EP1763589A1 - Biopuces modeles - Google Patents

Biopuces modeles

Info

Publication number
EP1763589A1
EP1763589A1 EP05771898A EP05771898A EP1763589A1 EP 1763589 A1 EP1763589 A1 EP 1763589A1 EP 05771898 A EP05771898 A EP 05771898A EP 05771898 A EP05771898 A EP 05771898A EP 1763589 A1 EP1763589 A1 EP 1763589A1
Authority
EP
European Patent Office
Prior art keywords
enzyme
attached
dna
enzymes
starting material
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05771898A
Other languages
German (de)
English (en)
Inventor
Jörn GLÖKLER
Philipp Angenendt
Jürgen KREUTZBERGER
Hans Lehrach
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
RiNA Netzwerk RNA-Technologien GmbH
Original Assignee
RiNA Netzwerk RNA-Technologien GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by RiNA Netzwerk RNA-Technologien GmbH filed Critical RiNA Netzwerk RNA-Technologien GmbH
Priority to EP05771898A priority Critical patent/EP1763589A1/fr
Publication of EP1763589A1 publication Critical patent/EP1763589A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01166—Heparanase (3.2.1.166)
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—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
    • B01J2219/0061—The surface being organic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00612—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports the surface being inorganic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00614—Delimitation of the attachment areas
    • B01J2219/00617—Delimitation of the attachment areas by chemical means
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00614—Delimitation of the attachment areas
    • B01J2219/00617—Delimitation of the attachment areas by chemical means
    • B01J2219/00619—Delimitation of the attachment areas by chemical means using hydrophilic or hydrophobic regions
    • 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
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—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
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/0074—Biological products

Definitions

  • This invention relates to a method for producing a surface with one or more products attached thereto, comprising the steps of: (a) putting a first and a second surface on top of each other, wherein one or more starting materials or one or more enzymes are attached to distinct sites on said first surface; (b) contacting said one or more starting materials attached to said first surface with one or more enzymes, or contacting said one or more enzymes attached to said first surface with one or more starting materials, wherein said contacting is effected under conditions permitting generation of one or more products by said enzyme(s) using said starting material(s); and (c) attaching said product(s) to distinct sites on said second surface, thereby obtaining said surface with one or more products attached thereto; wherein (i) step (a) is effected prior to step (b); (ii) step (b) is effected prior to step (a); or (iii) steps (a) and (b) are carried out simultaneously.
  • said first surface with one or more starting materials attached thereto is effected prior
  • biochip embraces a miniaturized device (a chip) carrying one or more distinct biological species such as biological molecules attached to distinct sites of its surface.
  • the arrangement of said sites may be regular, such arrangement being also referred to as an array. Accordingly, this type of biochip is also referred to as array or microarray.
  • DNA microarrays have been produced. Different DNA molecules, be it oligonucleotides or cDNA molecules, are fairly homogeneous with regard to their physicochemical properties as compared to peptides and proteins.
  • DNA is either devoid of a defined tertiary structure or at least a tertiary structure, if present, is not relevant for the type of interaction typically assayed on a DNA microarray, viz. hybridisation.
  • DNA may be immobilized via a terminal group on the surface of a chip without loss or without significant loss of the capability to hybridize.
  • cDNA libraries are available as are methods for the generation thereof, which provide a fair coverage of the transcriptome of a given sample and permit, if needed, to perform a normalization, whereby the cDNA copies of a primary library are equalized so that each original mRNA transcript is represented in the normalized library to the same extent as all the rest of the clones.
  • Manufacture of DNA arrays may be accomplished by performing on-chip synthesis of the DNA molecules. This is effected by a photolithographic process and is typically applied for oligonucleotides (Fodor et al. (1993), Pease et al. (1994)).
  • said longer probes are obtained, for example, from a library and subsequently transferred and attached to the surface of a carrier (Schena et al. (1995)). Transfer is typically performed by a printer, also referred to as arrayer. Attachment may involve the formation of covalent bonds or may be effected by non-covalent interaction.
  • a printer also referred to as arrayer. Attachment may involve the formation of covalent bonds or may be effected by non-covalent interaction.
  • knowledge of the oligonucleotide sequence is a prerequisite, whereas in the second process, the identity (such as the clone the cDNA originates from), if not the sequence, is known. Also known (as a consequence of the process of manufacture) are the coordinates on the array where a given DNA is attached.
  • DNA microarrays have been used to monitor the expression of cells, tissues and even entire organisms.
  • Proteins for microchips are typically expressed in simple recombinant organisms such as E.coli and subsequently purified. As many proteins are produced by the host in a non-soluble form, purification is effected under denaturing conditions in many cases. Even functional native proteins may suffer from immobilizing on a chip surface, from running dry of the surface they are attached to, or from long-term storage of the chip in that their activity is lost, decreased or modified.
