DE10116428A1 - Preparing libraries of selected molecules, particularly gene chips, useful for DNA sequencing, by interaction of the library with loading chip then transfer to carrier - Google Patents

Abstract

Method for preparing selected molecule libraries (55), especially gene or bio-chips, is new. Method for preparing selected molecule libraries (55), especially gene or bio-chips, comprises first preparing (i) a loading chip (10) with a defined oligonucleotide (ON) library (15) and (ii) a molecule library (25) in which at least one molecule includes an oligonucleotide segment complementary to at least one region of an ON in (15), to allow stable interaction between the molecule and ON (especially formation of a stable hybrid). Then (25) is applied to (15) so that interaction between the components of the two libraries occurs, forming a hybrid library (35). (35) is positioned next to a target (50), interaction between ON and molecules of (25) is undone and molecules that were previously bound to ON are transferred to (50) in a spatially ordered fashion, and immobilized as a (55). An Independent claim is also included for an apparatus for preparing (55), especially gene chips.

Description

The invention relates to a method and a device for producing high-density, spatially resolved, solid phase-immobilized selected molecule libraries or Molecular grid, in particular the invention relates to a method for producing Gene or bio chips.

It is known to use gene chips for determining sequence fractions of a test DNA or Use RNA at the DNA level. Usually as a double strand present DNA is here as a single strand in short pieces (oligonucleotide probe that 25 nucleotides long) is attached to the micro-fields of a chip. The determination of Sequence portions are carried out using the hybridization technique. Both the test DNA or RNA (for example of the tissue) as well as the chip DNA / RNA are under the selected conditions single-stranded. The test DNA or RNA is then included coupled fluorescence marker placed on the chip. Among the chosen Hybridization conditions bind only the complementary DNA single strands together. The checkerboard-like fields of the chip are then marked with an optical one Scanner checked. The scanner makes the result visible by looking for the different fields the degree of hybridization - d. H. the agreement of the test DNA or RNA with the RNA / DNA strands of the gene chip - in the form of Reproduces light intensity.

When it comes to the production of gene chips or micro-arrays, the important thing is that special single strands in short pieces at precisely defined positions on a hurry Pin the straps. For this purpose it is known after the "light-directed" synthesis proceed, cf. Fodor, S.A. et al., 1991 "Light-directed, spatially addressable parallel chemical synthesis ", Science 251: 767-773. This technique uses precise photolithographic masks to define the positions at which individual, specific nucleotides added to growing single-stranded nucleic acid chains become. Individual positions are activated or released by light, so that now couple a specific nucleotide to this precisely defined location, d. H. can "pin". This nucleotide is then placed on the support and adheres everywhere where a position had previously been activated by the light beam. After that further positions on this support are activated and now another nucleotide applied to the carrier that adheres to the now activated locations. This process  is continued until all positions with the oligonucleotide probe of choice are occupied.

This manufacturing process is very lengthy and requires many individual ones Manufacturing steps. Several photolithographic masks have to be made be placed one after the other very precisely positioned on the carrier have to. There is always the risk that the positions of the Slightly move photolithographic masks during the light activation phase and so that the entire chip in production is unusable.

In addition to the long time it takes to make a chip, the process is too very inflexible. To build a special architecture on a chip, you have to First, several photolithographic masks are made. These masks define the desired positions of the individual nucleotides of a special chip. If you want to build a chip with a different architecture - for example because if you want to additionally test a new virus on this chip - then you have to complete new set of photolithographic masks can be made. Moreover the method is not suitable, for. B. protein chips or mixed biomolecule chips manufacture.

The object of the present invention is therefore a production method and a Providing device for the production of gene or bio chips with the gene or Bio-Chips manufactured faster and with greater flexibility in architecture can be.

This task is independent of the method or the device Claims resolved. Special developments are in the dependent claims Are defined.

