EP1196539A4 - Substrats permettant d'immobiliser des acides nucleiques sur des supports solides - Google Patents

Substrats permettant d'immobiliser des acides nucleiques sur des supports solides

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Publication number
EP1196539A4
EP1196539A4 EP00947091A EP00947091A EP1196539A4 EP 1196539 A4 EP1196539 A4 EP 1196539A4 EP 00947091 A EP00947091 A EP 00947091A EP 00947091 A EP00947091 A EP 00947091A EP 1196539 A4 EP1196539 A4 EP 1196539A4
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
substrate
dna
activated ester
glass
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Withdrawn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
EP00947091A
Other languages
German (de)
English (en)
Other versions
EP1196539A1 (fr
Inventor
Ikunoshin Kato
Kiyozo Asada
Masanori Takayama
Mary-Anne Watt
Vladimir G Budker
Paul M Slattum
James E Hagstorom
Jon A Wolff
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.)
Takara Bio Inc
Arrowhead Madison Inc
Original Assignee
Takara Bio Inc
Mirus Corp
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 Takara Bio Inc, Mirus Corp filed Critical Takara Bio Inc
Publication of EP1196539A1 publication Critical patent/EP1196539A1/fr
Publication of EP1196539A4 publication Critical patent/EP1196539A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
    • C40B50/18—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support using a particular method of attachment to the solid support
    • C—CHEMISTRY; METALLURGY
    • C03—GLASS; MINERAL OR SLAG WOOL
    • C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
    • C03C17/00—Surface treatment of glass, not in the form of fibres or filaments, by coating
    • C03C17/34—Surface treatment of glass, not in the form of fibres or filaments, by coating with at least two coatings having different compositions
    • C03C17/3405—Surface treatment of glass, not in the form of fibres or filaments, by coating with at least two coatings having different compositions with at least two coatings of organic materials
    • 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
    • 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
    • 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
    • B01J2219/00529—DNA chips
    • 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
    • B01J2219/00608—DNA chips
    • 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
    • B01J2219/00617—Delimitation of the attachment areas by chemical means
    • 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
    • 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

Definitions

  • This innovation is concerned with the substrate(s) for nucleic acid(s) immobilization used to produce DNA chip(s). In addition, this innovation is also concerned with the method(s) for the immobilization of nucleic acid onto such substrates and the detection method(s) for the target nucleic acids on that substrate.
  • DNA chip(s) DNA microarray
  • a DNA chip is a microarray with many different genes or fragments (DNA fragments) immobilized on the surface of a solid substrate such as a glass slide.
  • a DNA chip (DNA microarray) is useful in the analysis of the expression, mutation, and polymorphism of gene(s).
  • the method of immobilization of target nucleic acids onto the substrate is the core technology required for the production of DNA chips.
  • One method involves the chemical synthesis of the nucleic acids on the tip a linker covalently bound to the substrate, for instance, described in Science, Vol. 251, 767-773 (1991) and Nucleic Acid Research, Vol. 20, 1679-1684 (1992).
  • DNA is limited to oligdnucleotides and a special apparatus is necessary to control the reaction. Thus, it is not a general method.
  • DNA or DNA prepared by PCR Polymerase Chain Reaction
  • PCR amplified products are covalently or non-covalently bound to the substrate, for example, written in Science, Vol. 270, 467-470 (1995) and Nucleic Acid Research, Vol. 22, 5456-5465 (1994).
  • the DNA (or RNA) is dissolved in a salt solution such as SSC (sodium chloride/sodium citrate) with or without a denaturing process.
  • SSC sodium chloride/sodium citrate
  • the DNA is spotted onto a glass slide coated with basic polycation such as polylysine, polyethylenimine, or silanized with an amine-containing silane compound (3- aminopropyltriethoxysilane for example), and is immobilized by UV-irradiation. All types of DNA (or RNA) should be able to be immobilized by this method.
  • oligonucleotides and short length DNAs are difficult to immobilize using this method. Even when long length DNA (greater than 0.3 Kb) are attached non-covalently, the processes of washing and hybridization can remove DNA from the substrate, which may cause a reduction in detectability of the target nucleic acid.
