WO2000039344A2 - Dispositif d'essai a sondes mixtes - Google Patents

Dispositif d'essai a sondes mixtes Download PDF

Info

Publication number
WO2000039344A2
WO2000039344A2 PCT/US1999/031149 US9931149W WO0039344A2 WO 2000039344 A2 WO2000039344 A2 WO 2000039344A2 US 9931149 W US9931149 W US 9931149W WO 0039344 A2 WO0039344 A2 WO 0039344A2
Authority
WO
WIPO (PCT)
Prior art keywords
probes
detection elements
substrate
target
complementary
Prior art date
Application number
PCT/US1999/031149
Other languages
English (en)
Other versions
WO2000039344A3 (fr
Inventor
Glenn Hoke
Adam B. Steele
John G. Hartwell
Original Assignee
Gene Logic Inc.
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Gene Logic Inc. filed Critical Gene Logic Inc.
Priority to EP99967727A priority Critical patent/EP1141413A2/fr
Priority to CA002356946A priority patent/CA2356946A1/fr
Priority to AU23959/00A priority patent/AU2395900A/en
Publication of WO2000039344A2 publication Critical patent/WO2000039344A2/fr
Publication of WO2000039344A3 publication Critical patent/WO2000039344A3/fr

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • 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
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • 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
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00585—Parallel processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • 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
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • 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
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • 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
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00612—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports the surface being inorganic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • 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
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00614—Delimitation of the attachment areas
    • B01J2219/00621—Delimitation of the attachment areas by physical means, e.g. trenches, raised areas
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • 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
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00623—Immobilisation or binding
    • B01J2219/00626—Covalent
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • 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
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00632—Introduction of reactive groups to the surface
    • B01J2219/00637—Introduction of reactive groups to the surface by coating it with another layer
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • 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
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00639—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium
    • B01J2219/00641—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium the porous medium being continuous, e.g. porous oxide substrates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • 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
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • 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
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00702—Processes involving means for analysing and characterising the products
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • 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