  • WO-A1 0170399 describes a microhybridization chamber. Aiming at improved throughput of chip experiments, preferably of DNA chip experiments, two chips are put on top of each other with a spacer in between, such that a chamber is formed, allowing the simultaneous hybridization of two chips with the same hybridization cocktail. A transfer of material from on chip to the other is not envisaged.
  • WO-A1 0214860 relates to a method of producing a protein array starting from DNA (or mRNA) using cell-free in vitro synthesis of the protein.
  • the protein is generated in situ in wells or on the surface of beads. Capture of the protein occurs on the surface of the same well, or the synthesized protein is transferred to a well on another surface and immobilized there. Transfer using a gridding robot is envisaged. A transfer by direct superposition of two surfaces is not envisaged.
  • US-A1 20030087292 describes methods and apparatus for promoting interactions between an array of probes deposited on a microarray substrate and target molecules in a target liquid.
  • the apparatus comprises a microarray and a cover forming a reaction chamber, whereby the microarray and the cover movable relative to each other. Moving the cover and the microarray relative to each other brings the target liquid more efficiently into contact with the probes on the microarray.
  • protein chip technology is facing in particular the following problems: (i) the provision of an expression library with a sufficient number of different clones for the recombinant expression of proteins, (ii) adequate protein expression in recombinant organisms and purification to homogeneity for the purpose of immobilizing the proteins on microchips, (iii) structural differences between proteins and different optimal conditions of different proteins, noting that uniform conditions are applied to all samples during a conventional microarray assay, and (iv) limited stability of proteins, which may be further reduced by attaching proteins to the surface of a chip.
  • this invention relates to a method for producing a surface with one or more products attached thereto, comprising the steps of: (a) putting a first and a second surface on top of each other, wherein one or more starting materials or one or more enzymes are attached to distinct sites on said first surface; (b) contacting said one or more starting materials attached to said first surface with one or more enzymes, or contacting said one or more enzymes attached to said first surface with one or more starting materials, wherein said contacting is effected under conditions permitting generation of one or more products by said enzyme(s) using said starting material(s); and (c) attaching said product(s) to distinct sites on said second surface, thereby obtaining said surface with one or more products attached thereto; wherein (i) step (a) is effected prior to step (b); (ii) step (b) is effected prior to step (a); or (iii) steps (a) and (b) are carried out simultaneously.
  • the surfaces according to the invention may be any surface.
  • the surface materials may be the same for both the first and the second surface, or they may be distinct.
  • the surface may be a coating applied to a carrier, or the surface of the carrier itself may be used.
  • Carrier materials commonly used in the art and comprising glass, plastic, gold and silicon are envisaged for the purpose of the present invention.
  • Coatings according to the invention, if present, include poly-L-lysine- and amino- silane-coatings as well as epoxy- and aldehyde-activated surfaces.
  • the phrase "putting a first and a second surface on top of each other” denotes an arrangement whereby two surfaces are superimposed in such a manner that (i) the first surface with one ore more starting materials or one or more enzymes attached thereto faces the second surface where the products are to be attached to and, as a consequence, (ii) the arrangement of said distinct sites on said second surface is a mirror image of the arrangement of said distinct sites on said first surface.
  • the term "mirror image” refers to an arrangement with the same spacings between the centers of distinct sites on both surfaces. At the same time, a mirror image on the second surface of an individual distinct site on the first surface and the site on the first surface may be, but are not necessarily of the same size.
  • both surfaces are of the same size and shape, a sandwich-like arrangement is obtained upon putting the two surfaces on top of each other (see also Example enclosed herewith).
  • the second surface with one or more products attached thereto could also be referred to as a blueprint of the first surface with one or more starting materials or enzymes attached thereto, whereby it is of note that there is an intermediate enzymatic reaction converting starting materials into products.
  • the preferred distance between the two surfaces put on top of each other will depend on the nature of the surfaces (e.g. smooth or structured) and the amount of substance attached thereto. Preferred distances may be implemented by the use of spacers of defined thickness.
  • ..distinct sites denotes spatially separated regions of said surfaces. These sites may be adjacent, virtually without surface patches not carrying material in between, or the distinct sites may be separated from each other by uncharged surface patches.
  • the surfaces may both be planar. Alternatively, the surfaces may exhibit a curvature.
  • the first surface may be convex, such as a cylinder, sphere or microsphere, and the second surface may be concave.
  • the term "product" according to the invention relates to one or more molecular species formed from one or more molecules of the starting material(s) by the action of one or more enzymes.
  • Enzymes according to the invention may be any enzyme.
  • the enzymes according to the invention may fall in any of the established categories consisting of oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases.
  • one or more enzymes may be used for the method of the invention. If more than one enzyme is being used, the enzymes may fall into the same category or into different categories.
  • starting material is not exhaustive, i.e., the addition of further reactants or educts may be necessary in order to establish the conditions permitting generation of product.