  • non-covalent immobilization process Another limitation of the non-covalent immobilization process is increased background signal due to nonspecific binding between residual basic functional groups (cationic) on the substrate surface and the probe nucleic acid (anionic). It is known that the residual amino groups may be blocked by acetylation (conversion of amines to carboxylic acids using an anhydride such as succinic anhydride). However, the conversion is not always complete.
  • a substrate which is applicable for the supersensitive detection of the target nucleic acid, and suitable for the immobilization of many types of nucleic acid, including both short and long length nucleic acids, single stranded nucleic acids, double stranded nucleic acids, DNA, and RNA is desirable.
  • the primary aims of this invention are: (1) preparation of a novel substrate for nucleic acid immobilization, capable of immobilizing nucleic acids with high efficiency; (2) the substrate immobilized with nucleic acid (i.e. prepared DNA microarrays); (3) production method for the immobilizing nucleic acids; and (4) detection method for nucleic acid using the substrate immobilized with nucleic acid.
  • the invention is primarily concerned with the substrate for nucleic acid immobilization; specifically a polyanion based surface for attachment of nucleic acids.
  • the polyanion is polyacrylic acid, however any polyanion may be used.
  • Another aspect of the invention involves the immobilization of the nucleic acid to the substrate via a covalent bond.
  • the preferred method involves transforming the carboxylic acid functionalities into activated esters.
  • the activated esters are then reacted with amine modified nucleic acids.
  • the substrate is a non-porous surface.
  • a preferred form being glass.
  • the third aspect of the embodiment is concerned with the substrate immobilized with nucleic acid(s); i.e. the substrate immobilized with nucleic acid, in which nucleic acid is covalently bound to the substrate through the activated ester version of polyanion.
  • the polyanion is polyacrylic acid as a preferable form.
  • a preferred activated ester is the pentafluorophenol ester prepared from the polyanion and pentafluorophenol via a carbodiimide coupling.
  • the fourth embodiment is concerned with the method(s) for nucleic acid immobilization to the substrate; i.e. the method for nucleic acid immobilization to the substrate where the substrate coated with an activated ester derivative of polyanion is contacted with amine-modified nucleic acid.
  • the polyanion is polyacrylic acid and the substrate is non-porous as the preferable form; glass is the best non-porous substrate.
  • the nucleic acid(s) is modified with functional group(s) that has reactivity to the activated ester derivative, in the preferable form the nucleic acid is modified with amino groups.
  • the fifth embodiment is concerned with the detection method(s) for target nucleic acids in the specimen(s) (or sample(s)) including but not limited to a: a) Process, in which the substrate for nucleic acid immobilization is the activated ester derivative of the polyanion. The polyanion has been previously covalently attached to the surface of the glass slide. b) Reaction process between the activated ester derivative mentioned above and the nucleic acid modified with a functional group that has reactivity to the ester derivative. c) Hybridization process between nucleic acid immobilized on the substrate (target nucleic acid) and complementary nucleic acid (probe nucleic acid). d) Detection process for hybridized nucleic acids.
  • the sixth embodiment is concerned with the detection method(s) for nucleic acid in the specimen including but not limited to a: a) Reaction between the activated ester derivative of the substrate and nucleic acid modified with a functional group that has reactivity to the activated ester derivative. b) Hybridization process between nucleic acid immobilized on the substrate and complementary nucleic. c) Detection process for hybridized nucleic acid.
  • a polyanion is a polymer that contains two or more negative charges.
  • nucleic acid includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in the form of an oligonucleotide messenger RNA, anti-sense, plasmid DNA, parts of a plasmid DNA or genetic material derived from a virus (viral DNA) linear DNA, or chromosomal DNA.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • a humidostat is any chamber capable of maintaining a fixed humidity range.
  • the substrate(s) for nucleic acid immobilization is not restricted if it is applicable for DNA chips or biosensors.
  • the substrate consists of a solid support having a non-porous and smooth surface, materials such as glass slides or silica beads are good to use, in the preferable form.
