WO2003038122A2 - Asymmetrical probes - Google Patents

Asymmetrical probes 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)
French (fr)
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WO2003038122A3 (en
Inventor
Peer F. STÄHLER
Ralf Mauritz
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Febit Ag
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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
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    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • 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
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    • 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
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    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00511—Walls of reactor vessels
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—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
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    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—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
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    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—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
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    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—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

Abstract

The invention relates to a method for the production of a support for the determination of nucleic acid analytes by hybridisation, comprising the in-situ synthesis of receptors on the support which hybridise with the nucleic acid analytes under test. Polymeric probes are the result which have binding properties thermodynamically favouring such probes, representing full-length products, more than just through the fact of being full-length products.

Description

Asymmetrische SondenAsymmetric probes
Beschreibungdescription
Die Erfindung betrifft ein Verfahren zur Herstellung eines Trägers für die Bestimmung von Nukleinsäureanalyten durch Hybridisierung, umfassend die in situ Synthese von Rezeptoren auf dem Träger, die mit dem zu bestimmenden Nukleinsäureanalyten hybridisieren. Das Ergebnis sind Polymersonden mit einem Bindungsverhalten, bei dem solche Sonden thermodynamisch bevorzugt sind, die Volllangenprodukte darstellen, mehr als nur alleine durch die Tatsache, dass sie Volllangenprodukte sind.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.
Biochemische und biologische Analyseverfahren lassen sich durch Miniaturisierung und Parallelisierung in ihrer Effizienz und Aussagekraft enorm steigern. Solche Miniaturisierungen betreffen z.B. die Analyse von genetischem Material mit Hilfe von Hybridisierungsexperimenten. DurchBiochemical 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. By
Entwicklung von Mikroarrays aus geeigneten Hybridisierungssonden lassen sich ganze Genome und Transkriptome untersuchen. Mikroreaktionstechnik kann mit solchen Mikroarrays gekoppelt werden, um auch beiDevelopment of 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
Probenvorbereitung oder Herstellung des Arrays zu schnellen und effizienten Systemen zu kommen. Dies schließt auch die Verwendung von mikrofluidischen Verfahren mit ein. Neben den recht weit verbreitetenSample preparation or fabrication of the array to come to fast and efficient systems. This also includes the use of microfluidic processes. In addition to the quite widespread
DNA-Mikroarrays sind auch Verfahren miniaturisiert und parallelisiert worden, die dem Screening nach besonderen Moleküleigenschaften dienen, z.B. Ribozyme. Weitere Polymersonden sind Proteine und solche Moleküle, die in der Natur nicht vorkommen, wie z.B. Peptidnukleinsäuren (PNA) .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).
Solche Mikroarrays werden auch als Biochips bezeichnet, d.h. Träger, auf denen eine Vielzahl unterschiedlicher Rezeptorsonden immobilisiert ist. Eine besondere Klasse von Mikroarrays wird durch in situ Synthese der benötigten Polymersonden direkt auf dem Träger hergestellt. Diese Reaktionsträger können z.B. zum Einsatz kommen in der akademischen Forschung, der Grundlagenforschung, der industriellen Forschung, in der Qualitätskontrolle, in der Pharmaforschung, in der Biotechnologie, in der klinischen Forschung, in Screening-Verfahren, in der patientenindividuellen Diagnostik, in klinischen Studien, in der Forensik, für genetische Tests, wie Elternschaftsbestimmungen, in der Tier- und Pflanzenzucht oder im Umweltmonitoring.Such 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. These 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.
Für die in situ Synthese von Polymersonden-Arrays auf einem Träger stehen verschiedene Verfahren zur Verfügung. Sie haben das gemeinsame Ziel, einen eleganten Weg zur Herstellung dieser Arrays zu eröffnen, der ressourcenschonend und ökonomisch ist und meist ein besonders gut definiertes Substrat für die nachfolgenden Analysen liefert. Außerdem können durch in situ Verfahren Arrays mit einer besonders hohen Zahl an unterschiedlichen Rezeptorsonden auf einem Reaktionsträger erzeugt werden.Various methods are available for the in situ synthesis of polymer probe arrays on a support. They have the common goal of opening an elegant way of producing these arrays that is resource-saving and economical and usually provides a particularly well-defined substrate for the subsequent analyzes. In addition, arrays with a particularly large number of different receptor probes can be generated on a reaction carrier by in situ methods.
Ein wesentlicher Nachteil solcher Verfahren ist aber, dass bei einem in situ Synthese-Prozess das Produkt der Synthese nicht anschließend auf gereinigt werden kann. Zur Vermeidung dieses Nachteils werden bei sogenannten "off chip" Synthesen der Polymersonden die entsprechenden DNA- Moleküle mit konventionellen Methoden hergestellt und anschließend dergestalt gereinigt, dass nahezu ausschließlich das Volllängenprodukt der Synthese vorliegt. Nur diese Molekülpopulation wird anschließend auf dem Substrat als Array angeordnet. Ein Nachteil dieses Verfahrens ist jedoch, dass die Anordnung der fertigen Polymersonden auf dem Array sehr aufwendig ist.A major disadvantage of such processes is, however, that in an in situ synthesis process, the product of the synthesis cannot subsequently be purified. To avoid this disadvantage, 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, however, is that the arrangement of the finished polymer probes on the array is very complex.