  • incubation times are adjusted to the times needed for enzymatic reaction to occur or to essentially reach equilibrium and are in the range between 1ms and 1 day, preferably between 0.1 sec and 2 hours.
  • Reaction may be performed in the dark, or at daylight, or under irradiation with electromagnetic radiation.
  • Preferred wavelengths of said electromagnetic radiation are between 1 and 10000 nm, preferably between 10 and 2000 nm, and most preferred between 200 and 1000 nm.
  • the reaction temperature is between 250 and 400 K, preferably between 273 and 313 K, and most preferred in the interval between 290 and 310 K, i.e., comprising ambient temperature.
  • the preferred temperature interval may be centered around the optimal growth temperature of the source organism. Temperature is maintained by means of a thermostat. Alternatively, and for example in case the temperature is ambient temperature, fluctuations of the temperature may occur. Said fluctuations, as measured on the timescale of the reaction, do not exceed 2 degrees, preferably 0.5 degree, and most preferred 0.1 degree from the average temperature.
  • the reaction may be exposed to air, or performed under a protective atmosphere provided by inert gas, such as nitrogen or argon. In a further preferred embodiment, further reactants may be provided which are gaseous, such as NO or acetylene.
  • Conditions in step (b) preferably comprise an aqueous solution. More preferred, said aqueous solution is buffered. Buffers are well known in the art and the skilled person is aware of appropriate buffers in dependency of enzyme(s), starting material(s) and product(s).
  • ionic strength may be adjusted, e.g., by the addition of sodium chloride and/or potassium chloride.
  • concentration of sodium chloride is between 0 and 2 M, preferably between 100 and 200 mM.
  • Examples comprise PBS (phosphate buffered saline) containing 1.37 M NaCI, 27 mM KCI, 43 mM Na 2 HPO 4 -7H 2 O and 14 mM KH 2 PO 4 in the 10-fold aqueous stock solution, which is adjusted to pH 7.3; SSC containing 3 M NaCI and 0.3 M sodium citrate in 20-fold aqueous stock solution, which is adjusted to pH 7.0; and STE (Saline Tris EDTA) containing 10 mM Tris base, 10 mM NaCI and 1mM ETA (acid).
  • PBS phosphate buffered saline
  • SSC containing 3 M NaCI and 0.3 M sodium citrate in 20-fold aqueous stock solution, which is adjusted to pH 7.0
  • STE
  • sodium chloride is absent 5 from the buffer preparation.
  • Examples for common buffer preparations without sodium or potassium chloride are TAE (Tris acetate EDTA) containing 2 M Tris acetate and 0.1 M EDTA in the 50-fold aqueous stock solution at pH 8.5; TBE (Tris borate EDTA) containing 0.89 M Tris base, 0.89 M Boric acid and 0.02 M EDTA in the 10-fold aqueous stock solution at pH 8.0; and TE (Tris EDTA) containing 10 mM 10 Tris base and 1 mM EDTA (acid) at pH 7.5.
  • TAE Tris acetate EDTA
  • TBE Tris borate EDTA
  • TE Tris EDTA
  • attaching in step (c) of the method of the invention refers to any method of attaching. It may involve the formation of one or more covalent bonds between the product and the surface. Alternatively, it may involve no bond formation and be 30 effected by non-covalent interactions. Attachment may involve a specifically derivatized surface on the one side and/or a tagged product on the other side. Alternatively, sufficient attachment may be provided by unspecific interaction between product and surface. Attachment by also be effected by surface tension, i.e., by adhesion of liquid or solution comprising the product to the surface.
  • the first and the second surface according to the invention may be put on top of each other in a first step, followed by delivery of enzyme(s) or starting material(s) to the volume between the two surfaces.
  • enzyme(s) or starting material(s) may be brought into contact with the first surface with starting material(s) or enzyme(s) attached thereto prior to putting the second surface on top of this arrangement.
  • a second surface may be covered with enzyme(s) or starting material(s), either at distinct sites or throughout, and then put on top of the first surface with starting material(s) or enzyme(s) attached thereto, which amounts to performing steps (a) and (b) simultaneously.
  • said enzyme(s) or starting material(s) are attached to distinct sites forming a mirror image of the pattern of distinct sites in the first surface or part thereof. This embodiment permits multiplexing, i.e. different treatment of different enzyme(s) and/or starting material(s), thereby allowing to account for different optimal conditions for activity of different enzymes.
  • said starting material(s) serve(s) as (a) template(s).
  • template refers to a molecular species which determines the chemical nature of the product(s).
  • a template is usually not subjected to change, or subjected only to a transient change, during an enzymatic reaction.
  • a template is not converted into product, but governs the de-novo synthesis of product from educts, for example building blocks such as monomers, in that it determines the type of monomer to be used at any given position of a product to be made from building blocks.
  • the number of building blocks constituting the template and the number of building blocks constituting the product are the same or substantially the same.