  • the substrate is treated with 3 -aminopropyltriethoxy silane to provide the surface of the substrate with functional groups capable of reacting with the polyanion (amino groups in this case).
  • Any silane capable of introducing functionality onto the surface of a glass substrate can be used in this innovation.
  • the polyanion can then be covalently attached to the silanated surface of the substrate via a carbodiimide coupling reaction.
  • Polyanions contain acidic functional groups such as carboxyl groups, phosphate groups, sulfate groups, and so on which have the effect of preventing non-specific binding (electrostatic) with probe nucleic acids.
  • the polyanion is covalently attached to the surface of the substrate; polyacrylic acid, polyglutamic acid, polyaspartic acid, and polyphosphate, are the preferable forms.
  • These polyanions are attached to the silanated surface of the substrate through carbodiimide (or other activated ester chemistry) chemistry.
  • an activated ester For example pentafluorophenol and imidazole esters are activated ester derivatives available for the carboxyl group and phosphate group, respectively.
  • an activated ester There are no special restrictions placed on the nucleic acid to be immobilized on the substrate. All of the synthesized oligonucleotides, polynucleotides, and their derivatives can be useful. Both single and double stranded forms of nucleic acids (DNA or RNA) are available. The derivatives are not restricted if they have modifications enabling immobilization onto the substrate surface.
  • the derivative(s) modified with amino group, thiol group, phosphate group, and aldehyde group at DNA 5' terminus are examples.
  • the derivatives modified at 5' terminus with a cross linker or linker as the spacer such as alkylamine are also available.
  • the modification at the 5' terminus is especially effective for the immobilization of short nucleic acids such as oligonucleotide. Any nucleic acid covalently modified to introduce various functional or signaling groups may also be used, Minis' LabellT reagents may be used to modify nucleic acids.
  • Modified nucleic acids mentioned previously are dissolved in the appropriate solution for immobilization, such as 20 mM MOPS (3-morpholino propane sulfate) buffer (pH 7.5) or 50 mM carbonate buffer (pH 9.5) at the concentration of 0.01-2.0 mg/ml, preferably 0.1-1.0 mg/ml.
  • MOPS 3-morpholino propane sulfate
  • carbonate buffer pH 9.5
  • the desired nucleic acid can be immobilized onto the substrate with activated ester functional group by surface treatment, by contact with it. That is, using a micropipette or DNA chip preparation apparatus (DNA arrayer), the constant volume of DNA solution mentioned above was spotted onto the substrate with activated ester functional group by surface treatment as described.
  • the substrate immobilized with nucleic acid is soaked into 0.3 M NaOH at room temperature for 5 min or boiling water for 2 min to denature the nucleic acid, washed with distilled water and absolute ethanol, and dried.
  • the activated ester groups not reacted with nucleic acid in the are hydrolyzed back to carboxylic acid moieties (i.e. negatively charged).
  • the acid groups prevent nonspecific electrostatic binding between probe nucleic acid and the substrate.
  • the succinic anhydride blocking process necessary for polylysine slides can be omitted. These acid groups also lower the background signal.
  • the quantity of nucleic acid immobilized on the substrate can be measured by using fluorescently-labeled nucleic acid. Labeled target nucleic acid can be covalently immobilized and residual fluorescent signal detected.
  • the immobilization method in this innovation is capable of supersensitive detection of the expression levels, mutations, polymorphisms, etc, in the gene(s), because this method provides for a high efficiency of immobilization. Furthermore the immobilized nucleic acid will not come off under the hybridization conditions (because the nucleic acid is covalently attached to the substrate).
  • the general hybridization technique is used for the detection of target nucleic acid covalently attached to the substrate (such as DNA chip).
  • the probe nucleic acid is labeled with a fluorophore (or other signaling group) and is denatured by alkali or heat.
  • the labeled probe nucleic acid is added to the hybridization solution.
  • a 5 ⁇ l to 20 ⁇ l portion of the hybridization solution is dropped onto the surface of array, overlaid with a cover glass taking care to prevent air bubbles from forming under the cover glass. This is kept in the humidistat at the appropriate temperature and time.