Definitions

  • the invention provides improved devices and methods for detecting analytes in a sample, such as nucleic acids.
  • Assay arrays have widespread application for efficient and rapid characterization of a test sample. Arrays derive their efficiency from combining a large number of singular, discrete tests into one, complex test. Recently, the value of arrays confined within small dimensions, so called “microarrays,” also has been recognized.
  • microarrays for biological assays has expanded rapidly.
  • the promise of rapid sample analysis by detecting binding between one or more components of a test sample, for example a pool of cDNA, and an array of immobilized binding reagents, such as DNA or oligonucleotides, has led to the development of a variety of methodologies useful in characterizing a test sample.
  • microarrays have been used in protocols for gene discovery, gene expression, gene mapping and mutation detection.
  • a traditional approach to characterizing a test sample is to perform a series of separate, discrete tests on the sample.
  • the test sample or target compound is divided into separate portions or aliquots, and each portion then is evaluated by a different assay, where each assay utilizes a particular binding reagent with a defined specificity.
  • Blood bank analyzers typically split each blood sample into small aliquots that are analyzed separately to determine each measured property.
  • This strategy becomes cumbersome as the number of tests increase and suffers from a dilution-by-aliquoting effect. For example, the amount of sample available for each assay diminishes in direct proportion to the number of assays to be performed on the sample. Therefore, a thorough characterization of a test sample by this strategy requires relatively large sample volumes.
  • This strategy also requires separate analysis channels for each test, thereby increasing the complexity of the analytical instrumentation in proportion to the number of tests examined. In sum, this approach limits the amount of sample available to each test, requires additional sample manipulation and increases apparatus complexity.
  • An alternative approach to characterizing a test sample is to use microarrays to perform multiple tests on the sample simultaneously.
  • Devices employing this strategy separate specific binding reagents or "probes" into discrete locations in a defined pattern on a surface and expose all of the probes to the sample at the same time. Since all of the binding probes access the sample in a single step, there is no need to divide the sample, and the dilution-by-aliquoting effect discussed in the previous approach is avoided.
  • This strategy simplifies the testing process and reduces the complexity of the apparatus and, consequently, its cost. The benefits of this approach become more apparent as the number of tests performed on a sample increases.
  • the quantity of binding probe that may be applied to the surface of the substrate is limited by the amount of surface per unit area. This parameter governs the yield of bound target and, thus, the detection sensitivity. Since a goal of such microarrays is to determine the presence of subtle changes in the levels of particular targets in a test sample, the sensitivity of a microarray largely determines its usefulness.
  • diffusion is the rate-limiting step of binding. On a flat surface, the time required for target molecules to diffuse over relatively long distances before encountering complementary binding probes can be up to several hours. Such time-consuming hybridizations reduce the efficiency of the assay and preclude use of such microarrays in point-of-care testing. Rapid, on-site analysis, while the patient is still in the office, allows for immediate treatment or counseling decisions.
  • Microarrays utilizing microchannels overcome some of the inherent limitations observed in two dimensional arrays.
  • MicroChannel devices have been described and are defined as comprising a substrate having oppositely facing first and second major surfaces, a multiplicity of discrete channels extending through said substrate from said first major surface to said second major surface and a binding reagent immobilized on the walls of said channels was described. (See Beattie, U.S. Patent No. 5,843,767).
  • Microchannels improve detection sensitivity due to the vastly increased surface area within the channels, which increases the quantity of binding reagent bound per cross sectional area.
  • microchannels also permits a flow-through strategy to be employed which ⁇ -in- ⁇ riizes the rate-limiting diffusion step preceding the hybridization reaction by reducing the time required for the average target molecule to encounter a surface- tethered probe from hours to milliseconds.
  • microarrays have been instrumental in decoding complex biological systems and have improved the characterization of target molecules, improvements in accessibility and sensitivity are required to maximize the benefit of microarray technology.
  • An important component in the effectiveness of microarrays is exposure of target molecules to as many binding probes as possible. Consequently, binding probes that are not presented adequately to target molecules do not add value to the array.
  • the processes employed to construct arrays are extremely complex, requiring highly technical machinery and lengthy manufacturing times. The high costs associated with these aspects are prohibitive and prevent the widespread use of the technology.
  • cDNA may be prepared from cellular mRNA, and then regions of the cDNA subjected to amplification by the polymerase chain reaction.
  • the cDNA may be fractionated by incorporation of a digestible nucleotide during the reverse transcription step from the mRNA.
  • a proportion of the dT nucleotides added to the reverse transcription step can be replaced with dU, providing a reverse transcript containing a controllable proportion of dU residues.
  • cRNA may be prepared that provides multiple RNA copies of certain regions of the original nucleic acid of interest. See for example US Patent Nos. 5,545,522 and 5,514,545.
  • This fractionation is used to produce a variety of short nucleic acid targets that are representative of the DNA or RNA pools that are of interest.
  • These shorter nucleic acid targets are detectably labeled and applied to the array which is comprised of a solid support with attached oligonucleotide-based probes.
  • the probes located within each individual spot have a single nucleic acid sequence that is specific for a particular locus within a gene or mRNA of interest.
  • the generation of shorter nucleic acid targets is a random event which produces targets having a variety of sequences derived from the original gene or RNA of interest. These multiple sequences usually are of varying length, and many of the sequences are not perfectly complementary to the nucleic acid probes contained within a spot. As a result, the majority of the labeled targets do not stably hybridize and cannot produce signals for detection.
  • an improved assay device that reduces background and enhances the probability or generating a signal from low abundance target molecules.
  • the present invention provides improved devices for detecting the presence of target molecules.
  • the devices use arrays of detection elements disposed in separate and spatially distinct sites, where each detection element can contain a plurality of probe molecules that hybridize to the target molecules. These devices allow improved detection of target molecules compared to conventional arrays.
  • each spatially isolated detection element contains a single probe that is complementary to a specific target molecule.
  • the nucleic acid sources When such arrays are used to detect, for instance, gene expression in a tissue or cell ("the nucleic acid sources"), a mixture of target nucleic acids first is prepared that is representative of the amount and types of the various mRNA molecules produced from the nucleic acid source. For example, reverse transcription-polymerase techniques (RT-PCR) have been used to prepare double stranded cDNA segments that correspond to portions of the mRNAs produced in the nucleic acid source.
  • cRNA may be prepared that provides multiple RNA copies of certain regions of the original nucleic acid of interest, as described, for example in US Patent Nos. 5,545,522 and 5,514,545.
  • these techniques would provide a single detectable target molecule that is representative of the identity and quantity of that mRNA.
  • these techniques tend to produce a mixture of target moieties, each of which can be considered representative of the original mRNA.
  • the mixture may contain target molecules of differing length, representing different regions of the original mRNA. These regions may be overlapping, may only overlap by a few nucleotides, may be juxtaposed, or may not overlap at all and be separated by many nucleotides. Accordingly, a detection element containing a single probe cannot efficiently hybridize to every target molecule that represents an original mRNA molecule, resulting in a potential loss of information.
  • One potential way to overcome this in a conventional array is to dedicate several different detection elements to probes for detecting target molecules representative of the original mRNA.