In particular, the object is achieved by a method for producing selected molecular libraries ( 55 ), in particular gene chips, comprising the steps:

• a) production of a loading chip ( 10 ) with a defined oligonucleotide library ( 15 );
• b) Provision of a molecule library ( 25 ), in which at least one molecule (25.i) of the molecule library ( 25 ) has an oligonucleotide section (26.i) which extends to at least one region (16.i) of an oligonucleotide (15.i) the oligonucleotide library ( 15 ) contained on the loading chip ( 10 ) is complementary to the formation of stable interactions between the molecule (25.i) of the molecule library ( 25 ) and the oligonucleotide (15.i) of the oligonucleotide library ( 15 ), in particular that Enable formation of a stable hybrid (35.i);
• c) the molecule library ( 25 ) with the oligonucleotide library ( 15 ) contained on the loading chip ( 10 ) to form stable interactions between oligonucleotides (15.i) of the oligonucleotide library ( 15 ) and molecules (25.i) of the molecule library ( 25 ) in Brought interaction, resulting in a hybrid library ( 35 );
• d) the hybrid library ( 35 ) is positioned adjacent to a target ( 50 );
• e) the hybrid library ( 35 ) is dissolved by solving the stable interaction between the molecules (25.i) of the molecule library ( 25 ) and the oligonucleotides (15.i) of the oligonucleotide library ( 15 ) and those previously bound to the oligonucleotide library ( 15 ) Molecules (25.i) of the molecular library ( 25 ) transferred spatially ordered to the target ( 50 ) and immobilized there permanently or temporarily as a selected molecular library ( 55 ).

The selected molecule library represents a molecular grid that is unique is spatially resolved. The individual positions of these molecular grids are thus in space or in the x-y direction, particularly preferably in the x-y-z direction, defined and known, so that later the location of the molecule positioned there can be clearly identified can. These molecular libraries are preferably arranged densely around the Make the information content on the gene or bio chip as high as possible. More than 900 different ones are particularly preferred Oligonucleotide probes arranged per square centimeter. Those are also preferred solid-phase immobilized selected molecule libraries, d. H. on a solid support or a target stored spatially fixed.

The loading chip ( 10 ) is equipped with a defined oligonucleotide library ( 15 ) and can be produced by conventional methods. This loading chip serves as a "master chip" for the following steps and represents a kind of negative form for the selected molecule library ( 55 ) to be produced. From this negative form, a special chip can then be obtained as the selected molecule library by selecting the molecular library ( 25 ). In the special case that only DNA or RNA sequences of the same length are involved, the loading chip ( 10 ) is the direct complement of the selected molecular library ( 55 ) to be produced.

The molecule library ( 25 ) is preferably a synthetically produced molecule library which has the molecules which are later to be found in the selected molecule library ( 55 ), ie the gene chip to be produced. These individual molecules of this molecular library have anchor segments or oligonucleotide sections which enable them to be able to couple to special oligonucleotides of the oligonucleotide library provided on the loading chip. These segments are designed to be complementary to the segments to which they are to be coupled. Through the choice of the different anchor segments, special molecules of this molecular library are coupled to specially defined locations on the loading chip and thus sorted spatially.

By choosing both the oligonucleotide library on the loading chip as well their spatial alignment and, moreover, with a special molecular library selected anchor segments can have a direct influence on the Architecture of the gene chips to be produced. First, about Choice of anchor segments of the oligonucleotide library can be determined, what on Can dock the loading chip at all. Then about the spatial division this oligonucleotide library on the loading chip specified at which locations can connect anything at all. Now the composition of the Molecular library predefined which molecules will end up on the gene chip at all can be positioned and the choice of anchor segments of each Molecules in the molecular library can be determined to which others Oligonucleotides on the loading chip and thus at what location on the end gene chip to be manufactured these molecules are positioned. The architecture of the gene Chips can therefore be flexible by choosing the loading chip with the one chosen Oligonucleotide library in its spatial orientation and the selection of one of several molecular libraries can be designed with corresponding anchor segments.

