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WO2003038122A2 - Sondes asymetriques - Google Patents

Sondes asymetriques Download PDF

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Publication number
WO2003038122A2
WO2003038122A2 PCT/EP2002/011911 EP0211911W WO03038122A2 WO 2003038122 A2 WO2003038122 A2 WO 2003038122A2 EP 0211911 W EP0211911 W EP 0211911W WO 03038122 A2 WO03038122 A2 WO 03038122A2
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Prior art keywords
building blocks
nucleic acid
receptors
carrier
synthesis
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PCT/EP2002/011911
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German (de)
English (en)
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WO2003038122A3 (fr
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Peer F. STÄHLER
Ralf Mauritz
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Febit Ag
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Publication of WO2003038122A3 publication Critical patent/WO2003038122A3/fr

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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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • 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
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • 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
    • C12Q1/6832—Enhancement of hybridisation reaction
    • 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
    • B01J2219/00511—Walls of reactor vessels
    • 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
    • B01J2219/00614—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
    • B01J2219/00614—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
    • B01J2219/00657—One-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
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00675—In-situ synthesis on the substrate
    • 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
    • B01J2219/00704—Processes involving means for analysing and characterising the products integrated with the reactor apparatus
    • 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
    • B01J2219/00709—Type of synthesis
    • B01J2219/00711—Light-directed synthesis
    • 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
    • B01J2219/00709—Type of synthesis
    • B01J2219/00713—Electrochemical synthesis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides

Definitions

  • the invention relates to a method for producing a support for the determination of nucleic acid analytes by hybridization, comprising the in situ synthesis of receptors on the support which hybridize with the nucleic acid analyte to be determined.
  • the result is polymeric probes with a binding behavior in which those probes that are full length products are thermodynamically preferred, more than just by the fact that they are full length products.
  • Biochemical and biological analysis methods can be enormously increased in their efficiency and meaningfulness by miniaturization and parallelization.
  • Such miniaturizations concern e.g. the analysis of genetic material with the help of hybridization experiments.
  • microarrays from suitable hybridization probes can be used to examine entire genomes and transcriptomes.
  • Microreaction technology can be coupled to such microarrays in order to
  • DNA microarrays have also been miniaturized and parallelized for screening special molecular properties, e.g. Ribozymes.
  • Other polymer probes are proteins and such molecules that do not occur in nature, e.g. Peptide nucleic acids (PNA).
  • PNA Peptide nucleic acids
  • microarrays are also referred to as biochips, ie carriers on which a large number of different receptor probes are immobilized.
  • a special class of microarrays is produced directly on the support by in situ synthesis of the required polymer probes.
  • reaction carriers can be used, for example, in academic research, basic research, industrial research, in quality control, in pharmaceutical research, in biotechnology, in clinical research, in screening processes, in patient-specific diagnostics, in clinical studies, in forensics, for genetic tests, such as parenting regulations, in animal and plant breeding or in environmental monitoring.
  • a major disadvantage of such processes is, however, that in an in situ synthesis process, the product of the synthesis cannot subsequently be purified.
  • the so-called "off chip” syntheses of the polymer probes are produced using conventional methods and then purified in such a way that the full-length product of the synthesis is almost exclusively present. Only this population of molecules is then arranged on the substrate as an array.
  • a disadvantage of this method is that the arrangement of the finished polymer probes on the array is very complex.
  • the proportion of the full-length product after X synthesis cycles can fall below a critical value, so that the analysis result is not influenced by this full-length product at all.
  • the coupling rate of the individual addition steps is below 95% (Beier, M., Hoheisel, JD, Production by quantitative photolitogaphic synthesis of individually quality checked DNA microarrays, Vol. 28, No. 4, P. 1-6, 2000).
  • Such methods can only be used to produce DNA polymer probes up to a length of 25 bases.
  • the present invention describes a method for improving the use of in situ synthesis methods in the production of polymer probe arrays by increasing the contribution of full-length products from the synthesis process to the analysis result. This is achieved through an asymmetrical configuration of the polymer probes.
  • modified building blocks are used that differ from the previously used building blocks in certain thermodynamic properties, such as, for example, the binding stability.
  • the same effect can be achieved by a suitable modification of the distal end of the polymer probes, for example with a hybridization amplifier.
  • Such a molecule is, for example, a so-called "minor groove binder” (Epoch Biosciences 2000 Annual Report, pages 4-5), which significantly increases the stability of the binding to the last 4-5 bases of the polymer probe.
  • “minor groove binders” are some natural antibiotics with a shape that allows folding into the "minor groove” of a DNA helix. This substitutes for the lack of purification of the polymer probes in in situ syntheses prior to application in a polymer probe array. The quality disadvantage of in situ synthesis processes is partially or completely compensated for in this way.
  • the method according to the invention improves the usability of polymer probe arrays synthesized in situ with regard to the quality and informative value of the analysis in comparison with the prior art.
  • Modified synthesis building blocks are, for example, ribonucleoside analogs, such as LNA's (locked nucleic acids), modified purine or pyrimidine bases, such as superstabilizing adenosine analogs (for example 2,4-diamine no adenosine), pyrazo lopyri midinee (e.g. P PG ) and phosphate backbone analogs, such as Methylphosphonates, phosphorothionates, phosphoramidates etc.
  • LNA's locked nucleic acids
  • modified purine or pyrimidine bases such as superstabilizing adenosine analogs (for example 2,4-diamine no adenosine)
  • pyrazo lopyri midinee e.g. P PG
  • phosphate backbone analogs such as Methylphosphonates, phosphorothionates, phosphoramidates etc.
  • duplex stabilizers that can be used are building blocks that can lead to triple helix formation by a third strand of nucleic acid or peptide, and stabilizing molecules, such as e.g. Intercalators, which are embedded between the base stacking of a DNA double strand.
  • Another aspect of the invention is the combination of the asymmetric probe design with in situ cleaning methods in which the termination products of the probe synthesis are removed in situ.
  • post-synthetic array optimization is made possible by the modified building blocks at the end of the polymer probes, which have been extended to the end. Shorter probes mostly do not carry any such modified building blocks and can be removed using suitable methods, such as chemical or / and enzymatic digestion.
  • the invention thus relates to a method for producing a support for the determination of nucleic acid analytes by hybridization, comprising the steps: (a) providing a support body and
  • Receptors selected from nucleic acids and nucleic acid analogues on the support by location-specific and / or time-specific immobilization of receptor building blocks at respectively predetermined positions on or in the support body, several different sets of synthesis building blocks being used for the synthesis of the receptors in order to obtain asymmetric, i.e. to receive receptors consisting of several different types of receptor building blocks.
  • the different sets of building blocks are selected so that the individual building blocks are the same in terms of specificity for complementary nucleic acid building blocks from the analyte, but have a different affinity for complementary nucleic acid building blocks from the analyte, so that the preference for full-length products of one synthesized in situ Polymer probe arrays are achieved through a targeted distribution of different types of building blocks along the polymer probes during the synthesis.
  • the preference for full-length products of an in situ synthesized polymer probe array is preferred by a targeted distribution of different types of building blocks along the polymer probes - 1 - reached during synthesis.
  • sets of synthesis building blocks are used for the method according to the invention, which behave in the same way with regard to certain parameters, but differ from one another in certain, for example thermodynamic, properties.
  • the distribution of the building blocks along the growing polymer during the in situ synthesis is chosen such that the full-length products with the number of building blocks n or at least the synthesis products from the last addition steps of the polymer extension contain modified building blocks.
  • a set of synthetic building blocks is used in the construction of the receptors, which has a higher affinity for complementary nucleic acid building blocks from the analyte than those previously used.
  • the one set of synthetic building blocks used for example, for the last step or steps in the construction of the receptors additionally has a higher resistance to degradation reagents, for example enzymes, such as nucleases and / and chemical reagents, such as acids or bases, in comparison to has the set of synthetic building blocks used for the first steps in the construction of the receptors.