  • some probes could be complementary to a 5' region of the mRNA, while others could be complementary to an internal sequence or a 3' sequence.
  • This approach has several disadvantages, however.
  • use of multiple detection elements for detecting different targets representative of a single mRNA significantly reduces the number of targets that can be assayed with a single substrate.
  • the amount of target bound at each detection element must exceed the lower limit of sensitivity for the detection system used to identify binding. If target molecules representative of a single mRNA are dispersed between several detection elements, the level of binding at each detection element might not be detectable, even where the aggregate amount of target nucleic acid would comfortably exceed this detection limit.
  • each detection element can comprise a plurality of probe molecules.
  • the probe molecules typically are nucleic acids such as DNA or RNA, but the invention is not so limited.
  • the probe molecules can be other binding moieties such as antibodies or their antigens, receptors or their ligands, or other binding molecules known in the art.
  • the resulting arrays of detection elements are effective for carrying out analytical tasks such as analysis of patterns of gene expression by hybridization of mRNA, cDNA, or cRNA to gene-specific probes, immunochemical analysis of protein mixtures, assay of receptor-ligand interactions and profiling of cellular populations involving binding of cell surface molecules to specific ligands or receptors. Other analytical tasks will be apparent to those skilled in the art.
  • the probes within an individual detection element are each complementary to different sequences within a target molecule representative of a single expressed gene.
  • a variety of target molecules that would be missed by a single probe, but that are target molecules that are representative of an expressed gene are captured at a single detection element.
  • This enhanced capture reduces the number of detection elements that are needed to analyze a given set of expressed genes.
  • the method can improve the sensitivity of detection of the target molecules, because the detectable signal from the mixture of target molecules is additive and is more likely to exceed the sensitivity limit of the detector. With this increased signal, the capacity to detect low abundance targets is increased.
  • Another advantage of the present invention is that it allows relatively short probe molecules to be used, as well as capturing target molecules that are representative of all regions of the expressed gene.
  • probes such as full length cDNAs or significantly longer nucleic acid probes are used, then there is an increased potential for non-specific hybridization, which increases background or produces false positive results.
  • hybridization is specific and random partial hybridization events are less likely to occur, reducing background.
  • the probes are about less than 200 bases long, although the skilled artisan will recognize and determine the optimum length of the probes that would be useful in the present invention using techniques well known to persons in the art.
  • a target molecule is "representative" of an expressed gene when it contains a contiguous sequence of nucleotides that is identical to a sequence found within the expressed gene.
  • the contiguous sequence is at least 15 nucleotides in length. More preferably, the contiguous sequence is at least 30, 50, 100, 200 or 500 bases in length.
  • the target molecule preferably is cDNA or mRNA.
  • the probe molecules contained within a particular detection element are complementary to different sequences within the expressed gene. These different species may have any degree of overlap: they may overlap completely or only slightly, they may be juxtaposed, or they may not overlap at all.
  • the particular probe sequences used, and the proportion of the individual probes contained within an individual detection element can be optimized depending on the method used to prepare the mixture of target molecules. For example, when the target molecules are cRNA prepared according to the methods of US Patent No. 5,716,785, the composition of the target molecule mixture tends to be weighted towards the 3' end of the expressed gene. Accordingly, the individual detection elements may contain a majority of probes complementary to sequences contained at the 3' end of the expressed gene. Optimization of the mixture of probes at a particular detection element can be carried out, if desired, using target molecule mixtures of known composition, by measuring the detectable signal obtained with particular probe mixtures.
  • Genomic DNA contains the messenger RNA sequence interspersed with intron sections.
  • the presence of genomic DNA in a sample is undesirable because it can diminish the measurable differential gene expression between two samples due to its presence at a constant level in both samples.
  • Probes can be designed for one or more of the intron regions for a selected gene and placed within a single spot, thereby providing a means for detecting genomic DNA contaudination in a sample.
  • the mixed probe sets contained within individual detection elements are useful in essentially any of the known microarray formats used for assaying gene expression, and also in array formats that are yet to be discovered.
  • the mixed probe sets are useful in microchannel arrays of the type described in US Patent No. 5,843,767, and also in arrays where the probes are confined to a two- dimensional substrate surface, such as those described in US Patent No. 5,744,305.
  • the substrate may be fabricated from any material that is capable of being stably affixed with nucleic acid probe molecules.
  • the substrate may be fabricated from silicon, nylon, or glass, or may be any material that can be coated with a gold film to which thiol-containing nucleic acid probes can be affixed.
  • the method of affixation can be covalent or non-covalent, provided that the probe molecule is retained on the substrate during hybridization conditions.
  • Other suitable substrates are described at columns 7-8 of US Patent No. 5,482,867, which patent is hereby incorporated by reference in its entirety.
  • target preparations are derived from the 3' end of m-RNAs, although preparations derived from the 5' end, and from internal portions of the mRNA sequence, can be used.
  • a set of probes that is unique to the mRNA is chosen using sequence analysis techniques that are well known in the art. The probes are prepared as single compounds using known methods.
  • the probes that make up an individual detection element may then be mixed and affixed to the substrate, or may be affixed sequentially or a combination thereof.
  • each probe typically is affixed using the same process, for example via the same type of covalent bond.
  • each probe may contain a 5' or 3' terminal thiol moiety which can react covalently with a surface that contains N-ethyl maleimide residues.
  • the affixation method can be varied for each probe, if necessary. Sequential application in this fashion permits use of affixation methods that cannot be carried out in a single step.
  • base-catalyzed and acid-catalyzed affixation steps can be carried out sequentially, but cannot be carried out simultaneously.
  • Other methods of affixation are known in the art and also are described in more detail below.
  • Synthesis of the probes as individual molecules also allows optimization of the binding probes.
  • the probes can be prepared with or without linker molecules used for affixation to the substrate surface, and the nature and length of the linkers can be varied.
  • the probes can be prepared to allow their affixation in either possible orientation (5'-»3' or 3' ⁇ 5').
  • a thiol group used to affix the probe to a surface may be placed at either te ⁇ ninus of the probe or, indeed, may be placed at an internal site, if desirable.
  • Each probe sequence may be optimized against a target molecule mixture before affixation to the substrate.
  • the probes within single detection element can have different chemical structures.
  • the probes may be DNA, RNA, phosphorothioate-linked oligonucleotides, 2'-O-methoxy linked oligonucleotides, or peptide nucleic acids.
  • the skilled artisan will recognize that any molecular structure capable of binding to DNA or RNA in a sequence-specific manner can be used as a probe molecule in the invention.
  • Probes can be produced from cDNAs by fragmentation and incorporation of a reactive chemistry, for example by incorporation of one or more nucleotides contaming a reactive moiety such as an amine group. Probes also can be produced for novel or unknown targets that are isolated from a target mixture by, for example, gel electrophoresis.
  • Reverse transcription ("RT") of an mRNA or PCR amplification of a DNA can be used to prepare a probe from a nucleic acid isolated from a band on a gel (or from a peak detected in a capillary electrophoresis) by extension of a primer.
  • the extended sequences resulting from the RT or PCR steps are randomly terminated by incorporation of a terminating nucleotide (e.g. dideoxyNTP) in the RT or PCR step.