The coupling of the molecules from the molecule library to the oligonucleotides on the loading chip is preferably achieved, in particular by the formation of stable interactions between this molecule (25.i) of the molecule library ( 25 ) and the oligonucleotide (15.i) of the oligonucleotide library ( 15 ) the formation of a stable hybrid (35.i).

This coupling is initiated in that the molecules of the molecular library ( 25 ) are brought into interaction with the oligonucleotide library ( 15 ) provided on the loading chip, thereby creating a hybrid library ( 35 ). Since this precisely couples the molecules to the desired positions on the loading chip that have the appropriate anchor segments, the molecules present in the molecule library can be spatially precisely defined and ordered and are now available as a hybrid. This hybrid consists of the oligonucleotide that adheres to the loading chip and the molecule from the molecule library bound to it via the anchor segment. Since only exact matches are taken into account, an exact selection of the molecules in a spatially defined orientation is possible.

The hybrid library ( 35 ) that is precisely arranged and sorted in this way is now positioned adjacent to a target ( 50 ). This target is preferably a secondary solid phase, such as nylon, polyamide, nitrocellulose, cellulose, modified celluloses, glass, polyimide, polyacrylamide, glass, silicon, silicone, precious metals or combinations of these materials. The target can particularly preferably also be a capillary array, into which the separated molecules can be placed in an orderly manner and thus be spatially defined.

The target preferably consists of a checkerboard arrangement of alternating electromagnetic fields, in which the molecules alone due to their electromagnetic properties are recorded.

Thereupon, the molecules (25.i) of the molecule library ( 25 ) contained in the hybrid library and previously bound to the oligonucleotide library ( 15 ) are transferred spatially ordered to the target ( 50 ) and there z. B. by forming covalent bonds, salt-like bonds or hydrophobic interactions or by focusing in alternating electromagnetic fields as a selected molecular library ( 55 ) immobilized. The release of the stable interaction between the molecules (25.i) of the molecule library ( 25 ) and the oligonucleotides (15.i) of the oligonucleotide library ( 15 ) can take place in various ways, which depend on the nature of the interactions.

The immobilization causes the molecules transferred to the target there remain positioned and their position cannot be expected due to future influences Giving up more, but sufficiently accessible for biochemical or biophysical Reactions or investigations remain. The biochemical remain Properties of the molecules largely preserved.

Another method according to the invention additionally comprises the step:

• a) the loading chip ( 10 ) is regenerated and used again in step (c).

The regeneration can e.g. B. by strongly denaturing agents such as urea or Formamide solutions or by heat.  

In this way it is possible to return the loading chip over and over again To use making a gene chip. Through this repeated use of the Loading chips becomes the time to manufacture a new gene chip dramatically reduced. During the production of a loading chip without modified nucleotides takes about 10 to 12 hours according to conventional methods (5-8 minutes per nucleotide x 4 per nucleotide, then chain extension: with 25 nucleotides approx. 10-12 hours), can the gene chips according to the inventive method with an existing Loading chip within a few minutes - or depending on the acceleration of the Hybridization in less than a minute.

A further method according to the invention additionally comprises the step between step (c) and step (e): d) Excess and / or incomplete but nevertheless non-selectively or non-specifically coupled molecules (25.i) of the molecular library ( 25 ) are removed from the hybrid library ( 35 ) Cut. This makes it possible to remove excess molecules from the loading chip, which could otherwise also undesirably get onto the gene chip to be produced. In addition, incorrect couplings to anchor segments or positions not provided for this purpose can also be corrected.

The conditions for the removal of the individual excess or incorrectly positioned molecules can be chosen with a defined, predetermined stringency, so that only stable interacting molecules remain at the respective positions of the loading chip ( 10 ) or the oligonucleotide library ( 15 ), e.g. , B. by varying the temperature, salinity and / or electrical potential.

Another method according to the invention additionally comprises the step:

• a) Excess molecules (25.i) of the molecule library ( 25 ) are used again after their removal in step (d) in step (c) which has been carried out again. This makes it possible to use the molecules of the molecule library which were not required for the hybrid formation on the loading chip in the first cycle in a next cycle. This saves resources.