  • the building blocks constituting the template on one side and the product on the other side may be of the same compound class or belong to different compound classes. It is understood that the conditions permitting the generation of one or more products according to the invention involve the addition of further reactants or educts such as the building blocks needed for the de novo synthesis of product using the template as guide.
  • said templates are attached to said first surface.
  • steps (a) to (c) are effected more than once with the same first surface with one or more starting materials or one or more enzymes attached thereto, thereby obtaining multiple surfaces with one or more products attached thereto.
  • said starting materials attached to the first surface are templates, which are read multiple times, whereby steps (c), i.e. attachment of product to the second surface, occur between subsequent readings of the templates.
  • steps (c) i.e. attachment of product to the second surface, occur between subsequent readings of the templates.
  • starting materials which for example are educts to be converted by the enzymes, may be added multiple times.
  • steps (a) to (c) are effected more than once and said surface with one or more products attached thereto obtained in a step (c) serves as a first surface with one or more starting materials or one or more enzymes attached thereto in a subsequent step (a).
  • This embodiment relates to a chain of two or more blueprint processes, wherein the one or more enzymes and/or the one or more starting materials used in a particular blueprint process may be distinct from the one ore more enzymes and/or the one or more starting materials used in a previous blueprint process.
  • the products of a previous blueprint process may be enzymes to be used in the subsequent blueprint process.
  • the products of a previous blueprint process may serve as templates for a subsequent blueprint process.
  • DNA chips can be copied in a blueprint process according to the invention to yield another DNA chip.
  • the DNA obtained thereby can be used to create an RNA chip.
  • the latter can be used to make a protein chip.
  • several such processes can be performed in a series before spots start to blur significantly.
  • the generation of one or more products in step (b) involves an amplification.
  • This embodiment relates to methods wherein the starting materials serve as templates.
  • the templates may be read multiple times, thereby obtaining more than one product molecule per template molecule during one blueprint process.
  • the amplification factor achieved is at leastiO.
  • said starting material is DNA.
  • said first surface with one or more starting materials attached thereto is a DNA chip.
  • the DNA according to the invention may be genomic DNA or cDNA.
  • derivatives of DNA such PNAs or DNA-PNA chimera.
  • DNA-PNA chimera are molecules comprising one or more DNA portions and one or more PNA portions.
  • PNA stands for "peptide nucleic acid".
  • a PNA is nucleic acid, wherein the sugar-phosphate backbone has been replaced with an amide backbone.
  • PNA oligomers are synthetic DNA-mimics with an amide backbone (Nielsen et al. (1991), Egholm et al. (1993)) that exhibit several advantageous features.
  • PNA oligomers are stable under acidic conditions and resistant to nucleases as well as proteases. Their electrostatically neutral backbone increases the binding strength to complementary DNA compared to the stability of the corresponding DNA duplex.
  • PNA oligomers can be shorter than oligonucleotides when used as hybridisation probes.
  • mismatches have a more destabilising effect, thus improving discrimination between perfect matches and mismatches.
  • PNA also permits the hybridisation of DNA samples at low salt or no-salt conditions, since no inter-strand repulsion as between two negatively charged DNA strands needs to be counteracted. As a consequence, the target DNA has fewer secondary structures under hybridisation conditions and is more accessible to the probe molecules.
  • said DNA is an aptamer.
  • Aptamers are DNA (or RNA) molecules that have been selected from random pools based on their ability to bind other molecules. Aptamers have been selected which bind nucleic acid, proteins, small organic compounds, and even entire organisms. A database of aptamers is maintained at http://aptamer.icmb.utexas.edu/.
  • said enzyme(s) comprise(s) (an) enzyme(s) catalyzing the replication of DNA.
  • said enzyme(s) is/are (a) DNA polymerase(s).
  • DNA polymerase(s) The use of any DNA polymerase is envisaged. More preferred, said DNA polymerase(s) is/are selected from the group consisting of thermophilic DNA polymerases, mesophilic DNA polymerases derived from phages like phi29, T7, T4, and E.coli polymerases.
  • said enzyme(s) comprise(s) (an) enzyme(s) transcribing DNA into RNA.
  • This embodiment relates to in vitro transcription of DNA.
  • said enzymes comprise (an) enzyme(s) transcribing DNA into RNA and (an) enzyme(s) translating RNA into protein.
  • An example of the latter is the in vitro mix known in the art which comprises enzymes and educts for both in vitro transcription and in vitro translation.
  • the latter embodiments provide a plurality of recombinant proteins or (poly)peptides, whereby an expression library is not needed. As the proteins or (poly)peptides are synthesized in vitro, problems pertinent to protein expression in recombinant organisms, such as formation of inclusion bodies, are avoided.
  • said enzyme(s) transcribing DNA into RNA is/are (a) RNA polymerase(s).