  • the cover glass is removed, the slide is washed thoroughly, moisture is removed, and finally fluorescent signal is detected by the fluoroscanner (fluorescence reader).
  • the polyanion coated glass slides specified in this innovation are able to immobilize nucleic acids eflFiciently.
  • the three-dimensional matrix formed on the surface of the substrate by the covalently attached polyanion allows for the immobilization of increased numbers of nucleic acids.
  • the substrate and the immobilized nucleic acid in this innovation provide supersensitive detection signal and low background signal detection of nucleic acid. Because the activated ester derivative of polyanion that does not react with the target nucleic acid reverts to polyanion when the activated ester is hydrolyzed, nonspecific binding of negatively charged probe nucleic acid to the substrate is inhibited.
  • target nucleic acids including oligonucleotides
  • the blocking process can be omitted after immobilization
  • the nonspecific binding between the target nucleic acid and the substrate is kept to a minimum
  • Example (Illustration) #1 Two kinds of phage DNA fragments (1.0 Kb and 0.3 Kb, respectively, latter is the part of former) shown as Sequence #1 and #2, respectively, in Sequence Listing, were prepared by PCR as the standard DNA.
  • PCR was performed by using phage DNA as a template and with following two combinations of primers; that of Primer S and Primer A 1000 shown as Sequence #3 and #4, respectively, and that of Primer S and Primer A300 as Sequence #5, in Sequence
  • Each DNA fragment was dissolved in 20 mM MOPS buffer (pH 7.5), the array was made by GMS 417 arrayer (Genetic MicroSystems) on the glass slide with amino groups on its surface, which is commercially available.
  • the glass slide used was poly-L- lysine coated POLY-PREP-SLIDES (Sigma: abbreviated as PLL coated slide glass) and MAS, amino alkyl silane family, coated slide (Matsunami Glass Ind.: abbreviated as MAS coated slide glass), these slides are widely used for the preparation of DNA chips.
  • the spots were established with the size and intervals of 150 Hi and 375 Hi, respectively. Five spots of same DNA were arrayed.
  • each slide was kept in the incubator controlled at 37 degrees C and 90% humidity for 1 h, and then, UV cross-linked by 60 mj/cm 2 .
  • Slide(s) was washed once with 0.2% SDS and twice with distilled water, removed moisture by centrifugation at low speed (ca.1,000 rpm), followed by air drying.
  • the pre-treated slide mentioned above was washed with distilled water thoroughly, boiled in water bath for 3 min, and exposed in ice cold ethanol to denature DNA. Ethanol was removed by low speed centrifugation. After that, slide was air dried and stored in a desiccator.
  • a portion of slide(s) was treated with absolute succinic anhydride to block the free amino group(s) on the substrate surface by succinylation.
  • the blocking solution was prepared by the mixing of 1.5 g succinic anhydride resolved in 89.5 ml of N-methyl-2- pyrrolidone with 9 ml of 1 M borate buffer (pH 8.0), the slide was soaked in the blocking solution for 20 min at the room temperature. The treated slide was washed with distilled water thoroughly, boiled in water bath for 3 min, and exposed to ice cold ethanol to denature DNA. Ethanol was removed by low speed centrifugation. After that, slide was air dried and stored in a desiccator.
  • This synthesized DNA was spotted onto glutaraldehyde treated slides using GMS 417 arrayer in the same manner mentioned in Example Illustration #1-(1). After that, slide glass was washed with 0.2% SDS for 2 min, continuously washed with distilled water. To reduce the Schiff-base, formed by covalent binding between aldehyde group on the substrate and amino group on DNA, into stable amine, the slides were soaked into 0.25% sodium borohydride (Wako Pure Chemical Industries) containing PBS- 100% ethanol (volumetric ratio 3: 1) for 5 min, and air dried. (3) Covalent attachment of polyanion to the solid support.
  • the polyacrylic acid coated slides were converted into activated ester coated slides in the following manner. 1.8 g pentafluorophenol, 2.0 g EDC, and 13 mg dimethylamino- pyridine were dissolved into 50 ml dimethylformamide.