For the preparation of the oligonucleotide library ( 15 ) of the loading chip ( 10 ), oligonucleotide probes ( 17 ) with a length of 3 to 100 nucleotides, preferably with a length of 5 to 50 nucleotides, particularly preferably with a length of 15 to 25 nucleotides, are used, which are particularly preferably by a covalent one chemical bond are coupled to the loading chip.

The stability of the bonds increases with the length of the nucleotides. On the other hand, nucleotides can be produced less and less with increasing length, so that the synthesis unit decreases with longer nucleotides. The selectivity of the individual oligonucleotides, i.e. the correct coupling to a correct counterpart, is low in the case of particularly short nucleotides (length <5) and in the case of particularly long nucleotides (length <45), while in the area in between is a maximum of 15 to 30 nucleotides Selectivity is present. The particularly preferred range thus represents a range in which the bond is sufficiently stable, can be produced sufficiently selectively and is still pure enough.

In a further method according to the invention, more than 900 different oligonucleotide probes ( 17 ) are used per square centimeter. This makes it possible to display high-density structures on the gene chip.

In a further preferred method, modified oligonucleotide probes ( 17 ') (= ONS) and particularly preferably chimeric modified oligonucleotide probes ( 17 ") are used to produce the oligonucleotide library ( 15 ) of the loading chip ( 10 ) and / or the molecular library ( 25 ) allow a more specific interaction with the molecular library ( 25 ) without interacting with the biologically relevant parts of the molecular library ( 25 ).

In a preferred method, oligonucleotide analogs of the following classes are used as chimeric oligonucleotide probes ( 17 '/ 17 "): PNA (Peptide Nucleic Acids), LNA (Locked Nucleic Acids), 2'-5' linked 3'-deoxyribonucleotides, DNA / RNA , DNA / PNA, DNA / LNA, DNA / 20Mc-RNA, p-RNA (pyranosyl-DNA / RNA) or a mixture of molecules of these molecular classes The oligonucleotide library ( 15 ) is preferably based on the so-called "codon-triplet synthon" Processes have been made to reduce the number of syntheses required.

The molecule library ( 25 ) can preferably be produced by combinatorial chemistry and / or by the "split and pool" method.

The molecule library ( 25 ) is preferably a chimeric molecule library ( 25 ) which also contains molecules (25.i) which have an anchor segment (26.i) for interaction with oligonucleotides (15.i) of the oligonucleotide library ( 15 ) of the loading chip ( 10 ) and / or which have a link molecule (26.i) for interaction with a catch molecule (54.i) of the target ( 50 ). By providing an anchor segment, it is possible for the molecule to adhere to the oligonucleotides on the loading chip, while the link molecule ensures the connection to the target via a catch molecule present there. It is also conceivable that the target is such that no catch molecule is required, since the material of the target offers a sufficient binding possibility for the molecules of the molecule library, such as nylon for oligonucleotides or by setting appropriate physical properties so that the molecules can be held permanently or temporarily. Temporary anchoring offers the advantage that the target can be reused several times.

The anchor segment (26.i) is preferably a molecule which interacts strongly with the representatives of the oligonucleotide library ( 15 ) and this molecule can be assigned to the following substance classes: peptides, proteins, "aptamer oligonuleotides", "molecular beacon" oligonucleotides, single-stranded DNA, double-stranded DNA , Single-stranded RNA, self-complementary DNA / RNA chimera or a mixture of molecules of these molecular classes. Also particularly preferred is cDNA and tagged cDNA. This enables a clear assignment of the molecules from the molecule library to the oligonucleotides on the loading chip. The chimeric molecular libraries ( 25 ) are preferably produced by biochemical or molecular biological methods. Preferably a can be used on so-called "magnetic beads" coupled molecule library (25) (25) as a molecule library. This enables the interaction to be released by magnetic or electrical effects. "Magnetic beads" can be placed on microstructured surfaces in micro "holes" and thus support the selection on the target. In such a molecular library, representatives of exactly one molecule (25.i) are preferably coupled to exactly one "magnetic bead" (27.i).