Die mangelnde Auf reinigung kann im Zusammenspiel mit der spezifischen Ausbeute der in situ Synthese zu deutlichen Einbußen bei derThe lack of purification combined with the specific yield of the in situ synthesis can lead to significant losses in the
Analysequalität führen, wenn der Anteil an Volllängenprodukt vergleichsweise niedrig ist. Dies spielt insbesondere bei DNA-Mikroarrays eine wesentliche Rolle, da die Länge eines immobilisierten DNA- Sondenmoleküls über die Spezifität der potentiellen Hybridisierungsreaktion mit einem Probenmolekül entscheidet. Diese Spezifität ist wiederum ein entscheidender Parameter für das analytische Potential eines DNA- Mikroarrays.Lead analysis quality if the proportion of full-length product is comparatively low. This is particularly important with DNA microarrays play an important role since the length of an immobilized DNA probe molecule determines the specificity of the potential hybridization reaction with a sample molecule. This specificity is in turn a crucial parameter for the analytical potential of a DNA microarray.
Bisher bekannte Verfahren zur in situ Herstellung von Polymersonden- Arrays beinhalten, dass alle aufeinanderfolgenden Additionsschritte mit einzelnen Bausteinen dieser Sonden durchgeführt werden. Keiner dieser Schritte weist eine Kopplungsrate von 100 % auf. Für den Fachmann ist klar, dass eine chemische serielle. Kondensation wie im Fall einer in situ Synthese von Polymersonden nicht zu 100 % Volllängenprodukt führen kann. In der DNA-Synthese sind die Kopplungsraten für die konventionelle Säulenmethodik nach vielen Jahren der Optimierung und unter Verwendung der effizientesten bekannten chemischen Methode (Phosphitamid-Methode nach Caruthers) immer noch unter 100 %.Previously known methods for the in situ production of polymer probe arrays include that all successive addition steps are carried out with individual components of these probes. None of these steps have a 100% coupling rate. It is clear to the person skilled in the art that a chemical serial. Condensation as in the case of in situ synthesis of polymer probes cannot lead to a 100% full length product. In DNA synthesis, the coupling rates for conventional column methodology are still below 100% after many years of optimization and using the most efficient known chemical method (Caruthers' phosphitamide method).
Mit vergleichsweise niedrigen Ausbeuten bei der seriellen Ankopplung von Synthesebausteinen kann der Anteil des Volllängenproduktes nach X Synthesezyklen unter einen kritischen Wert fallen, so dass das Analyseergebnis gar nicht von diesem Volllängenprodukt geprägt wird. Bei der photolitographischen in situ Synthese von DNA mit MeNPOC- Schutzgruppen ist z.B. beschrieben, dass die Kopplungsrate der einzelnen Additionsschritte unter 95 % liegt (Beier, M., Hoheisel, J.D., Production by quantitative photolitogaphic synthesis of individually quality checked DNA microarrays, Vol. 28, No. 4, P. 1 -6, 2000). Mit solchen Verfahren lassen sich sinnvollerweise nur DNA-Polymersonden bis zu einer Länge von 25 Basen erzeugen. Auf solch einem Array stehen nur noch ca. 27 % Volllangenprodukte, sofern die Rate tatsächlich 95 % beträgt. Mit einer Kopplungsrate von 90 % je Additionsschritt ergibt sich nur noch ein Wert von 7 %. Bislang kann nur die Synthese von Polymersonden vor der Anordnung auf dem Array unter Verwendung eines geeigneten Reinigungsschrittes für nahezu 100 % Volllängen-Sonden sorgen. Diese Vorgehensweise ist jedoch - wie bereits ausgeführt - mit anderen Nachteilen behaftet.With comparatively low yields in the serial coupling of synthesis building blocks, 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. In the case of the photolitographic in situ synthesis of DNA with MeNPOC protective groups, it is described, for example, that 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. Only about 27% of full-length products are left on such an array, provided the rate is actually 95%. With a coupling rate of 90% per addition step, there is only a value of 7%. So far, only the synthesis of polymer probes before placement on the array using a suitable cleaning step can provide nearly 100% full length probes. However, as already stated, this procedure has other disadvantages.