  • the polyacrylic acid coated glass slides prepared in Example Illustration #l-(3) were soaked in this solution for 5 h, rinsed with dimethylformamide and methylene chloride, dried under vacuum, and finally stored in a desiccator.
  • Activated ester coated glass slides spotted with DNA were kept in the incubator (Tokyo Rikakikai: Eyela model KC-1000) controlled at 37 degrees C and 90% humidity for 30 min, and then, incubated in 0.2% SDS at room temperature to wash out the excess of salt, followed by the wash with distilled water. After that, the slides was soaked in 0.3 N NaOH for 5 min to denature DNA, washed with distilled water thoroughly, dried by centrifugation at about 1,000 rpm, and stored in a desiccator.
  • the 1.0 Kb lambda phage DNA fragments prepared in Example #1-(1) were labeled with Cy5TM using Label IT ® Cy5TM Labeling Kit (Minis) according to instruction manuals.
  • the labeled nucleic acid was purified by ethanol precipitation.
  • This Cy5TM labeled nucleic acid probe was diluted with pre-hybridization buffer to the final concentrations of 20, 2 and 0.2 ng/ml, heated at 95 degrees C for 2 min, cooled to room temperature. Insoluble matter was removed by centrifugation. About 5 ul of the hybridization solution was dropped onto the nucleic acid array. It was overlaid with a glass coverslip taking care to avoid air bubble formation, and incubated at 42 degrees C for 20 h in the humidistat.
  • the cover glass was removed in the 2x SSC solution at room temperature, washed twice at 65 degrees C for 5 min and at 55 degrees C for 30 min with 0.2% SDS containing 2x SSC. Then, it was washed with 0.05x SSC at room temperature for 10 min, removed moisture by centrifugation at about 1,000 rpm, and air-dried.
  • the nucleic acid array was read using a GMS 418 array scanner (GMS) with emission wavelength as 635 nm and excitation wavelength as 660 nm.
  • GMS GMS 418 array scanner
  • the detected signal strength was analyzed by the image analyzing software ImaGene (Biodiscovery). The result obtained was summarized in Table 1.
  • Two other oligonucleotides of 24 nucleotides in length were also synthesized; one oligonucleotide having a complementary sequence to the first oligonucleotide shown as Sequence #7, with Cy3TM at 5' terminus (Cy3-R Oligo): and another oligonucleotide having unrelated sequence shown as Sequence #14, with alkyl amino linker at 5' terminus (NH 2 -N Oligo).
  • OH-F Oligo, NH 2 -F Oligo, and NH 2 -N Oligo were dissolved in 20 mM MOPS buffer (pH7.5) at a final concentration of 5, 50, and 500 DM, and then arrayed onto activated ester coated glass slide activated by the method in Example #l-(4). At that time, seven spots of each DNA sequence were arrayed. After spotting, the slide was washed and dried as described in Example #2, finally it was treated as oligonucleotide chip. As the control, the same oligonucleotide array was prepared on the glutaraldehyde treated slide as described in Example #l-(2). (2) Hybridization of oligonucleotide chip
  • the oligonucleotide chips made on each slide were hybridized with separately prepared Cy3-R Oligos as a probe, and the resulting fluorescent signals were observed. Briefly, about 5 ul of the hybridization solution, consisting of 1 mM Cy3-R Oligo, 0.2% SDS, 5x Denhardts solution, 0.1 mg/ml salmon sperm DNA, and 6x SSC, was added to the oligonucleotide array. It was overlaid with a glass coverslip taking care to avoid air bubble formation and incubated at 42 degrees C for 20 h in the humidistat. The cover glass was removed in the 2x SSC solution at room temperature and washed twice for 10 min in 2x SSC at room temperature.
  • Example (Illustration) #4 Three-dimensional effect of polyacrylic acid The hybridization signals from nucleic acid arrays, on which the fluorescently labeled nucleic acids of different length were spotted, were calculated as signal per nucleic acid length immobilized.
  • a cDNA fragment of human transferrin receptor (abbreviated as TFR) was prepared by the following method. The extraction of mRNA from human cell line K562 (Dainippon Pharmaceutical) and the preparation of cDNA of TFR were performed according to the normal method. With the above cDNA as template, a 4,738 bp PCR product was prepared, using TFR (GeneBank No.