  • said RNA polymerase(s) is/are (a) viral RNA polymerase(s) such as SP6 RNA polymerase or T7 RNA polymerase.
  • RNA polymerase III is RNA polymerase III.
  • siRNA molecules are short (typically 21 to 23 nucleotides long) double-stranded RNA molecules acting as mediators of RNA silencing (Tijsterman et al. (2002)). They are capable of reducing or causing to virtually vanish the amount of their cognate transcript.
  • RNA polymerase III transcribes small, noncoding transcripts that are not capped or polyadenylated at the 5' and 3' ends, respectively.
  • RNA polymerase III initiates transcription at defined nucleotides, and terminates transcription when it encounters a stretch of four or five thymidines. Consequently, it is possible to design small RNAs synthesized by RNA polymerase III that carry 3' overhangs of one to four uridines, a structural feature resembling that defined for siRNAs to be effective in vitro (see also Shi (2003)).
  • double-stranded RNA is formed during or upon transcription, and that said enzymes comprise furthermore an endonuclease such as RNase III.
  • Means for ensuring the formation of double-stranded RNA are known in the art and reviewed in Shi (2003).
  • RNase III Diicer cleaves the double-stranded RNA to yield siRNA molecules.
  • said surface with one or more products attached thereto is a siRNA chip.
  • siRNA chips obtained by the method of the invention may be used directly after generation. Problems arising from limited stability of siRNAs attached to a surface are thereby obviated.
  • siRNA chips may be used for studying the phenotypes of genes knocked down by RNAi.
  • cells to be transfected with the siRNA molecules may be provided in arrayed form and brought into contact with a siRNA chip according to the invention, thereby obtaining a transfected cell array.
  • said starting material is RNA.
  • said enzyme(s) comprise(s) (an) reverse transcriptase(s).
  • This embodiment of the method of the invention yields cDNA, when mRNA is provided as a template.
  • said RNA is an aptamer.
  • said enzyme(s) comprise(s) (an) enzyme(s) translating RNA into protein.
  • This embodiment is directed to in vitro translation of RNA.
  • said enzyme(s) translating RNA into protein is/are comprised in extracts from E.coli, wheat germ, or rabbit reticulocytes, or are purified recombinant components of the translational apparatus.
  • said surface with one or more products attached thereto is a protein chip.
  • (poly)peptide chips are also embraced.
  • the term "(poly)peptide” as used herein describes fragments of proteins as described herein above and refers to a group of molecules which comprise the group of peptides, consisting of up to 30 amino acids, as well as the group of polypeptides, consisting of more than 30 amino acids.
  • the term ,,protein refers to any protein. It includes soluble proteins, for example enzymes such as kinases, proteases, phosphatases, oxidases and reductases as well as non-enzyme soluble proteins, for example soluble proteins comprising immunoglobulin domains such as soluble antibodies.
  • Membrane proteins comprising peripheral membrane proteins and integral membrane proteins are also included.
  • membrane proteins include receptors.
  • Receptors include growth factor receptors, G-protein coupled receptors and receptors with seven, but also with less or more than seven transmembrane helices.
  • the term "protein” refers to single-domain proteins as well as to multi-domain proteins. Further embraced are embodiments, wherein the protein in its natural environment functions as part of a metabolic pathway and/or a signal transduction pathway.
  • the protein may consist of or comprise protein domains known to function in signal transduction and/or known to be involved in protein-protein interaction.
  • Examples for such domains are Ankyrin repeats; arm, Bcl-homology, Bromo, CARD, CH, Chr, C1 , C2, DD, DED, DH, EFh, ENTH, F-box, FHA, FYVE, GEL, GYF, hect, LIM, MH2, PDZ, PB1, PH, PTB, PX, RGS, RING, SAM, SC 1 SH2, SH3, SOCS, START, TIR, TPR, TRAF, tsnare, Tubby, LJBA, VHS, W, WW, and 14-3-3 domains. Further information about these and other protein domains is available from the databases InterPro (http://www.ebi.ac.uk/interpro/, Mulder et al., 2003), Pfam
  • DNA chips can be stored effectively over long time periods, which is in general not possible for protein chips without loss of activity or function.
  • the protein or (poly)peptide chips obtainable by the method of the invention may be used directly after generation. Problems arising from limited stability of proteins or (poly)peptides attached to a surface are thereby obviated.
  • the generated protein comprises an affinity tag.
  • the affinity tag allows specific and affine attachment. Attachment via a tag may involve covalent and/or non-covalent interactions. Alternatively, tags are absent from the proteins and attachment is unspecific.
  • said affinity tag is selected from the group consisting of a His tag, a streptavidin binding tag, a cellulose binding domain, glutathione-S-transferase, cutinase or a tag comprising the sequence LPXTG, wherein X is any one of the naturally occurring L-amino acids.
  • said attaching in step (c) is preferably effected by Ni-NTA, streptavidin, cellulose, glutathione, phosphonate or oligo-gycine.