Vor diesem Hintergrund ist es wünschenswert, die geschilderten Nachteile zu vermeiden, die sich aus einer Population von unterschiedlich langen Molekülen auf den einzelnen Positionen eines Mikroarrays ergeben können, ohne die Nachteile einer "off chip" Synthese in Kauf nehmen zu müssen.Against this background, it is desirable to avoid the disadvantages described, which can result from a population of molecules of different lengths on the individual positions of a microarray, without having to accept the disadvantages of an "off chip" synthesis.
Die vorliegende Erfindung beschreibt ein Verfahren zur Verbesserung der Nutzung von in situ Syntheseverfahren in der Herstellung von Polymersonden-Arrays, indem der Beitrag von Volllängenprodukten aus dem Syntheseprozess zum Analyseergebnis erhöht wird. Dies wird erreicht durch eine asymmetrische Konfiguration der Polymersonden. Insbesondere in den letzten Syntheseschritten werden hierzu modifizierte Bausteine verwendet, die sich in bestimmten thermodynamischen Eigenschaften, wie z.B. der Bindungsstabilität, von den vorher verwendeten Bausteinen unterscheiden. Alternativ oder zusätzlich kann der gleiche Effekt erzielt werden durch eine geeignete Modifikation des distalen Endes der Polymersonden, z.B. mit einem Hybridisierungsverstärker. Ein solches Molekül ist z.B. ein sogenannter "Minor Groove Binder" (Epoch Biosciences 2000 Annual Report, Seiten 4-5), der die Stabilität der Bindung an die letzten 4-5 Basen der Polymersonde deutlich erhöht. Beispiele für solche "Minor Groove Binders" sind einige natürliche Antibiotika mit einer Gestalt, die eine Faltung in die "Minor Groove" einer DNA-Helix erlaubt. Damit wird die bei in situ Synthesen fehlende Aufreinigung der Polymersonden vor der Aufbringung in einem Polymersonden-Array substituiert. Der Qualitätsnachteil von in situ Syntheseverfahren wird auf diese Weise teilweise oder ganz ausgeglichen. Durch das erfindungsgemäße Verfahren wird die Nutzbarkeit von in situ synthetisierten Polymersonden-Arrays in Bezug auf die Qualität und Aussagekraft der Analyse im Vergleich zum Stand der Technik verbessert. Insbesondere für die Anwendung von Analysen, die mit sehr genauem Analyseergebnis arbeiten müssen, wie z.B. die Bestimmung von Punktmutationen in der klinischen molekularen Diagnostik, wird damit ein Zugang zu breiterer Anwendung und die Grundlage für bessere Ersetzbarkeit der in situ synthetisierten Polymersonden-Arrays geschafffen.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. In the last synthesis steps in particular, modified building blocks are used that differ from the previously used building blocks in certain thermodynamic properties, such as, for example, the binding stability. Alternatively or additionally, 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. Examples of such "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. In particular for the use of analyzes that have to work with a very precise analysis result, such as the determination of point mutations in clinical molecular diagnostics, this creates access to broader application and the basis for better replaceability of the polymer probe arrays synthesized in situ.
Verfahren und Moleküle für die Synthese von Polymersonden mit modifizierten thermodynamischen Eigenschaften unter Verwendung von modifizierten Nukleotidbausteinen sind aus der Patentschrift US 6,1 56,501 A bekannt. Auf die dort offenbarten Bausteine wird ausdrücklich Bezug genommen. Darüber hinaus sind in der Literatur Modifikationen an der fertigen Polymersonde bekannt, die die Bindungseigenschaften der Polymersonden verändern, z.B. eine Einlagerung von "Minor Groove Binders" (MGB). Modifizierte Synthesebausteine sind beispielsweise Ribonukleosidanaloga, wie LNA's (locked nucleic acids), modifizierte Purin- bzw. Pyrimidinbasen, wie superstabilisierende Adenosinanaloga (z.B. 2,4- D i ami n o aden osi n ) , Pyrazo lopyri mid ine ( z . B . P PG) sowie Phosphatrückgratanaloga, wie z.B. Methylphosphonate, Phosphorthionate, Phosphoramidate etc.Methods and molecules for the synthesis of polymer probes with modified thermodynamic properties using modified nucleotide building blocks are known from US Pat. No. 6,156,501. Reference is expressly made to the components disclosed there. In addition, modifications to the finished polymer probe which alter the binding properties of the polymer probes are known in the literature, e.g. a storage of "Minor Groove Binders" (MGB). 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.
Weitere verwendbare Duplexstabilisatoren sind Bausteine, die zu einer Tripelhelixbildung durch einen dritten Nukleinsäure- oder Peptidstrang führen können, sowie stabilisierende Moleküle, wie z.B. Interkalatoren, die sich zwischen die Basenstapelung eines DNA-Doppelstranges einlagern.Other 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.