  • This procedure will produce a variety of probes of varying length, each having a common 5' end (the primer sequence), but where all the probes recognize a single gene.
  • Optimal procedures for attachment of nucleic acids to silicon dioxide surfaces can be based on well-established silicon chemistry (Parkam et al. , Biochem. Biophys. Res. Commun., 1:1-6 (1978); Lund et al , Nucl. Acids Res. 16:10861-10880, (1988)).
  • This chemistry is used to introduce a linker group onto the glass bearing a terminal epoxide moiety that specifically reacts with a terminal primary amine group on the oligonucleotide.
  • This versatile approach (using epoxy silane) is inexpensive and provides a dense array of monolayers that can be readily coupled to terminally modified (amino- or thiol-derivatized) oligonucleotides.
  • linker groups for example an amine or a mercaptan
  • the linker group should be reactive toward moieties that can be incorporated into a biomolecule, or toward a cross- linking agent that is reactive with a moiety that can be incorporated into a biomolecule. See for example U.S. Patent No. 5,077,210, which is herein incorporated by reference.
  • the density of probe attachment may be controlled over a wide range by mixing long chain amino alcohols with the amine-derivatized oligonucleotides during attachment to epoxysilanized glass.
  • This strategy essentially produces a monolayer of tethered DNA, interspersed with shorter chain alcohols, resulting in attachment of oligonucleotides down to about 5 run apart on the surface.
  • Variable length spacers are introduced optionally onto the ends of the oligonucleotides, by incorporation of triethylene glycol phosphoryl units during the chemical synthesis. These variable linker arms are useful for determining how far from the substrate surface oligonucleotide probes should be separated to be readily accessible for pairing with the target DNA strands.
  • Thiol chemistry adapted from the method of Whitesides and coworkers on the generation of monolayers on gold surfaces (Lee et al. Pure & Appl. Chem. 63:821-828 (1991) and references cited therein.), may be used for attachment of DNA to gold and platinum surfaces.
  • Dithiols e.g., 1 JO-decanedithiol
  • Dithiols are linked at one terminus to the metal surface, and also provide a terminal, reactive thiol moiety for reaction with bromoacetylated oligonucleotides.
  • the density of attachment of DNA to gold or platinum surfaces is controlled at the surface- activation stage by use of defined mixtures of mono- and dithiols.
  • linker groups for example an amine or a carboxylic acid
  • the linker group should be reactive toward moieties that can be incorporated into a biomolecule (for example aldehydes or amines), or toward a cross-linking agent that is reactive with a moiety that can be incorporated into a biomolecule.
  • Suitable cross-linking agents are known in the art and are available, for example, from Pierce Chemical Co. (Rockford, IL).
  • the silicon surface may be halogenated with, for example, a thionyl halide or a phosphoryl halide, or with an elemental halogen, to produce a halogenated surface.
  • the halogenated surface may then be derivatized, either directly with a nucleic acid probe containing a nucleophilic amine or thiol moiety, or with a linker that then can be used to couple to a probe molecule. See (Bergerson et al. J. Am. Chem. Soc. 121:454-455 (1999), which is incorporated herein by reference.
  • the halogen is chlorine.
  • the present invention requires precise application of the probes or probe mixtures to discrete locations on the substrate surface of a device. As the variety and replication of probes in a microarray may be very large, their accurate and precise application on the device is a considerable technical challenge.
  • a variety of devices have been used to make microarrays by spotting probes onto a surface.
  • Mechanical pen-like spotting devices have been developed that deliver the desired probes by contacting the substrate surface.
  • such a device has been used to spot 8,192 oligonucleotide probes onto a flat surface.
  • Another device used for spotting probes directly onto a surface is an ink jet-like dispensing device. Such a device has been employed to dispense oligonucleotide probes directly onto the surface of a charge-coupled device.
  • the device may be used in conjunction with detection technologies that are known in the art that are capable of discriminating between regions in which binding has taken place and those in which no binding has occurred.
  • the detection methodology is capable of quantitating the relative extent of binding in different regions.
  • DNA and RNA sequence detection advantageously may be used, although the skilled artisan will recognize that other detection methodologies, including methods to be developed in the future, may be used.
  • Autoradiography may be performed, for example, using 32 P or 35 S labelled samples, although the skilled artisan will recognize that other radioactive isotopes also may be used.
  • a highly preferred method of detection is a charge-coupled-device array or CCD array.
  • CCD array With the CCD array, a individual pixel or group of pixels within the CCD array is placed adjacent to each confined region of the substrate where detection is to be undertaken. Light attenuation, caused by the greater absorption of an illuminating light in test sites with bound molecules, is used to determine the sites where binding has taken place. Lens-based CCD cameras and microscope imaging also can be used.
  • a detection apparatus can be constructed such that sensing of changes in AC conductance or the dissipation of a capacitor placed contiguous to each conformed region can be measured.
  • RF radio-frequency
  • NCG Nanochannel glass
  • NCG materials are glass structures containing a regular geometric array of parallel holes or channels as small as several nm in diameter (e.g. 33 nm) or as large as a hundred micrometers or more in diameter. See Tonucci et al. , Science 258:783-785 (1992), and US Patent No. 5,234,594 which are incorporated herein by reference in their entireties.
  • These nanochannel glass structures can be fabricated in various array configurations to provide a high surface area to volume ratio, and can possess packing densities in excess of 3xl0 10 channels per square centimeter.
  • a variety of materials can be immobilized or fixed to the glass surfaces within the channels of the NCG array.
  • Nanochannel glass arrays are fabricated by arranging dissimilar glasses in a predetermined configuration where, preferably, at least one glass type is acid etchable.
  • a two-dimensional hexagonal close packing array is assembled from etchable glass rods (referred to as the channel glass) and an inert glass tube (referred to as the matrix glass).
  • the pair is then drawn under vacuum to reduce the overall cross-section to that of a fine filament.
  • the filaments are then stacked, re-fused and redrawn. This process is continued until appropriate channel diameters and the desired number of array elements are achieved.
  • the ratio of the diameter of the etchable glass rod to that of the outside dimension of the inert glass tubing, the center-to-center spacing of the rods and their diameters in the finished product become independently adjustable parameters. See Tonucci, supra.
  • the NCG material is wafered perpendicular to the direction of the channels with a diamond saw and then polished to produce sections of material having a defined thickness, for example, about 0.1 mm to about 1.0 mm.
  • the channel glass of the array structure is then etched away with an acid solution.
  • the opposing faces of the substrate need not be parallel, and the substrate may be thinner or thicker than about OJ mm to about 1.0 mm.
  • the thickness of the substrate also can range from about 10 ⁇ m to about 250 ⁇ m, from about 50 to about 500 ⁇ m, from about 250 ⁇ m to about 1.5 mm, or about 500 ⁇ m to about 2 mm thick.
  • the cross-sectional configuration of the channels may be varied.
  • the geometry of the channels may include, but is not limited to, a circular or hexagonal cross-section.
  • a hexagonal close packing arrangement of channel glasses which, after acid etching, contains typically 10 7 channels that are uniformly dispersed in the substrate.
  • the channel diameter is typically 450 nm and the center-to-center spacing is approximately 750 nm.
  • the channel diameter can be wider or narrower than 450 nm, and the center-to-center spacing also may be varied. Variation in the channel geometry allows for design of variation in the density of the channels in the substrate.
  • a second example of hexagonal array structure is one in which separated clusters of channels are formed during the fabrication process.
  • an open array structure with typical channel diameters of 300 nm in which the overall glass structure consists of an array of 18 ⁇ m diameter subarrays, spaced typically 25 ⁇ m apart from neighboring arrays.
  • Silicon designs containing channels are advantageously employed because of their adaptability to low cost mass production processes and their ability to incorporate in the fabrication process structural elements that function in fluidic entry and exit from the hybridization site and structures (e.g., electrodes) that may function in hybridization detection.