  • Ni-NTA designates nickel which is chelated by nitrilotriacetic acid (NTA), which is similar to EDTA.
  • LPXTG is a sequence recognised by sortase.
  • Sortase is an enzyme capable of catalyzing a transpeptidation reaction and has been described in the patent application W0-A2 0062804.
  • the N-terminal portion of the protein or (poly)peptide, ending with residue X 2 of the recognition site, is transferred from the enzyme to a second protein or (poly)peptide, and a peptide bond is formed.
  • the second protein or (poly)peptide envisaged by the inventors of WO-A2 0062804 is a pentaglycine peptide with a free N-terminus and part of a peptidoglycan occurring in the cell wall of a Gram-positive bacterium.
  • the recognition motif LPXiX 2 G is sufficiently rare such that unwanted cleavage hardly occurs. Therefore, tagging of the proteins or (poly)peptides obtained by the enzymatic reaction with said motif is envisaged.
  • transglutaminase can be used for linking proteins or (poly)peptides containing the amino acid glutamine to a surface carrying primary amino groups.
  • said protein chip is an antibody chip or antibody array.
  • Said antibody which is monoclonal antibody, polyclonal antibody, single chain antibody, or fragment thereof that specifically binds said peptide or polypeptide also including bispecific antibody, synthetic antibody, antibody fragment, such as Fab, a F(ab 2 )', Fv or scFv fragments etc., or a chemically modified derivative of any of these (all comprised by the term "antibody”).
  • Monoclonal antibodies can be prepared, for example, by the techniques as originally described in K ⁇ hler and Milstein, Nature 256 (1975), 495, and Galfre, Meth. Enzymol. 73 (1981), 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals with modifications developed by the art.
  • antibodies or fragments thereof to the aforementioned peptides can be obtained by using methods which are described, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988.
  • surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to an epitope of the peptide or polypeptide of the invention (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).
  • the production of chimeric antibodies is described, for example, in WO89/09622.
  • a further source of antibodies to be utilized in accordance with the present invention are so-called xenogenic antibodies.
  • the general principle for the production of xenogenic antibodies such as human antibodies in mice is described in, e.g., WO 91/10741 , WO 94/02602, WO 96/34096 and WO 96/33735.
  • Antibodies to be employed in accordance with the invention or their corresponding immunoglobulin chain(s) can be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art; see, e.g., Sambrook (1989), loc. cit..
  • polyclonal antibody also relates to derivatives of said antibodies which retain or essentially retain their binding specificity. Whereas particularly preferred embodiments of said derivatives are specified further herein below, other preferred derivatives of such antibodies are chimeric antibodies comprising, for example, a mouse or rat variable region and a human constant region.
  • scFv fragment single-chain Fv fragment
  • the antibody, fragment or derivative thereof according to the invention specifically binds the target protein, (poly)peptide or fragment or epitope thereof whose presence or absence is to be monitored.
  • the term "specifically binds" in connection with the antibody used in accordance with the present invention means that the antibody etc. does not or essentially does not cross-react with (polypeptides of similar structures. Cross-reactivity of a panel of antibodies etc. under investigation may be tested, for example, by assessing binding of said panel of antibodies etc. under conventional conditions (see, e.g., Harlow and Lane, (1988), loc. cit.) to the (poly)peptide of interest as well as to a number of more or less (structurally and/or functionally) closely related (poly)peptides.
  • said antibody or antibody binding portion is or is derived from a human antibody or a humanized antibody.
  • humanized antibody means, in accordance with the present invention, an antibody of non-human origin, where at least one complementarity determining region (CDR) in the variable regions such as the CDR3 and preferably all 6 CDRs have been replaced by CDRs of an antibody of human origin having a desired specificity.
  • CDR complementarity determining region
  • the non-human constant region(s) of the antibody has/have been replaced by (a) constant region(s) of a human antibody.
  • said starting material(s) is/are (a) substrate(s) of said enzyme(s) and said enzyme(s) convert(s) said substrate(s) into said product(s).
  • enzymes like horseradish peroxidase may convert starting materials like thyramide into reactive species which may be conjugated to a second surface, for example a second surface with tyrosine groups attached thereto (thyramide signal amplification).
  • the method of the invention further comprises prior to steps (a) and (b): (a 1 ) attaching to a first surface one or more starting materials or one or more enzymes, thereby obtaining said first surface with one or more starting materials or one or more enzymes attached thereto.
  • said attaching comprises transfer of said starting material(s) or said enzyme(s) to said first surface using an arrayer, wherein distinct pins of the arrayer transfer distinct solutions of said starting material(s) or of said enzyme(s).
  • array designates an apparatus comprising an array of pins attached to a robot capable of taking up a plurality of different solutions with different pins and delivering them to a surface, thereby generating an arrangement of distinct sites on the surface with different solutions being delivered to different sites.