Ein weiterer Aspekt der Erfindung ist die Kombination des asymmetrischen Sondendesigns mit in situ Reinigungsmethoden, bei denen die Abbruchprodukte der Sondensynthese in situ entfernt werden. Die postsynthetische Array-Optimierung wird in dieser Ausführungsform durch die modifizierten Bausteine am Ende der bis zuletzt verlängerten Polymersonden ermöglicht. Kürzere Sonden tragen überwiegend keine solchen modifizierten Bausteine und lassen sich mit geeigneten Verfahren, wie z.B. einem chemischen oder/und enzymatischen Verdau, entfernen.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. The In this embodiment, 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.
Ein Gegenstand der Erfindung ist somit ein Verfahren zur Herstellung eines Trägers für die Bestimmung von Nukleinsäure-Analyten durch Hybridisierung, umfassend die Schritte: (a) Bereitstellen eines Trägerkörpers undThe 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
(b) schrittweises Aufbauen eines Arrays von mehreren verschiedenen(b) gradually building an array of several different ones
Rezeptoren ausgewählt aus Nukleinsäuren und Nukleinsäureanaloga auf dem Träger durch orts- oder/und zeitspezifisches Immobilisieren von Rezeptorbausteinen an jeweils vorbestimmten Positionen auf dem oder im Trägerkörper, wobei man für die Synthese der Rezeptoren mehrere unterschiedliche Sätze von Synthesebausteinen verwendet, um asymmetrische, d.h. aus mehreren unterschiedlichen Arten von Rezeptorbausteinen bestehende Rezeptoren zu erhalten.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.
Die unterschiedlichen Sätze von Bausteinen werden dabei so ausgewählt, dass die einzelnen Bausteine in Bezug auf die Spezifität für komplementäre Nukleinsäurebausteine aus dem Analyten gleich sind, aber eine unterschiedliche Affinität für komplementäre Nukleinsäurebausteine aus dem Analyten aufweisen, so dass die Bevorzugung von Volllängenprodukten eines in situ synthetisierten Polymersonden-Arrays durch eine gezielte Verteilung unterschiedlicher Arten von Bausteinen entlang der Polymersonden während der Synthese erreicht wird.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.
Vorzugsweise wird die Bevorzugung von Volllängenprodukten eines in situ synthetisierten Polymersonden-Arrays durch eine gezielte Verteilung unterschiedlicher Arten von Bausteinen entlang der Polymersonden - 1 - während der Synthese erreicht. Dazu werden für das erfindungsgemäße Verfahren Sätze von Synthesebausteinen verwendet, die sich in Bezug auf bestimmte Parameter gleich verhalten, aber in bestimmten, z.B. thermodynamischen, Eigenschaften voneinander abweichen. Die Verteilung der Bausteine entlang des wachsenden Polymers während der in situ Synthese wird dabei so gewählt, dass die Volllangenprodukte mit der Bausteinzahl n oder aber zumindest die Syntheseprodukte aus den letzten Additionsschritten der Polymerverlängerung modifizierte Bausteine enthalten.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. For this purpose, 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.
In einer bevorzugten Ausführungsform wird zumindest für den letzten Schritt oder die letzten Schritte, z.B. die letzten zwei, drei oder vier Schritte, beim Aufbau der Rezeptoren ein Satz von Synthesebausteinen verwendet, der eine höhere Affinität für komplementäre Nukleinsäurebausteine aus dem Analyten aufweist als die vorher verwendeten.In a preferred embodiment, at least for the last step or steps, e.g. the last two, three or four steps, 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.
Weiterhin ist es bevorzugt, dass der einen, z.B. für den oder die letzten Schritte beim Aufbau der Rezeptoren verwendete Satz von Synthesebausteinen zusätzlich eine höhere Beständigkeit gegenüber Abbaureagenzien, z.B. Enzymen, wie Nukleasen oder/und chemischen Reagenzien, wie Säuren oder Basen, im Vergleich zu dem für die ersten Schritte des Aufbaus der Rezeptoren verwendeten Satz von Synthesebausteinen aufweist. In diesem Fall kann z.B. nach Beendigung der Rezeptorsynthese ein gezielter Abbauschritt durchgeführt werden, mit dem der Anteil von Nicht-Volllängenprodukten gegenüber dem Anteil der Volll ngenprodukte verringert wird. Der Einbau von "abbaubeständigen" Bausteinen und ein nachfolgender Abbauschritt können im Übrigen auch ein- oder mehrmals während früherer Schritte der Rezeptorsynthese erfolgen. Eine alternative oder ergänzende Vorgehensweise sieht die Erzeugung unterschiedlicher Hybridisierungsaffinitäten für einzelne Sätze von Bausteinen durch Verwendung von Modifikationen der Rezeptoren, z.B. mittels Hybridisierungsverstärkern, vor, wodurch ihre Eigenschaften in der gewünschten Art zugunsten der Volllangenprodukte verändert werden. Der Einbau von Hybridisierungsverstärkern erfolgt ortsspezifisch, d.h. es wird eine erhöhte Hybridisierungsaffinitätfür komplementäre Nukleotidbausteine aus dem Analyten für eine vorbestimmte Anzahl (d.h. einen Satz) einzelner Bausteine aus dem Rezeptor vorgesehen. Vorzugsweise wird der Hybridisierungsverstärker an das distale Ende des Rezeptors angefügt, wobei z.B. die letzten 3-5 Basen des Rezeptors bezüglich der Hybridisierungsaffinität modifiziert werden.Furthermore, it is preferred that 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. In this case, 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. Incidentally, "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.