The interactions in step (c) preferably correspond to Watson-Crick double helices, in particular antiparallel double helices. In step (c), it is particularly preferred to form antiparallel or parallel double helices between a 2'-5'-oligonucleotide (15.i) of the loading chip ( 10 ) and an RNA anchor segment (26.i) of the molecular library ( 25 ). In order to increase the selectivity, parallel duplexes or triplexes are preferably formed as interactions. To increase the stability, antiparallel duplexes or triplexes are preferably formed.

In a preferred process step (c) and / or (d) are process used to accelerate the formation of the interaction and / or the serve increased discrimination of different interactions. Training the Interactions can be achieved through the use of initiator sites, e.g. lipophilic  Anchor, be accelerated. Matches are found faster and two matching molecules can be in this fast connecting part an optimal starting position for the following hybridization.

Above all, the increased discrimination of different interactions serves Activities where the temperature during the formation of the interactions is changed in a controlled manner; and / or an electrical field is created and checked is changed; and / or a magnetic field when using "magnetic beads" is created and changed in a controlled manner. These activities serve the increased Discrimination between the quality of the individual bonds. This allows Bonds that are not fully or between not fully complementary Oligonucleotides were formed to be resolved.

The target ( 50 ) particularly preferably comprises nylon, polyamide, nitrocellulose, cellulose, modified celluloses, glass, polyimide, polyacrylamide, quartz, silicon, silicone, noble metals or combinations of these materials. As a result, a carrier is provided as a target on which the selected molecular library can be stored precisely and reproducibly in a spatially clearly defined manner. The order of the selected molecular library can also be three-dimensional and thus represent the target ( 50 ) complex spatial structures and contain further integrated functionalities. These integrated functionalities can be, for example, those with which an evaluation or analysis of the test sequences coupled to this point or to this molecule is already possible. For this purpose, microelectrodes are preferably used which, after the sample has been added, already measure the result on the target, for example using the quartz-crystal microbalance method.

The target ( 50 ) is preferably set up in a number of sub-steps using a number of sub-libraries ( 25 ). Either the same loading chip can be brought into interaction with different molecular libraries ( 25 , 25 ', 25 ",...) One after the other and these can be loaded onto the target one after the other, or several loading chips ( 10 , 10 ', 10 ",. .) are used to successively load the target from different or the same molecule library in order to generate a gene chip or the selected molecule library ( 55 ). Complex molecular libraries ( 25 ) are particularly preferably transferred to the target ( 50 ) in several substeps.

These complex molecular libraries are e.g. B. mixtures of sub-libraries, where z. B. each sub-library is optimized for a complex disease. It can  also be mixtures of human and pathogenic sub-libraries. It is also conceivable that they are mixtures of nucleotides, peptide epitopes, antibodies, Proteins and small molecules (systems biology).

In a further method according to the invention, the transfer from step (f) to the target ( 50 ) takes place in several steps due to the spatial reduction.

These individual steps could be multiple sub-libraries in multiple steps or one Subtraction by pre-assigning a partial area of the loading chips to the then no molecule can couple to another sub-library.

In a further preferred method according to the invention, an interphase ( 40 ) is used in step (e) to establish physical contact between the loading chip ( 10 ) and the target ( 50 ). Such an interphase simplifies the transport of the molecules onto the target. Water, solutions, etc. are conceivable as interphase. A buffered aqueous solution and / or a highly viscous solution and / or a gel are particularly preferably used as interphase ( 40 ). This interphase also serves to minimize diffusions during transfer, to protect the molecules from environmental influences and to avoid direct mechanical contact between the loading chip and the target. The interphase can preferably also remain on the finished bio or gene chip in this way.