  • Stable, open-cell materials containing channels between first and second surfaces of the material are used to accomplish enhancements and to introduce qualitatively new features in these devices, whereby the surface area of discrete and isolated binding regions comprises groups of channels that are increased by a factor of 100 to 1000 relative to a two- dimensional surface.
  • Thin-film processing technology can be used to deposit chemically inert and thermally stable microchannel materials. Materials and processing methods are selected to achieve low-cost semiconductor batch fabrication of integrated semiconductor detectors.
  • the microchip device provides in situ multi-site analysis of binding strength as ambient conditions are varied. Silicon materials containing channels are fabricated in oriented arrays with charmel diameters selected over the range from 2 nm to several micrometers. Random, interconnected pore arrays also can be made.
  • Porous silicon is produced most easily through electrochemical etching. It can be processed into two important channel structures, interconnected networks and oriented arrays.
  • the channel diameter is tailored from approximately 2 nm to micrometer dimensions by selection of doping and electrochemical conditions.
  • etching is thought to proceed through a tunneling mechanism in which electrons are injected into the channel surface through field concentration effects. In the case of p-material the mechanism seems to be through moderation of carrier supply at the electrolyte/silicon interface.
  • the following structures can be fabricated for use as suitable substrates for the present invention: i) dense oriented arrays of channels oriented with axis along ⁇ 100 > direction and with channel diameters in the range of 10 to 100 nm.
  • Characterization can be undertaken by scanning electron microscopy.
  • the surface wetting properties are varied using vapor treatment with silylation materials and chlorocarbons.
  • Oriented porous silicon also can be produced by track etching similar to that used to produce high channel-density dielectrics which function as molecular sieves are produced by nuclear track etching. While nuclear track etching is used to produce these molecular sieves in a wide range of inorganic materials, it is most often used with dielectrics such as mica and sapphire. In this method, described in U.S. Patent No. 3,303,085 (Price, et al.
  • a substrate is first bombarded with nuclear particles (typically several MeV alpha particles) to produce disturbances or "tracks" within the normal lattice structure of the material and then wet-etched to produce channels which follow the tracks caused by the nuclear particles.
  • nuclear particles typically several MeV alpha particles
  • Price et al. disclose that the exposure of a mica substrate to heavy, energetic charged particles will result in the formation of a plurality of substantially straight tracks in its lattice structure and that these tracks can be converted into channels by wet etching the substrate. The same approach can be used to form oriented channels in silicon substrates.
  • Channel sizes and density of the channels are variably controllable using track etching techniques with channels typically 0.2 ⁇ m in diameter and densities on the order of 10 9 /cm 2 , although narrower or broader channels can be generated, leading to greater or smaller channel densities.
  • Particle track depths are energy dependent on the incident particle beam. With typical beam energy, channels can be extended, for example, through an entire 500 ⁇ m-thick substrate. Incorporation of these materials on the device discussed above is readily accomplished.
  • the use of implantation-etched dielectrics as the sensor element has advantages versus the silicon approach since the material is hydrophilic.
  • the size of the silicon array wafers may be modified in a variety of ways without departing from the spirit of the invention.
  • binding reagent Delivery of binding reagent to defined locations within or upon a substrate is accomplished in certain embodiments using micro-spotting devices, as illustrated below.
  • a Hamilton Microlab 2200 robotic fluid delivery system equipped with special low volume syringes and 8-position fluid heads, capable of delivering volumes of 10-100 nl at 500 ⁇ m xyz stepping and a few percent precision.
  • 40-nl samples of probe solution are placed into the wells of the high density NCG wafer.
  • a piezoelectrically controlled substage custom fitted for the Microlab 2200 permits xy positioning down to submicron resolution. Custom fabricated needles are employed.
  • the eight-needle linear fluid head is operated in staggered repetitive steps to generate the desired close spacing across the wafer.
  • the system has a large stage area and rapid motion control, providing capacity to produce hundreds of replicate hybridization wafers.
  • a microjet system capable of delivering subnanoliter probe solutions to the wafer surface is employed as follows to form oligonucleotides and other assays: For placement of probe into individual hybridization sites within ultra-high density wafers, with volumes of one nl (corresponding to a 130 ⁇ m sphere diameter or 100 ⁇ m sphere or 100 ⁇ m cube) commercially available dispensing equipment using ink-jet technology as the microdispensing method for fluid volume below is employed.
  • the droplets produced using ink-jet technology are highly reproducible and can be controlled so that a droplet may be placed on a specific location at a specific time according to digitally stored image data.
  • Typical droplet diameters for demand mode ink-jet devices are 30-100 ⁇ m, which translates to droplet volumes of 14-520 pi.
  • Droplet creation rates for demand mode ink-jet devices are typically 2,000-5,000 droplets per second.
  • both the resolution and throughput of demand mode ink-jet microdispensing are in the ranges suitable for the ultrahigh density hybridization wafer.
  • the microdispensing system is modified from a MicroFab drop-on-demand ink-jet type device, hereafter called a Micro Jet device such that this type of device can produce 50 ⁇ m diameter droplets at a rate of 2000 per second.
  • a MicroFab drop-on-demand ink-jet type device hereafter called a Micro Jet device
  • the operating principles of this type of device are known (Wallace, "A Method of Characteristics Model of a Drop-On-Demand Ink- Jet Device Using an Integral Drop Formation Method," ASME publication 89-WA/FE-4, December 1989) and used to effect the modification.
  • To increase throughput eight of these devices are integrated into a line array less than 1 inch (25mm) long. The eight devices are loaded with reagent simultaneously, dispense sequentially, and flush simultaneously. This protocol is repeated until all of the reagents are dispensed.
  • cycle time is associated with loading and flushing reagents, limiting the advantages of a complex of parallel dispensing capability.
  • Typical cycle time required is as on the following order: 1 minute for flush and load of 8 reagents; 30 seconds to calibrate the landing location of each reagent; 15 seconds to dispense each reagent on one location of each of the 16 genosensors, or 2 minutes to dispense all 8 reagents.
  • Total time to load and dispense 8 reagents onto 16 sensors is thus 3.5 minutes.
  • Total time for 64 reagents onto 16 sensors would be 28 minutes.
  • the microdispensing system will consist of the subsystems listed below:
  • a Beckman Biomec is modified to act as the multiple reagent input system. Between it and the MicroJet dispense head are a system of solenoid valves, controlled by the system controller. They provide pressurized flushing fluid (deionized water or saline) and air to purge reagent from the system and vacuum to load reagent into the system.
  • pressurized flushing fluid deionized water or saline
  • X-Y Positioning System A commercially available precision X-Y positioning system, with controller, is used. Resolution of 0.2 ⁇ m and accuracy of 2 ⁇ m are readily obtainable. The positioning system is sized to accommodate 16 sensors, but MicroJet dispense head size, purge station, and the calibration station represent the main factors in dete ⁇ r-ining overall size requirements. 4. Vision System - A vision system is used to calibrate the "landing zone" of each MicroJet device relative to the positioning system. Calibration occurs after each reagent loading cycle. Also, the vision system locates each dispensing site on each sensor when the 16 sensor tray is first loaded via fiducial marks on the sensors. For economy, a software based system is used, although a hardware based vision system can be advantageously employed.
  • System Controller A standard PC is used as the overall system controller.
  • the vision system image capture and processing also reside on the system controller.
  • a stock solution of epoxysilane is freshly prepared with the following proportions: 4 ml 3-glycidoxypropyl-trimethoxysilane, 12 ml xylene, 0.5 ml N,N- diisopropylethylamine (Hunig's base). This solution is flowed into the channels of the wafer, followed by soaking for 5 hours in the solution at 80°C, followed by flushing with tetrahydrofuran, drying at 80°C, and drying in a vacuum desiccator over Drierite or in a desiccator under dry argon.