In einer bevorzugten Ausführungsform des erfindungsgemäßen Verfahrens wird ein Nukleinsäure-Array, ausgewählt aus DNA- oder RNA-Arrays, insbesondere ein DNA-Array, aufgebaut, wobei ein erster Satz von Synthesebausteinen, bestehend aus unmodifizierte DNA- oder RNA- Synthesebausteinen, die zweckmäßigerweise in Form geeigneter Derivate mit Phosphoramidite, H-Phosphonate etc. vorliegen, verwendet wird. Als zweiter Satz für den oder die letzten Schritte des Rezeptoraufbaus wird dann ein Satz von Synthesebausteinen, ausgewählt aus N3'-P5'- Phosphoramidat (NP) Bausteinen, Locked Nukleinsäure (LNA) Bausteinen, Morpholinophosphordiamidat (MF) Bausteinen, 2'-O-Methoxyethyl (MOE) Bausteinen, 2'-Fluoro,arabino-Nukleinsäure (FANA) Bausteinen oder Peptidnukleinsäure (PNA) Bausteinen verwendet wird. Selbstverständlich ist das erfindungsgemäße Verfahren jedoch auch für den Aufbau von modifizierten Nukleinsäure-Arrays geeignet, wobei als erster Satz von Bausteinen ein erster modifizierter Bausteinsatz und als zweiter Satz ein zweiter modifizierter Bausteinsatz verwendet wird, wobei sich die beiden Bausteinsätze - wie zuvor beschrieben - hinsichtlich der Affinität für komplementäre Nukleinsäurebausteine des Analyten und gegebenenfalls zusätzlich hinsichtlich der Beständigkeit gegenüber Abbaureagenzien unterscheiden.In a preferred embodiment of the method according to the invention, 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. Of course, 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.
Das erfindungsgemäße Verfahren umgeht die Reinigungsproblematik für in situ Polymersonden durch eine asymmetrische Konfiguration der Sonden, die zu einem erhöhten Beitrag der Volllangenprodukte zur Bindungsenergie im Doppelstrang bei einer späteren Anwendung auf dem Biochip führt. Damit kann die Analysequalität eines in situ hergestellten Polymersonden- Arrays in denselben Bereich gebracht werden wie bei Verwendung von gereinigten Sonden aus der "off chip" Synthese, während gleichzeitig die Vorteile der in situ Synthese zum Tragen kommen. Darüber hinaus ist das erfindungsgemäße Verfahren auch zur Herstellung von RNA-Arrays, z.B. Ribozym-Arrays geeignet.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. In addition, the method according to the invention is also suitable for the production of RNA arrays, e.g. Ribozyme arrays are suitable.
Das erfindungsgemäße Verfahren eignet sich zum Nachweis oder/und zur Isolierung von Nukleinsäuren, z.B. zur Durchführung von De-novo- Sequenzierungen, Re-Sequenzierungen und Punktmutationsanalysen, z.B. SNP-Analysen und den Nachweis neuer SNPs. Weiterhin kann das Verfahren für die Analyse von Genomen, Genomvariationen, Genominstabilitäten und Chromosomen sowie zur Genexpressions- bzw. Transkriptomanalyse oder zur Analyse von cDNA-Bibliotheken eingesetzt werden. Das Verfahren eignet sich auch für die Herstellung von substratgebundenencDNA-BibliothekenodercRNA-Bibliotheken. Außerdem können Arrays für die Herstellung synthetischer Nukleinsäuren, Nukleinsäuredoppelstränge und synthetischer Gene erzeugt werden. Desweiteren können auch Arrays von PCR-Primem, Sonden für homogene Assays, Molecular Beacons und Haarnadel-Sonden hergestellt werden. Schließlich können auch Arrays für die Herstellung, Optimierung oder Entwicklung von Antisense-Molekülen erzeugt werden.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. Furthermore, 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. Furthermore, arrays of PCR primers, probes for homogeneous assays, molecular beacons and hairpin probes can also be produced. Finally, arrays for the production, optimization or development of antisense molecules can also be created.
Das erfindungsgemäße Verfahren eignet sich besonders für die Herstellung von Trägerkörpern mit Kanälen, z.B. mit geschlossenen Kanälen. Die Kanäle sind vorzugsweise Mikrokanäle mit einem Querschnitt von z.B. 10- 1000 μm. Beispiele für geeignete Trägerkörper mit Kanälen sind in WO00/1301 8 beschrieben. Vorzugsweise wird ein Trägerkörper verwendet, der zumindest teilweise im Bereich der mit Rezeptoren zu bestückenden Positionen optisch transparent oder/und elektrisch leitfähig ist.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 μm. Examples of 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.