  • a targeted degradation step can be carried out, for example, after the end of the receptor synthesis, with which the proportion of non-full-length products is reduced compared to the proportion of full-length products.
  • "degradable" building blocks and a subsequent dismantling step can also be installed one or more times during earlier steps of receptor synthesis.
  • An alternative or additional procedure provides for the generation of different hybridization affinities for individual sets of building blocks by using modifications of the receptors, for example by means of hybridization amplifiers, whereby their properties are changed in the desired manner in favor of the full-length products.
  • the installation of hybridization amplifiers is site-specific, ie an increased hybridization affinity for complementary nucleotide building blocks from the analyte is provided for a predetermined number (ie a set) of individual building blocks from the receptor.
  • the hybridization enhancer is preferably added to the distal end of the receptor, the last 3-5 bases of the receptor, for example, being modified with regard to the hybridization affinity.
  • a nucleic acid array selected from DNA or RNA arrays, in particular a DNA array is built up, a first set of synthesis building blocks consisting of unmodified DNA or RNA synthesis building blocks which are expediently in Form of suitable derivatives with phosphoramidites, H-phosphonates, etc. is used.
  • a set of synthesis building blocks selected from N3'-P5'-phosphoramidate (NP) building blocks, locked nucleic acid (LNA) building blocks, morpholinophosphorodiamidate (MF) building blocks, 2'-O- Methoxyethyl (MOE) building blocks, 2'-fluoro, arabino-nucleic acid (FANA) building blocks or peptide nucleic acid (PNA) building blocks is used.
  • NP N3'-P5'-phosphoramidate
  • LNA locked nucleic acid
  • MF morpholinophosphorodiamidate
  • MOE 2'-O- Methoxyethyl
  • FANA arabino-nucleic acid
  • PNA peptide nucleic acid
  • the method according to the invention is, however, also suitable for the construction of modified nucleic acid arrays, the first set of building blocks being a first modified building block insert and the second set being a second modified building block insert, the two building block inserts - as described above - with respect to Affinity for complementary nucleic acid building blocks of the analyte and optionally distinguish additionally in terms of resistance to degradation reagents.
  • the method according to the invention circumvents the cleaning problem for in situ polymer probes by an asymmetrical configuration of the probes, which leads to an increased contribution of the full-length products to the binding energy in the double strand in a later application on the biochip.
  • the analysis quality of an in situ produced polymer probe array can thus be brought into the same range as when using cleaned probes from the "off chip” synthesis, while at the same time the advantages of the in situ synthesis come into play.
  • the method according to the invention is also suitable for the production of RNA arrays, e.g. Ribozyme arrays are suitable.
  • the method according to the invention is suitable for the detection and / or isolation of nucleic acids, e.g. for performing de novo sequencing, re-sequencing and point mutation analyzes, e.g. SNP analysis and detection of new SNPs.
  • the method can be used for the analysis of genomes, genome variations, genome stabilities and chromosomes as well as for gene expression or transcriptome analysis or for the analysis of cDNA libraries.
  • the method is also suitable for the production of substrate-bound cDNA libraries or cRNA libraries.
  • Arrays can also be generated for the production of synthetic nucleic acids, nucleic acid duplexes and synthetic genes.
  • arrays of PCR primers, probes for homogeneous assays, molecular beacons and hairpin probes can also be produced.
  • arrays for the production, optimization or development of antisense molecules can also be created.
  • the method according to the invention is particularly suitable for the production of carrier bodies with channels, for example with closed channels.
  • the Channels are preferably microchannels with a cross section of, for example, 10-1000 ⁇ m.
  • suitable carrier bodies with channels are described in WO00 / 1301 8.
  • a carrier body is preferably used which is at least partially optically transparent and / or electrically conductive in the region of the positions to be equipped with receptors.
  • the method according to the invention is furthermore particularly suitable as an integrated synthesis analysis method, i.e. the finished support is used in situ for the analyte determination and then optionally for further synthesis-analysis cycles as described in WO00 / 1301 8.
  • the invention also relates to a carrier for the determination of analytes which contains a large number, preferably at least 100 and particularly preferably at least 500, of different immobilized receptors, the receptors each consisting of a number of different, e.g. two or more sets of synthetic building blocks are constructed, and the individual synthetic building blocks are the same in terms of specificity for complementary nucleic acid building blocks from the analyte, but have a different affinity for complementary nucleic acid building blocks from the analyte.
  • a carrier for the determination of analytes which contains a large number, preferably at least 100 and particularly preferably at least 500, of different immobilized receptors, the receptors each consisting of a number of different, e.g. two or more sets of synthetic building blocks are constructed, and the individual synthetic building blocks are the same in terms of specificity for complementary nucleic acid building blocks from the analyte, but have a different affinity for complementary nucleic acid building blocks from the analyte.
  • the invention further relates to a reagent kit comprising a carrier body and at least two different sets of building blocks for the synthesis of receptors on the carrier.
  • the reagent kit can also contain reaction liquids.
  • the invention also relates to a device for integrated synthesis and analyte determination on a support, comprising a programmable light source matrix, a detector matrix, one preferably arranged between the light source and detector matrix Carriers and means for supplying fluids into the carrier and for discharging fluids from the carrier and optionally reservoirs for synthesis reagents and samples.
  • the programmable light source or exposure matrix can be a reflection matrix, a light valve matrix, for example an LCD matrix, or a self-emitting exposure matrix. Such light matrices are disclosed in WOOO / 1301 8.
  • the detector matrix for example an electronic CCD matrix, can optionally be integrated in the carrier body.
  • the construction of the receptors on the carrier can comprise fluid-chemical synthesis steps, photochemical synthesis steps, electrochemical synthesis steps or combinations of two or more of these steps.
  • An example of the electrochemical synthesis of receptors on a support is described in DE 101 20 663.1.
  • An example of a hybrid method comprising the combination of fluid chemical steps and photochemical steps is described in DE 1 01 22 357.9.
  • the invention is further illustrated by the following example.
  • a DNA microarray to a length of the DNA probes of 25 building blocks is synthesized.
  • an analogue with suitable properties is condensed to the probe instead of a natural nucleotide.
  • This can be an LNA (locked nucleic acid) building block, which is known to be able to be produced for all four bases of the DNA (and thus a set of suitable building blocks is available), and for all four bases with significantly higher ones Melting temperature hybridized to its complementary target molecule.
  • LNA locked nucleic acid

