type II) is apparent at birth. Unlike other DNA repair diseases, Cockayne syndrome is not linked to cancer. Two genes defective in Cockayne syndrome, CSA and CSB, have been identified so far. Both genes code for proteins that interacts with components of the transcriptional machinery and with DNA repair proteins. Defects in the XPB, XPD, and XPG genes can result in three different syndromes Cockayne syndrome, xeroderma pigmentosum, or trichothiodystrophy, depending on the specific mutation involved (van Gool AJ, van der Horst GT, Citterio E, Hoeijmakers JH. Cockayne syndrome: defective repair of transcription? EMBO J 1997 Jul 16; 16 (14):4155-62).
  • Glaucoma is a general term used for a group of diseases that can lead to damage to the eye's optic nerve and result in blindness.
  • the most common form of the disease is open-angle glaucoma, which affects about three million Americans, half of whom don't know they have it.
  • Glaucoma has no symptoms at first but over the years can steal its victims' sight, with side vision being effected first. It is estimated that nearly 100,000 individuals in the US suffer from glaucoma due to a mutation in the GLC1A gene, found on chromosome 1.
  • PCG Primary congenital glaucoma
  • CYPIBI cytochrome P4501B1
  • SRY (sex-determining region Y gene) is found of the Y chromosome. In the cell, it binds to
  • Tuberous sclerosis is an hereditary disorder characterized by benign, tumor-like nodules of the brain and/or retinas, skin lesions, seizures and/or mental retardation. Patients may experience a few or all of the symptoms with varying degrees of severity.
  • Two loci for tuberous sclerosis have been found: TSCl on chromosome 9, and TSC2 on chromosome 16. It took four years to pin down a specific gene from the TSCl region of chromosome 9: in 1997, a promising candidate was found.
  • TSC2 codes for a protein called tuberin, which, through database searches, was found to have a region of homology to a protein (GAP3, a GTPase- activation protein) found in pathways that regulate the cell (The European Chromosome 16 Tuberous Sclerosis Consortium. Identification and characterization of the tuberous sclerosis gene on chromosome 16.
  • Deletions involving chromosome 9 occur in more than 50% of human bladder cancers of all grades and stages.
  • a critical region of deletion at 9q34 between the markers D9S149 and D9S66, an interval which contains the TSC 1 gene acts as a bladder tumour suppressor gene (Hornigold N, Devlin J, Davies AM, Aveyard JS, Habuchi T, Knowles MA. Mutation of the 9q34 gene TSCl in sporadic bladder cancer. Oncogene 1999 Apr 22;18(16):2657-61).
  • Werner syndrome is a premature aging disease. Its physical characteristics may include short stature (common from childhood on) and other features usually developing during adulthood: wrinkled skin, baldness, cataracts, muscular atrophy and a tendency to diabetes mellitus, among others. The disorder is inherited and transmitted as an autosomal recessive trait.
  • the gene for Werner disease (WRN) was mapped to chromosome 8 and cloned: by comparing its sequence to existing sequences in GenBank, it is a predicted helicase (DNA unwinder important for DNA replication) belonging to the RecQ family.
  • Miscellaneous other genes involved in clinical disorders include but are not limited to beta-globin, phenylalanine hydroxylase, alpha-antitrypsin, 21- hydroxylase, pyruvate dehydrogenase, dihydropteridine reductase, rhodopsin, nerve growth factor, superoxide dismutase, adenosine deaminase, beta-thalassemia, ornithine transcarbamylase, collagen, beta-hexosaminidase, topoisomerase II, hypoxanthine phosphoribosyltransferase, phenylalanine 4- monooxygenase, Factor VIII, Factor IX, nucleoside phosphorylase, glucose- 6-phosphate dehydrogenase, phosphoribosyltransferase.
  • CJD Creutzfeldt- Jakob disease

Abstract

La présente invention concerne des compositions et des procédés de criblage, de séquençage et/ou de mesure quantitative d'un acide nucléique intéressant au moyen de l'hybridation de cet acide nucléique avec un ensemble de sondes oligonucléotidiques qui sont couplées à des microparticules multicolores adressables par fluorescence. Ces microparticules sont en suspension dans un réseau liquide qui peut être placé dans des puits ou des cupules à réaction prédéterminés d'une plaque à microtitration. Pour des applications de séquençage, chaque réseau liquide de ce type comprend de préférence une quelconque combinaison des séquences possible pour une longueur donnée de la sonde. L'hybridation s'effectue par la reconnaissance complémentaire de l'analyte intéressant avec une sonde. La sonde, la cible et/ou la molécule concurrente sont marquées avec une molécule reporteur pour que, au moment de l'hybridation, les changements des paramètres du signal de fluorescence soient enregistrés et analysés.
EP00955728A 1999-08-20 2000-08-21 Technologie des reseaux liquides Withdrawn EP1248853A2 (fr)

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