Das erfindungsgemäße Verfahren eignet sich weiterhin besonders als integriertes Synthese-Analyse-Verfahren, d.h. der fertige Träger wird in situ für die Analytbestimmung und anschließend gegebenenfalls für weitere Synthese-Analyse-Zyklen eingesetzt wie in WO00/1301 8 beschrieben.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.
Weiterhin betrifft die Erfindung auch einen Träger für die Bestimmung von Analyten, der eine Vielzahl, vorzugsweise mindestens 100 und besonders bevorzugt mindestens 500, von unterschiedlichen immobilisierten Rezeptoren enthält, wobei die Rezeptoren aus jeweils mehreren unterschiedlichen, z.B. zwei oder noch mehreren Sätzen von Synthesebausteinen aufgebaut sind, und wobei die einzelnen Synthesebausteine in Bezug auf die Spezifität für komplementäre Nukleinsäurebausteine aus dem Analyten gleich sind, aber eine unterschiedliche Affinität für komplementäre Nukleinsäurebausteine aus dem Analyten aufweisen.Furthermore, 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.
Weiterhin betrifft die Erfindung einen Reagenzienkit, umfassend einen Trägerkörper und mindestens zwei unterschiedliche Sätze von Bausteinen für die Synthese von Rezeptoren auf dem Träger. Weiterhin kann der Reagenzienkit auch Reaktionsflüssigkeiten enthalten.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.
Gegenstand der Erfindung ist auch eine Vorrichtung zur integrierten Synthese- und Analytbestimmung an einem Träger, umfassend eine programmierbare Lichtquellenmatrix, eine Detektormatrix, einen vorzugsweise zwischen Lichtquellen- und Detektormatrix angeordneten Träger sowie Mittel zur Zufuhr von Fluids in den Träger und zur Ableitung von Fluids aus dem Träger und gegebenenfalls Reservoirs für Synthesereagenzien und Proben. Die programmierbare Lichtquellen- bzw. Belichtungsmatrix kann eine Reflexionsmatrix, eine Lichtventilmatrix, z.B. eine LCD-Matrix, oder eine selbstemittierende Belichtungsmatrix sein. Derartige Lichtmatrices sind in WOOO/1301 8 offenbart. Die Detektormatrix, z.B. eine elektronische CCD-Matrix, kann gegebenenfalls im Trägerkörper integriert sein.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.
Der Aufbau der Rezeptoren auf dem Träger kann fluidchemische Syntheseschritte, photochemische Syntheseschritte, elektrochemische Syntheseschritte oder Kombinationen von zwei oder mehreren dieser Schritte umfassen. Ein Beispiel für die elektrochemische Synthese von Rezeptoren auf einen Träger ist in DE 101 20 663.1 beschrieben. Ein Beispiel für ein Hybridverfahren, umfassend die Kombination von fluidchemischen Schritten und photochemischen Schritten, ist in DE 1 01 22 357.9 beschrieben.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.
Weiterhin soll die Erfindung durch das nachfolgende Beispiel erläutert werden.The invention is further illustrated by the following example.
Beispielexample
Es wird ein DNA-Mikroarray zu einer Länge der DNA-Sonden von 25 Bausteinen synthetisiert. Für den letzten Baustein wird statt einem natürlichen Nukleotid ein Analogon mit geeigneten Eigenschaften an die Sonde kondensiert. Dies kann ein LNA (locked nucleic acid) Baustein sein, von dem bekannt ist, dass er zum einen für alle vier Basen der DNA hergestellt werden kann (und damit ein Satz passender Bausteine vorhanden ist), zum anderen für alle vier Basen mit deutlich höherer Schmelztemperatur an sein komplementäres Zielmolekül hybridisiert. Die Diskriminierung zwischen einer Hybridisierung oder Bindung an das Volllängenprodukt mit 25 Bausteinen Länge im Vergleich zu den Abbruchprodukten mit 24 oder weniger Nukleotiden wird dadurch verbessert. Das wirkt sich positiv auf das Analyseergebnis aus. A DNA microarray to a length of the DNA probes of 25 building blocks is synthesized. For the last building block, 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. The discrimination between hybridization or attachment to the This improves the full length product with 25 building blocks in length compared to the termination products with 24 or fewer nucleotides. This has a positive effect on the analysis result.