  • the recombinant plasmid pT7TFR was obtained by the ligation of above PCR fragment with pT7Blue vector (Novagen).
  • Fluorescently labeled PCR fragments of different lengths were prepared by the PCR using one primer of the pairs labeled with Rhodamine X (PE Biosystems: abbreviated as ROX) at 5' terminus and pT7TFR as primer and template, respectively; i.e., the PCR was performed using the primer pairs of S7 (Sequence #8) labeled with ROX and AS4 (Sequence #10), AS3 (Sequence #11), AS2 (Sequence #12), or AS1 (Sequence #13). Resulting in 1.0, 0.5, 0.2, and 0.1 kb fragments, respectively, being prepared.
  • ROX Rhodamine X
  • each DNA fragment was dissolved in 20 mM MOPS buffer (pH 7.5) and aminated using the Amino Label IT ® reagent; i.e., the Amino Label IT ® reagent was dissolved in dimethyl sulfoxide and added into the purified DNA solution as 1/5 ratio (W/W) and incubated at 37 BE for 1 h.
  • Each aminated DNA was purified by ethanol precipitation method.
  • the amino Label IT reagent was prepared as described in the patent application W098/52961, with the displacement of fuluoro-group to amino group.
  • the aminated TFR fragment was dissolved in 20 mM MOPS buffer (pH 7.5) at a final concentration of 0.5 mg/ml, and then spotted seven points of each sequence by the method of Example #1-(1) onto activated ester coated slide glass activated as Example #l-(4). After spotting, it was washed and dried as described in Example #3.
  • the same array was prepared on the MAS coated slide glass as Example #1-(1), and treated with or without succinic anhydride blocking of free amino groups after immobilization.
  • the DNA chips made on each slide glass were hybridized with Cy5 labeled DNA probe, which has a different fluorescent wavelength than ROX, and observed their signals. That is, the 1.0 Kb TFR fragment prepared above was labeled with Cy5TM using Cy5 Label IT ® Kit (Minis) according to manufacturers recommendations.
  • the hybridization solution consisting of 20 ng/ml DNA probe labeled with Cy5, 0.2% SDS, 5x Denhardts solution, 0.1 mg/ml salmon sperm DNA, and 6x SSC, was added onto an area of DNA immobilized on the chip. It was overlaid with the cover glass and incubated at 65 degrees C for 20 h in the humidistat. The cover glass was removed in the 2x SSC solution at room temperature and washed twice at 65 degrees C for 5 min and at 55 degrees C for 30 min with 0.2% SDS containing 2x SSC. Then, it was washed with 0.05x SSC at room temperature for 10 min, removed moisture by low centrifugation at about 1,000 ⁇ m, and air-dried.
  • the signal strength of spots per DNA on the activated ester coated slide glass was higher than that on the MAS coated slide glass. This result indicates that the activated ester coated slide glass allows for more efficient hybridization.
  • the three-dimensional lattice created by the covalently attached polyanion may allow greater access to the attached target nucleic acids.
  • Example #2-(l) Two types of 1.0 Kb and 0.3 Kb lambda phage DNA fragments with amino group at their ends were prepared by the same method in Example #2-(l). A portion of the DNA was aminated using the Amino Label IT ® reagent as described Example #4. Each DNA fragment was dissolved in 20 mM MOPS buffer (pH 7.5) at a final concentration of 0.5 mg/ml, and spotted in seven different places onto activated ester coated slide glass and MAS coated slides.
  • Nucleic acid arrays made on each slide glass were hybridized with fluorescent Cy3-labeled DNA probes, and the resulting signals were analyzed. That is, a 1.0 Kb lambda phage fragment prepared above was labeled with Cy3 using Label IT ® Cy3 Labeling Kit (Minis) according to the manufacturers recommendations. Using this Cy3 labeled DNA probe, hybridization and washing were performed as the described in Example #4.
  • DNA chip fluorescence i.e. signal intensity
  • GMS 418 array scanner with emission wavelength as 532 nm and excitation wavelength as 570 nm.