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Abstract

La présente invention concerne un procédé de réalisation d'un support pour la détection d'analytes d'acide nucléique par hybridation, comprenant la synthèse in situ de récepteurs sur le support qui s'hybrident avec l'analyte d'acide nucléique à détecter. Il en résulte des sondes polymères ayant des propriétés de liaison qui favorisent d'un point de vue thermodynamique les sondes de ce type qui représentent des produits pleine longueur, plus que seulement le fait qu'elles soient des produits pleine longueur.
PCT/EP2002/011911 2001-10-26 2002-10-24 Sondes asymetriques WO2003038122A2 (fr)

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DE10152925A DE10152925A1 (de) 2001-10-26 2001-10-26 Asymmetrische Sonden
DE10152925.2 2001-10-26

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WO2003038122A2 true WO2003038122A2 (fr) 2003-05-08
WO2003038122A3 WO2003038122A3 (fr) 2004-03-25

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6156501A (en) * 1993-10-26 2000-12-05 Affymetrix, Inc. Arrays of modified nucleic acid probes and methods of use
CA2214430A1 (fr) * 1995-03-04 1996-09-12 Boehringer Mannheim Gmbh Detection d'acides nucleiques specifique aux sequences
US6130046A (en) * 1998-05-04 2000-10-10 Affymetrix, Inc. Techniques for synthesis integrity evaluation utilizing cycle fidelity probes
WO2000012123A2 (fr) * 1998-08-28 2000-03-09 Febit Ferrarius Biotechnology Gmbh Procede et dispositif de mesure pour la determination d'une pluralite d'analytes dans un echantillon
ATE314488T1 (de) * 1999-03-18 2006-01-15 Exiqon As Einstufige probezubereitung und bestimmung von nukleinsäure in biologischen proben

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WO2003038122A3 (fr) 2004-03-25
DE10152925A1 (de) 2003-05-08

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