Claims

Ansprüche Expectations
1 . Verfahren zur Herstellung eines Trägers für die Bestimmung von Nukleinsäure-Analyten durch Hybridisierung, umfassend die Schritte:1 . A method for producing a support for the determination of nucleic acid analytes by hybridization, comprising the steps:
(a) Bereitstellen eines Trägerkörpers und(a) providing a support body and
(b) schrittweises Aufbauen eines Arrays von mehreren unterschiedlichen Rezeptoren ausgewählt aus Nukleinsäuren und Nukleinsäureanaloga auf dem Träger durch orts- oder/und zeitspezifisches Immobilisieren von Rezeptorbausteinen an jeweils vorbestimmten Positionen auf dem oder im Träger, dadurch gekennzeichnet, dass man für die Synthese der Rezeptoren mehrere unterschiedliche Sätze von Synthesebausteinen verwendet, wobei die einzelnen Bausteine aus unterschiedlichen Sätzen in Bezug auf die Spezifität für komplementäre Nukleinsäurebausteine aus dem Analyten gleich sind, aber eine unterschiedliche Affinität für komplementäre Nukleinsäurebausteine aus dem Analyten aufweisen, so dass der Beitrag von Volllänge-Rezeptoren bezüglich der Bindung an Nukleinsäure-Analyten gegenüber dem Beitrag von Abbruchprodukt-(b) step-by-step construction of an array of several different receptors selected from nucleic acids and nucleic acid analogs on the carrier by location- and / or time-specific immobilization of receptor building blocks at respectively predetermined positions on or in the carrier, characterized in that several are synthesized for the receptors different sets of synthetic building blocks are used, the individual building blocks from different sets being the same in terms of specificity for complementary nucleic acid building blocks from the analyte, but having a different affinity for complementary nucleic acid building blocks from the analyte, so that the contribution of full-length receptors with regard to binding on nucleic acid analytes compared to the contribution of
Rezeptoren erhöht wird.Receptors is increased.
2. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, dass zumindest für den letzten Schritt beim Aufbau der Rezeptoren ein Satz von Synthesebausteinen verwendet wird, der eine höhere Affinität für komplementäre Nukleinsäurebausteine aus dem Analyten aufweist. 2. The method according to claim 1, characterized in that at least for the last step in the construction of the receptors, a set of synthetic building blocks is used which has a higher affinity for complementary nucleic acid building blocks from the analyte.
3. Verfahren nach Anspruch 1 oder 2, dadurch gekennzeichnet, dass zumindest für einen Schritt beim Aufbau der Rezeptoren ein Satz von Synthesebausteinen verwendet wird, der eine höhere Beständigkeit gegenüber Abbaureagenzien aufweist.3. The method according to claim 1 or 2, characterized in that at least for one step in the construction of the receptors, a set of synthesis building blocks is used, which has a higher resistance to degradation reagents.
4. Verfahren nach Anspruch 3, dadurch gekennzeichnet, dass nach einem Aufbauschritt unter Verwendung eines Satzes von beständigen Synthesebausteinen ein Abbauschrittdurchgeführt wird.4. The method according to claim 3, characterized in that after a set-up step using a set of durable synthesis building blocks, a dismantling step is carried out.
5. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass der erste Satz von Bausteinen aus unmodifizierten DNA- oder RNA-Synthesebausteinen und der zweite Satz aus N3'-P5'-5. The method according to any one of the preceding claims, characterized in that the first set of building blocks from unmodified DNA or RNA synthesis building blocks and the second set of N3'-P5'-
Phosphoramidat (NP) Bausteinen, Locked Nukleinsäure (LNA) Bausteinen, Morpholinophosphordiamidat (MF) Bausteinen, 2'-O- Methoxyethyl (MOE) Bausteinen, 2'-Fluoro,arabino-Nukleinsäure (FANA) Bausteinen oder Peptidnukleinsäure (PNA) Bausteinen ausgewählt wird.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 selected ,
6. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass ein Hybridisierungsverstärker derart an den Rezeptor angefügt wird, dass die Affinität für komplementäre Nukleotidbausteine aus dem Analyten für eine vorbestimmte Anzahl einzelner Bausteine aus dem Rezeptor erhöht wird.6. The method according to any one of the preceding claims, characterized in that a hybridization amplifier is added to the receptor such that the affinity for complementary nucleotide building blocks from the analyte is increased for a predetermined number of individual building blocks from the receptor.
7. Verfahren nach Anspruch 6, dadurch gekennzeichnet, dass der Hybridisierungsverstärker an das distale Ende des Rezeptors angefügt wird. 7. The method according to claim 6, characterized in that the hybridization amplifier is added to the distal end of the receptor.
8. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass man einen Trägerkörper mit Kanälen bereitstellt, Flüssigkeit mit Rezeptorbausteinen durch die Kanäle geleitet wird und die unterschiedlichen Rezeptoren an jeweils vorbestimmten Positionen in den Kanälen aufgebaut werden.8. The method according to any one of the preceding claims, characterized in that a carrier body with channels is provided, liquid with receptor building blocks is passed through the channels and the different receptors are built up at predetermined positions in the channels.