  • Oligonucleotide bearing 5'- or 3'-al-kylamine (introduced during the chemical synthesis) is dissolved at lO ⁇ M - 50 ⁇ M in water and flowed into the channels of the silica wafer. After reaction at 65°C overnight the surface is briefly flushed with water at 65 °C, then with lOmM triethylamine to cap off the unreacted epoxy groups on the surface, then flushed again with water at 65°C and air dried.
  • amine-derivatized oligonucleotides can be attached to epoxysilane-derivatized glass in dilute (eg., lOmM - 50mM) KOH at 37°C for several hours, although a higher background of nonspecific binding of target sample DNA to the surface (independent of base pairing) may occur during hybridization reaction.
  • dilute eg., lOmM - 50mM
  • a stock solution of mercaptosilane was freshly prepared with the following proportions: 1 ml 3-mercaptopropyl-trimethoxysilane, 49 ml toluene.
  • the wafer was incubated in this solution for 1 hour under ambient conditions, followed by rinsing with toluene and ethanol, drying at 80°C for 4 hours.
  • Derivatized wafers were stored in a desiccator cabinent until oligonucleotides are attached.
  • Oligonucleotides bearing 5'- or 3'- or internal alky lamine (introduced during the chemical synthesis) were dissolved at lOnM - 50 ⁇ M in lxSSC buffer and deposited into a group of channels on the wafer using one of the microdispensing methods described above. After reaction at room temperature in a light-tight dry-box for at least 4 hours, typically 12 - 24 hours, the surface was blocked with a 1 % solution of polyvinylpyroUidone and FicollTM and dried at 80°C for 1 hour.
  • a method of flowing the liquids through the wafer was used.
  • the wafer was packaged within a 2 mm x 4 mm polypropylene frame, which served as an upper reservoir and structure for handling.
  • a polypropylene vacuum chamber with a Delrin o- ring around its upper edge permitted clamping of the wafer onto the vacuum mamfold to form a seal.
  • a screw-drive device with feedback control was provided for control of fluid flow through the wafer.
  • the liquid delivery cartridge contained a fluid inlet and outlet, a window for in situ observation of the hybridization event, permitted temperature control by either an intrinsic or extrinsic mechanism, and optionally contained a mechanism for moving fluids within the chip-containing cavity (e.g. electrodes, valves or bladders).
  • Oligonucleotide probes were synthesized by phosphoramidite chemistry (Beaucage et al. Tet. Lett. 22:1859-1862 (1981)) using an segmented synthesis strategy that is capable of producing over a hundred oligonucleotides simultaneously (Beattie et al , Biotechnol. Appl. Biochem. 10:510-521 (1988); Beattie et al. , Nature 352:548-549 (1991)). The oligonucleotides were derivatized with the alkylamino function during the chemical synthesis, either at the 5 '-end or the 3' -end.
  • the end attachment placed the probes 50-100 nm apart on the surface, corresponding to up to 10 8 probes in a 50 ⁇ m x 50 ⁇ m area.
  • Approximately 10 10 -10" oligonucleotide probes were tethered within a 50 ⁇ m cube of silicon in the nanofabricated wafer.
  • the density of bound oligonucleotides per cross sectional area was estimated by end-labeling prior to the attachment reaction, then quantitating the radioactivity using the phosphorimager.
  • Known quantities of labeled oligonucleotides dried onto the surface were used to calibrate the measurements of binding density.
  • the target DNA was prepared by the polymerase chain reaction, incorporating [ 32 P]nucleotides into the product during the amplification or by using gamma- 32 P[ATP] + polynucleotide kinase to 5 '-label the amplification product. Unincorporated label was removed by Centricon filtration. Preferably, one of the PCR fragments was 5'-biotin-labeled to enable preparation of single strands by streptavidin affinity chromatography.
  • the target DNA was dissolved in hybridization buffer (50mM Tris-HCl, pH 8, 2mM EDTA, 3.3M tetramethylammonium chloride) at a concentration of at least 5nM (5 fmol/ ⁇ l) and specific activity of at least 5,000 cpm/ftnol.
  • hybridization buffer 50mM Tris-HCl, pH 8, 2mM EDTA, 3.3M tetramethylammonium chloride
  • Target cRNA also was prepared according to the methods described in US Patent No. 5,716,785.
  • the target cRNA was dissolved in 5x SSPE (50mM phosphate, pH 7.4, 0.75M sodium chloride, 5mM EDTA) at concentrations of 5 to 25 ⁇ g/ml.
  • 5x SSPE 50mM phosphate, pH 7.4, 0.75M sodium chloride, 5mM EDTA
  • hybridization can be carried out in buffer containing 1M KC1 or NaCl or 5.2M Betaine, in place of tetramethylammonium chloride.
  • the cRNA sample was flowed into the channels of the chip and incubated at 20 to 50°C for 15 minutes to 12 hours, then washed by flowing hybridization solution through the chip at room temperature for 15 minutes.
  • the temperature of hybridization and washing can be varied over the range 5°C to 50°C for hybridization with short oligonucleotides. Higher temperatures may be desired for hybridization using longer probes.
  • Part A Phosphorimager and film detection
  • the detection and quantitation of hybridization intensities was carried out using methods that are widely available: phosphorimager and film.
  • a Cyclone phosphorimager Packard Instruments
  • the Biorad phosphorimager having a sample resolution of about 100 ⁇ m that is capable of registering both beta emission and light emission from chemiluminescent tags also could be used.
  • Reagent kits for cherniluminescence detection available from Millipore and New England Nuclear, which produce light of 477 and 428 nm, respectively, also may be with the Biorad instrument.
  • Chemiluminescent tags are introduced into the target DNA samples (random-primed vector DNA or PCR fragments) using the procedures recommended by the supplier.
  • Radioactive tags 2 P and 33 P, incorporated by random priming and PCR reaction
  • Film exposure is used for comparison.
  • radioactive tags incorporated using polynucleotide kinase
  • CCD genosensor devices are capable of maximum resolution and sensitivity and were used with chemiluminescent, fluorescent and radioactive tags (Lamture et al. supra.
  • Part A Fabrication of Porous Silicon Wafer
  • Part B Formation of cDNA Array
  • a set of oligonucleotide probes is synthesized incorporating primary amines into the strands.
  • the probes corresponding to a single expressed gene are applied to single discrete locations of the wafer using a Hamilton Microlab 2200 fluid delivery system equipped with an 8-needle dispensing head. After all probes are dispensed, a slight vacuum is briefly applied from below to ensure that fluid has occupied the channels. Following incubation at room temperature overnight or at 60°C for 30-60 minutes, the wafer is flushed with warm water, then reacted with 50mM trie ylamine to cap off the unreacted epoxy groups on the surface, then flushed again with warm water and air dried.
  • Cytoplasmic RNA is extracted from cultured cells by the method of Chomczynski et al. , (Anal. Biochem. 162:156-159 (1993)), treated with DNAse I to remove DNA contamination, then extracted with phenol/chloroform and ethanol precipitated. Reverse transcriptions and PCR are performed as described in the "differential display" protocol of Nishio et al, (FASEB J., 8:103-106 (1994)). Prior to hybridization, PCR products are labeled by random priming in the presence of [A- 32 P]dNTPs, and unincorporated label is removed by Centricon filtration.
  • Part D Hybridization of Expressed Sequences to cDNA Array
  • a solution of 1% "Blotto" or 50mM tripolyphosphate is flowed through the channels of the wafer to minimize the nonspecific binding of target DNA, then the porous silicon array is washed with hybridization solution (50mM Tris-HCl, pH 7.5, lmM EDTA, 1M NaCl).
  • hybridization solution 50mM Tris-HCl, pH 7.5, lmM EDTA, 1M NaCl.
  • Labeled PCR fragments representing the 3 '-end of expressed genes are recovered from the Centricon filtration units in hybridization buffer, and the entire wafer is flooded with this DNA solution.
  • the hybridization module is placed at 65 °C and a peristaltic pump, connected to the lower vacuum chamber, is used to gradually flow the labeled DNA through the channels of the wafer over the course of 30-60 minutes.
  • the wafer is washed three times with hybridization buffer at 65°C.
  • the wafer is briefly dried, then placed onto the phosphor screen of a phosphorimager and kept in the dark for a period of time determined by the intensity of label.
  • the phosphor screen is then placed into the phosphorimager reader for quantitation of individual hybridization signals arising from each channel region in the array.
  • Total cytoplasmic mRNA is isolated from cells cultured under two conditions and subjected to the "differential display" procedure described above to prepare fragments representative of individual mRNA species present under the two conditions. These samples are hybridized to two identical probe arrays, to yield two hybridization signal patterns that are compared to determine the relative extent of hybridization at each detection element in the array. The resulting hybridization patterns represent the profile of expressed genes under the two different culture conditions (for example in the presence and absence of a drug or chemical that induces a change in the expression of some genes).