  • the pins may be arranged in a way such that solutions from the wells of a microtiter plate may be taken up.
  • An arrayer may be operated manually or computer-controlled or by a combination of both.
  • said starting material(s) or enzyme(s) are provided in a solution which is diluted such that molecules of said starting material(s) or enzyme molecules are separately attached to said first surface. It is envisaged that said first surface as a whole or significant parts thereof are suitable for the attachment of starting material(s) or enzyme(s), and that starting material(s) or enzyme(s) are delivered to the entire surface or significant parts thereof.
  • the solution of the starting material(s) or enzyme(s) is diluted such the probability of attachment of distinct molecular species at a distance below a certain threshold is sufficiently low.
  • the distance threshold depends on the fidelity of the blueprint process, in particular with regard to the size and spacing of sites with distinct molecular species attached thereto.
  • the distance threshold furthermore depends on the envisaged use of the surface with one or more products attached thereto, for example, on the chosen method for detecting products attached to said surface. It is understood that the arrangement of attached starting material(s) or enzyme(s) obtained thereby may not be regular, but random.
  • said first surface comprises an array of locations suitable for attaching a molecule of said starting material or an enzyme molecule, separated by areas not suitable for attaching.
  • an arrayed arrangement of starting material or enzyme is achieved, whereby no arrayer is used for that purpose.
  • the latter two embodiments comprise a method of making a biochip, i.e., of a first surface with starting material(s) or enzyme(s) attached thereto, without the need for a library of, for example, clones, whereby different clones are stored in different containers. It is understood that the blueprint method according to the invention may be used to amplify the single molecules of template attached to distinct, but randomly located sites of the first surface.
  • Said template molecules may be DNA molecules, which may subsequently be amplified using a DNA polymerase. This process can be considered as being analogous to, but more efficient than the plating and subsequent growth of colonies of a library on agar.
  • RNA may be generated and/or a protein chip be obtained therefrom.
  • an identification of the DNA either directly or via the transcription or translation product may be effected by methods known in the art.
  • both surfaces are smooth.
  • smooth denotes a surface which is unstructured, i.e., compartments such as wells or the like are absent from said surface.
  • the distance between the two surfaces is preferably below 100 ⁇ m in order to avoid convection phenomena. At distances below 100 ⁇ m dilution occurs mostly owing to diffusion only. The experiments shown in the Examples enclosed herewith have been performed with a distance less than 30 ⁇ m between the two surfaces.
  • said distinct sites are droplets.
  • the droplets provide compartmentalization of the reaction sites and attachment to the smooth surface.
  • the cohesion of the droplet provides compartmentalization, whereas the adhesion to the surface provides attachment. Adhesion on a normal glass or plastic surface is sufficient to keep the droplets in place and to prevent flowing together and/or spill-over.
  • the droplets are droplets of a liquid or solution comprising the starting material(s) or the enzyme(s).
  • said attaching may be achieved by the spotting of said droplets.
  • the term "spotting" designates the localized delivery of small amounts of liquid to a surface.
  • Devices for spotting including the simultaneous spotting of multiple samples, wherein said multiple samples, for example, have been picked up from one or more microtiter plates, are well known in the art. Delivery of material by spotting may involve a direct contact between the spotting pin and the surface. Alternatively, and similar to inkjet printers, such contact is not required. Preferably, spotting is done with a spotting robot comprising the spotting device and a computer controlling the function of the spotting device and providing a user interface. Distances between droplets are for example about 20 ⁇ m when working with contact printing arrayers. It is understood that the use of smooth surfaces and droplets requires a precise adjustment of the distance between the surfaces when the surfaces are put on top of each other.
  • a distance too large would not permit the transfer of product to the second surface, whereas a distance to small would render the droplet confluent.
  • Appropriate distances depend on the volume of the droplets and the surface properties, for example the surface tension of the droplets on said surface.
  • the method of the invention using droplets comprises the following further step after step (a 1 ) and prior to steps (a) and (b): (a") spotting of further droplets between droplets spotted in step (a'), such that droplets spotted in step (a') which are in the neighbourhood of said further droplets flow together.
  • step (b) may be performed in a way that step (a") is effected concomitantly, i.e., the step of contacting involves the spotting of starting material(s) or enzyme(s) in such a manner that flowing together of droplets of enzyme(s) or starting material(s), respectively, is achieved.
  • said flowing together may be confined to a subset of said droplets of starting material(s) or enzyme(s).
  • microfluidics structures are created on the surface without necessitating arduous surface modifications such as etching commonly performed in the manufacture of microfluidics devices.
  • gradients may be established by making a row or column of droplets confluent, whereby the first droplet contains a solution of a compound, which is absent from the remainder of the droplets in that row or column.
  • At least one of said surfaces has compartments.
  • said compartments are wells, for example, the wells of a microtiter plate.
  • said surface has hydrophobic and hydrophilic regions.