9. Verfahren nach Anspruch 8, dadurch gekennzeichnet, dass der Trägerkörper Mikrokanäle mit einem Durchmesser von 10 bis 1000 /m aufweist.9. The method according to claim 8, characterized in that the carrier body has microchannels with a diameter of 10 to 1000 / m.
10. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass man einen mindestens im Bereich der vorbestimmten Positionen für den Rezeptoraufbau optisch transparenten oder/und elektrisch leitfähigen Träger verwendet.10. The method according to any one of the preceding claims, characterized in that one uses at least in the region of the predetermined positions for the receptor structure optically transparent and / or electrically conductive carrier.
1 1 . Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass der Aufbau der Rezeptoren fluidchemische Syntheseschritte, elektrochemische Syntheseschritte oder/und photochemische Syntheseschritte umfasst.1 1. Method according to one of the preceding claims, characterized in that the structure of the receptors comprises fluid-chemical synthesis steps, electrochemical synthesis steps or / and photochemical synthesis steps.
1 2. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass der Rezeptorarray mindestens 100 unterschiedliche Rezeptoren umfasst. 1 2. The method according to any one of the preceding claims, characterized in that the receptor array comprises at least 100 different receptors.
1 3. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass man den Träger für einen oder mehrere integrierte Synthese- Analyse-Zyklen einsetzt.1 3. The method according to any one of the preceding claims, characterized in that the carrier is used for one or more integrated synthesis-analysis cycles.
14. Träger zur Bestimmung von Nukleinsäure-Analyten durch Hybridisierung, umfassend14. Carrier for the determination of nucleic acid analytes by hybridization, comprising
(a) einen Trägerkörper und(a) a support body and
(b) eine Vielzahl auf dem oder/und im Trägerkörper immobilisierte Rezeptoren ausgewäh lt aus Nuklei nsäuren u nd(b) a multiplicity of receptors immobilized on or / and in the carrier body selected from nucleic acids and
Nukleinsäureanaloga, dadurch gekennzeichnet, dass die Rezeptoren aus mehreren unterschiedlichen Sätzen von Synthesebausteinen aufgebaut sind, wobei die einzelnen Bausteine aus unterschiedlichen Sätzen in Bezug auf die Spezifität für komplementäre Nukleinsäurebausteine aus dem Analyten gleich sind, aber eine unterschiedliche Affinität für komplementäre Nukleinsäurebausteine aus dem Analyten aufweisen.Nucleic acid analogues, characterized in that the receptors are made up of several different sets of synthetic building blocks, the individual building blocks from different sets being the same in terms of specificity for complementary nucleic acid building blocks from the analyte, but having a different affinity for complementary nucleic acid building blocks from the analyte.
1 5. Reagenzienkit zur Herstellung eines Trägers, umfassend1 5. A reagent kit for making a carrier comprising
(a) einen Trägerkörper und(a) a support body and
(b) mi nd este n s zwei u ntersc hied l ic he Sätze vo n Synthesebausteinen zum Aufbau polymerer Rezeptoren ausgewählt aus Nukleinsäuren und Nukleinsäureanaloga auf dem Trägerkörper, wobei die einzelnen Bausteine aus unterschiedlichen Sätzen in Bezug auf die Spezifität für komplementäre Nukleinsäurebausteine aus dem Analyten gleich sind, aber eine unterschiedliche Affinität für komplementäre Nukleinsäurebausteine aus dem Analyten aufweisen. (b) with two different sets of synthetic building blocks for building polymeric receptors selected from nucleic acids and nucleic acid analogs on the carrier body, the individual building blocks from different sets being identical in terms of specificity for complementary nucleic acid building blocks from the analyte are, but have a different affinity for complementary nucleic acid building blocks from the analyte.
1 6. Vorrichtung zur integrierten Synthese und Analytbestimmung an einem Träger, umfassend1 6. Device for integrated synthesis and analyte determination on a carrier, comprising
(a) einen Träger nach Anspruch 14 oder einen Reagenzienkit nach Anspruch 1 5, (b) eine programmierbare Lichtquellenmatrix,(a) a carrier according to claim 14 or a reagent kit according to claims 1-5, (b) a programmable light source matrix,
(c) eine Detektionsmatrix,(c) a detection matrix,
(d) Mittel zur Zufuhr von Fluids in den Träger und zur Ableitung von Fluids aus dem Träger und(d) means for supplying fluids to and removing fluids from the carrier and
(e) gegebenenfalls Reservoirs für Synthesereagenzien und Proben.(e) if necessary, reservoirs for synthesis reagents and samples.
17. Vorrichtung nach Anspruch 16, dadurch gekennzeichnet, dass der Träger bzw. der Trägerkörper des Reagenzienkits zwischen Lichtquellenmatrix und Detektormatrix angeordnet ist. 17. The apparatus according to claim 16, characterized in that the carrier or the carrier body of the reagent kit is arranged between the light source matrix and the detector matrix.
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