Abstract

L'invention porte sur des dispositifs et procédés permettant de conduire des réactions de fixation. Lesdits dispositifs comportent un ensemble d'éléments de détection liés à un substrat et dont chacun est confiné dans un emplacement spatial distinct du substrat. Lesdits éléments de détection contiennent plusieurs réactifs de fixation, chacun de ceux d'un même élément de détection pouvant se fixer à une seule cible.
PCT/US1999/031149 1998-12-31 1999-12-30 Dispositif d'essai a sondes mixtes WO2000039344A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP99967727A EP1141413A2 (fr) 1998-12-31 1999-12-30 Dispositif d'essai a sondes mixtes
CA002356946A CA2356946A1 (fr) 1998-12-31 1999-12-30 Dispositif d'essai a sondes mixtes
AU23959/00A AU2395900A (en) 1998-12-31 1999-12-30 Assay device comprising mixed probes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US11458698P 1998-12-31 1998-12-31
US60/114,586 1998-12-31

Publications (2)

Publication Number Publication Date
WO2000039344A2 true WO2000039344A2 (fr) 2000-07-06
WO2000039344A3 WO2000039344A3 (fr) 2000-09-21

Family

ID=22356166

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/031149 WO2000039344A2 (fr) 1998-12-31 1999-12-30 Dispositif d'essai a sondes mixtes

Country Status (4)

Country Link
EP (1) EP1141413A2 (fr)
AU (1) AU2395900A (fr)
CA (1) CA2356946A1 (fr)
WO (1) WO2000039344A2 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001071039A2 (fr) * 2000-03-20 2001-09-27 Incyte Genomics, Inc. Sequences polynucleotidiques combinees en tant que resultats d'essais discrets
WO2003046214A2 (fr) * 2001-11-29 2003-06-05 Focusgenomics Gmbh Procede d'analyse de l'expression de genes commandee par translation
GB2409454B (en) * 2002-10-01 2007-05-23 Nimblegen Systems Inc Microarrays having multiple oligonucleotides in single array features
WO2015021080A2 (fr) 2013-08-05 2015-02-12 Twist Bioscience Corporation Banques de gènes synthétisés de novo

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6902702B1 (en) 2000-08-16 2005-06-07 University Health Network Devices and methods for producing microarrays of biological samples
CN108884462B (zh) * 2016-01-26 2022-11-25 日产化学株式会社 单链寡核苷酸