  • a grid is placed between the surfaces put on top of each other. It is envisaged that said grid provides compartmentalization during the transfer of product from the first to the second surface.
  • This embodiment of the method of the invention may be used when an arrayed arrangement of transferred product(s) is to be generated from a random arrangement of starting material(s) or enzyme(s).
  • the grid is finer than the expected dot size. This would result in a pixel-like resolution of the surface with positives situated in a neighbourhood.
  • the spacing of said grid matches the spacing of locations on said first surface with said starting material(s) or enzyme(s) attached thereto.
  • This embodiment relates a method wherein the arrayed arrangement on the first surface is to be maintained during a transfer of product(s) to the second surface under conditions where flowing together of the distinct sites might occur.
  • different locations on said first surface with said one or more starting materials attached thereto are provided with different enzymes, or wherein different locations on said first surface with said one or more enzymes attached thereto are provided with different starting materials.
  • This embodiment relates to a multiplex version of the method of the invention.
  • the term "multiplex" indicates that a plurality of sites on said surface can be addressed and handled individually.
  • the present invention also embraces a surface generated by the method of any of the preceding claims.
  • the Figures show:
  • Figure 1 "Donor" chip carrying template DNA of GFP.
  • the template DNA is immobilized to the donor chip and the expressed protein is detected both on the donor chip and the "acceptor” chip (shown in Figure 2) using GFP-specific polyclonal antibodies.
  • Figure 2 "Acceptor" chip obtained by a blueprint process according to the invention involving protein expression.
  • the acceptor chip is coated with Ni-NTA.
  • Bound GFP protein is detected using GFP-specific polyclonal antibodies.
  • control plasmid supplied by the cell-free transcription and translation mix was spotted in four columns per concentration on onto the amine glass slides at concentrations ranging from 100 ⁇ g/ml to 500 ⁇ g/ml using a QArray Spotting robot (Genetix, New Milton, UK). Additionally a negative control without DNA was performed.
  • the cell-free transcription and translation mix was prepared as recommended by the manufacturer.
  • a nickel chelate slide was placed in a humidified chamber and 100 ⁇ l of the diluted cell-free transcription and translation mix was dispensed onto the surface, avoiding the formation of bubbles.
  • the slide carrying the DNA was placed DNA-side down onto the nickel chelate slide and the chamber was closed and incubated at 3O 0 C for 4 hours. After expression of the proteins, the slide sandwich was opened in TBS containing 0.1 % Tween-20 (TBS-T) and both slides were rinsed with TBS and blocked in 3% (w/v) fat-free milk powder dissolved in TBST for 30 min.
  • TBS-T 0.1 % Tween-20
  • the slides were incubated in 0.3 ⁇ g/ml polyclonal anti-GFP antibodies derived from rabbit in blocking solution for 30 minutes at 4 0 C. After rinsing both slides with TBS, the slides were incubated in 0.1 mg/ml Cy3-labelled polyclonal anti-rabbit antibodies dissolved in blocking solution for 30 min at 4°C. The slides were rinsed with TBS and washed twice in TBS-T for 15 minutes each. Finally, the slides are rinsed again with TBS, dried by pressurized air and scanned.
  • Figures 1 and 2 clearly show a DNA-dependent expression of GFP. Although no decrease of signal intensity can be observed with decreasing DNA concentration, a mirror image of the spotting pattern can be seen on the acceptor chip, thereby providing a proof-of-principle of the method of the invention. Deficiencies caused by the expression and diffusion and visible in the upper part of the scans are likely to be solved, e.g. a reduction of the incubation time and/or by covalent immobilization of the DNA template.
  • PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules. Nature, 365, 566-568.

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Abstract

La présente invention a trait à un procédé pour la production d'une surface avec un ou des produits fixés à celle-ci, comprenant les étapes suivantes: (a) la superposition d'une première surface et d'une deuxième surface, où une ou des matières de départ ou une ou des enzymes sont fixées à des sites distincts sur ladite première surface; (b) la mise en contact desdites une ou des matières de départ fixées à ladite première surface avec une ou des enzymes, ou la mise en contact de ladite une ou desdites enzymes fixées à ladite première surface avec une ou des matières de départ, ladite mise en contact étant réalisée dans des conditions permettant la génération d'un ou de plusieurs produits par ladite/lesdites enzyme(s) au moyen de ladite/desdites matière(s) de départ; et (c) la fixation dudit/desdits produit(s) à des sites distincts sur ladite deuxième surface, permettant ainsi l'obtention de ladite surface avec un ou des produit(s) fixé(s) à celle-ci; selon lequel (i) les étapes (a) et (b) sont réalisées simultanément. Dans un mode de réalisation préféré, ladite surface avec un ou des produit(s) fixé(s) à celle-ci est une puce ADN. Dans un autre mode de réalisation préféré, ladite surface avec un ou des produit(s) fixé(s) est une puce à protéines.
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