  • the obtained images were analyzed and the values of the signal strength were converted to numerical data using the image analyzing software ImaGene.
  • the relative fluorescent strength was calculated by that of spot on MAS coated slide glass as 100. The result obtained was summarized in Table 4. Table 4
  • the signal strength from the activated ester coated slide glass was higher than that from the MAS coated slide glass. Especially the signal for the 300 bp short DNA fragment was much higher than on the MAS slides.

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Abstract

L'invention concerne un substrat et un procédé permettant d'immobiliser des acides nucléiques sur un support solide. Un polyanion contenant un ester activé est fixé par covalence sur un support solide. Dans un mode de réalisation préféré, ce polyanion est de l'acide polyacrylique.
EP00947091A 1999-07-07 2000-07-06 Substrats permettant d'immobiliser des acides nucleiques sur des supports solides Withdrawn EP1196539A4 (fr)

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US14270899P 1999-07-07 1999-07-07
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US476028 1999-12-31
PCT/US2000/018573 WO2001002538A1 (fr) 1999-07-07 2000-07-06 Substrats permettant d'immobiliser des acides nucleiques sur des supports solides

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WO2003027674A1 (fr) * 2001-09-21 2003-04-03 Takara Bio Inc. Support pour l'immobilisation des ligands
JP2004097173A (ja) * 2002-07-17 2004-04-02 Toyo Kohan Co Ltd 静電層を有する固体支持体及びその用途
JP4380631B2 (ja) * 2003-05-19 2009-12-09 東レ株式会社 選択結合性物質固定化担体
CN1735807A (zh) * 2003-12-19 2006-02-15 成都夸常医学工业有限公司 芯片检测方法及相关装置
US8048377B1 (en) 2004-03-08 2011-11-01 Hewlett-Packard Development Company, L.P. Immobilizing chemical or biological sensing molecules on semi-conducting nanowires
EP2476759B1 (fr) 2009-09-10 2018-04-04 Toyo Kohan Co., Ltd. Support pour la retenue d'acides nucléiques
CN101696449B (zh) * 2009-11-10 2017-05-17 苏州吉玛基因股份有限公司 核酸芯片、其制备方法及用途
CN101738425B (zh) * 2010-01-06 2013-03-13 天津科技大学 用于抗生素和心脏病标记物快速检测的适配子生物传感器的制作方法
JP6127520B2 (ja) 2013-01-09 2017-05-17 日本軽金属株式会社 バイオチップ用基板及びその製造方法
CN105219836B (zh) * 2014-05-27 2018-05-22 昆明寰基生物芯片产业有限公司 一种微阵列用活性醛基修饰基片
CN104805509A (zh) * 2015-04-08 2015-07-29 南京普东兴生物科技有限公司 一种羧基修饰的基因芯片基片及其制备方法
CN105220237A (zh) * 2015-10-29 2016-01-06 昆明寰基生物芯片产业有限公司 一种微阵列用活性醛基修饰基片及其制备方法
GB2580384B (en) * 2019-01-08 2021-01-27 Quantumdx Group Ltd Oligonucleotide deposition onto polypropylene substrates
CN112442101A (zh) * 2019-09-05 2021-03-05 华为技术有限公司 寡核苷酸的合成方法、合成装置

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US5492840A (en) * 1988-11-10 1996-02-20 Pharmacia Biosensor Ab Surface plasmon resonance sensor unit and its use in biosensor systems
WO1992003720A1 (fr) * 1990-08-17 1992-03-05 Fisons Plc Dispositif analyseur
WO1992021976A1 (fr) * 1991-06-04 1992-12-10 Fisons Plc Dispositif d'analyse
WO2000005582A2 (fr) * 1998-07-21 2000-02-03 Burstein Laboratories, Inc. Dispositifs de dosage utilisant des disques optiques et methodes correspondantes

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JP3727882B2 (ja) 2005-12-21
CN1203169C (zh) 2005-05-25
KR20020026473A (ko) 2002-04-10
EP1196539A1 (fr) 2002-04-17
JP2003504595A (ja) 2003-02-04
CN1379810A (zh) 2002-11-13

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