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995011995A1 (fr) * 1993-10-26 1995-05-04 Affymax Technologies N.V. Reseaux de sondes d'acide nucleique sur des microplaquettes biologiques
EP0721016A2 (fr) * 1994-10-21 1996-07-10 Affymax Technologies N.V. Banque d'acides nucléiques immobilisées, procédés pour leur fabrication et procédés de séquençage et de screening les utilisant
US5763263A (en) * 1995-11-27 1998-06-09 Dehlinger; Peter J. Method and apparatus for producing position addressable combinatorial libraries
WO1998041657A1 (fr) * 1997-03-20 1998-09-24 Affymetrix, Inc. Resequençage iteratif
EP0867506A1 (fr) * 1996-09-13 1998-09-30 Laboratory of Molecular Biophotonics Phase solide pour la detection d'acides nucleiques cibles, procede de production de cette phase et procede de detection d'acide nucleique cible
WO1998053103A1 (fr) * 1997-05-21 1998-11-26 Clontech Laboratories, Inc. Ensembles d'acide nucleique
WO2000011223A1 (fr) * 1998-08-24 2000-03-02 Affymetrix, Inc. Emploi de sondes groupees en analyse genetique

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995011995A1 (fr) * 1993-10-26 1995-05-04 Affymax Technologies N.V. Reseaux de sondes d'acide nucleique sur des microplaquettes biologiques
EP0721016A2 (fr) * 1994-10-21 1996-07-10 Affymax Technologies N.V. Banque d'acides nucléiques immobilisées, procédés pour leur fabrication et procédés de séquençage et de screening les utilisant
US5763263A (en) * 1995-11-27 1998-06-09 Dehlinger; Peter J. Method and apparatus for producing position addressable combinatorial libraries
EP0867506A1 (fr) * 1996-09-13 1998-09-30 Laboratory of Molecular Biophotonics Phase solide pour la detection d'acides nucleiques cibles, procede de production de cette phase et procede de detection d'acide nucleique cible
WO1998041657A1 (fr) * 1997-03-20 1998-09-24 Affymetrix, Inc. Resequençage iteratif
WO1998053103A1 (fr) * 1997-05-21 1998-11-26 Clontech Laboratories, Inc. Ensembles d'acide nucleique
WO2000011223A1 (fr) * 1998-08-24 2000-03-02 Affymetrix, Inc. Emploi de sondes groupees en analyse genetique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ERIK GENTALEN & MARK CHEE: "A novel method for determining linkage between DNA sequences: hybridization to paired probe arrays" NUCLEIC ACIDS RESEARCH, vol. 27, no. 6, 1999, pages 1485-1491, XP002139730 OXFORD UNIVERSITY PRESS, SURREY., GB ISSN: 0305-1048 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001071039A2 (fr) * 2000-03-20 2001-09-27 Incyte Genomics, Inc. Sequences polynucleotidiques combinees en tant que resultats d'essais discrets
WO2001071039A3 (fr) * 2000-03-20 2002-10-10 Incyte Genomics Inc Sequences polynucleotidiques combinees en tant que resultats d'essais discrets
WO2003046214A2 (fr) * 2001-11-29 2003-06-05 Focusgenomics Gmbh Procede d'analyse de l'expression de genes commandee par translation
WO2003046214A3 (fr) * 2001-11-29 2004-04-01 Focusgenomics Gmbh Procede d'analyse de l'expression de genes commandee par translation
GB2409454B (en) * 2002-10-01 2007-05-23 Nimblegen Systems Inc Microarrays having multiple oligonucleotides in single array features
WO2015021080A2 (fr) 2013-08-05 2015-02-12 Twist Bioscience Corporation Banques de gènes synthétisés de novo
EP3722442A1 (fr) 2013-08-05 2020-10-14 Twist Bioscience Corporation Banques de gènes synthétisés de novo
EP4242321A2 (fr) 2013-08-05 2023-09-13 Twist Bioscience Corporation Banques de gènes synthétisés de novo

Also Published As

Publication number Publication date
EP1141413A2 (fr) 2001-10-10
AU2395900A (en) 2000-07-31
CA2356946A1 (fr) 2000-07-06
WO2000039344A3 (fr) 2000-09-21

Similar Documents

Publication Publication Date Title
US7125674B2 (en) Microfabricated flowthrough porous apparatus for discrete detection binding reactions
AU700315B2 (en) Microfabricated, flowthrough porous apparatus for discrete detection of binding reactions
US20030044777A1 (en) Flowthrough devices for multiple discrete binding reactions
JP4334823B2 (ja) 多重化分子分析装置および方法
Beattie et al. Advances in genosensor research
US7056666B2 (en) Analysis of surface immobilized polymers utilizing microfluorescence detection
WO1999067641A2 (fr) Decodage de detecteurs matriciels a microspheres
WO1999060170A1 (fr) Reseaux lineaires de composes immobilises et procedes d'utilisation de ces derniers
Heise et al. Immobilization of DNA on microarrays
EP1643249A1 (fr) Biopuce
EP1151302A1 (fr) M thodes et compositions de criblage intrabille
O'Donnell-Maloney et al. Microfabrication and array technologies for DNA sequencing and diagnostics
CA2315296A1 (fr) Embouts en phase solide et utilisations correspondantes
EP1307283A2 (fr) Jeux ordonnes d'echantillons composites utilisant des microspheres equipees d'une chambre d'hybridation
US7473562B2 (en) Method for high-throughput integrated chemical and biochemical reactions
WO2000039344A2 (fr) Dispositif d'essai a sondes mixtes
CN109370891B (zh) 一种生物芯片及其制备方法
JP2003232791A (ja) プローブ固相化反応アレイ
CN110944743A (zh) 在晶圆上进行化学合成的系统和方法
JP2003526096A (ja) 核酸、ｄｎａ、ｒｎａ、ｐｎａ及び蛋白質などの分子化合物のハイブリダイゼーションを同時かつ多角的に検出し定量化するための方法及びシステム
JP2002281967A (ja) 印加によりその捕捉機能が制御できる捕捉手段を具備する生化学分析装置およびその装置を用いた生化学分析方法
Zhou et al. DNA microarray technology
JP2001255327A (ja) 多孔質支持体と遅延蛍光を用いる物質の検出及び／又は定量法
Matson DNA Arrayed on Plastic Devices
Eickhoff et al. Eckhard Nordhoff, Lajos Nyarsik, Günther Zehetner, Wilfried Nietfeld, and Hans Lehrach

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 23959/00

Country of ref document: AU

ENP Entry into the national phase in:

Ref country code: CA

Ref document number: 2356946

Kind code of ref document: A

Format of ref document f/p: F

Ref document number: 2356946

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 1999967727

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1999967727

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWW Wipo information: withdrawn in national office

Ref document number: 1999967727

Country of ref document: EP