In a preferred method, a spacer ( 45 ), ie a further physical layer, is introduced between the loading chip ( 10 ) and the target ( 50 ), the spacer ( 45 ) allowing a spatially high-resolution transfer. The spacer ( 45 ) is particularly preferably a microchannel system, for example a capillary array, or a capillary chip produced by a micromechanical method.

The spacer ( 45 ) preferably allows focusing or spatial reduction of the transfer process in step (f). As a result, even denser structures can be realized on the gene chip to be produced.

In a preferred method, in step (f) to dissolve the primary Interaction thermal and / or electrical and / or magnetic energy or Radiation used. In this way it is possible to detach the molecules of the Molecular library from the hybrids to control even more precisely and even optimized in time perform.  

In a further advantageous method of the present invention, stable interactions and / or covalent chemical bonds are established in step (f) between the target ( 50 ) and the transferring molecules (25.i) of the molecular library ( 25 ).

In a preferred method of the present invention, after Binding of the transferring molecules (25.i) that are no longer required Oligonucleotide sections (26.i) cleaved hydrolytically and / or enzymatically. On in this way it is possible to obtain the selected molecular library obtained, i. H. the genetic Chip to clean from the sections that are mainly connected to the Have served loading chip. As a result, potential defects in the Avoided using the gene chip.

The object is further achieved by a device for producing selected molecular libraries ( 55 ), in particular gene chips, comprising:

• a) a feed device for providing a loading chip ( 10 ) with a defined oligonucleotide library ( 15 );
• b) a storage device for providing a molecular library ( 25 ), in which at least one molecule (25.i) of the molecular library ( 25 ) has an oligonucleotide section (26.i) which connects to at least one area (16.i) of an oligonucleotide (15 .i) the oligonucleotide library ( 15 ) contained on the loading chip ( 10 ) is complementary in order to form stable interactions between the molecule (25.i) of the molecule library ( 25 ) and the oligonucleotide (15.i) of the oligonucleotide library ( 15 ) , in particular to enable the formation of a stable hybrid (35.i);
• c) an interaction device with which the molecule library ( 25 ) with the oligonucleotide library ( 15 ) contained on the loading chip ( 10 ) for the formation of stable interactions between oligonucleotides (15.i) of the oligonucleotide library ( 15 ) and molecules (25.i) of the molecule library ( 25 ) is brought into interaction, resulting in a hybrid library ( 35 );
• d) a positioning device with which the hybrid library ( 35 ) is positioned adjacent to a target ( 50 );
• e) a transfer device by means of which the hybrid library ( 35 ) is dissolved by releasing the stable interaction between the molecules (25.i) of the molecule library ( 25 ) and the oligonucleotides (15.i) of the oligonucleotide library ( 15 ) and which has previously been sent to the oligonucleotide library ( 15 ) bound molecules (25.i) of the molecular library ( 25 ) are spatially arranged on the target ( 50 ) and immobilized there as the selected molecular library ( 55 ).

This device makes it possible to carry out the method according to the invention in one Machine. The feed device is preferably a carrier for the Loading chip. This is particularly preferably displaceable in the x-y and z directions. In this way, the loading chip is held and for the further steps of the method placed in the appropriate positions.

The reserve device serves to mix and provide Molecule libraries. This retention device is designed so that different prefabricated basic components of the molecular libraries or on whole Molecular part libraries can be accessed and provided directly or can be mixed. This can preferably be microtiter plates or Act nanotiter plates, preferably it is a microtiter, or at "magnetic beads" around a magnetizable pin.

The interaction device serves the molecular library, which is created by the Provisioning device was provided in connection with the loading chip bring so that the molecules of the molecule library in the designated places of the loading chip can couple. Such an interaction device can be a Be a gripper arm with which the loading chip is immersed in the molecule library, particularly preferably under precise control of temperature, electrical or magnetic field or the like

With the positioning device, the loading chip is in contact with the target brought or aligned immediately adjacent. This can be a robot arm or a Carousel loading station.

The transfer device then serves to release the molecules and to transfer them of these molecules onto the target. This transmission device preferably comprises one Application device for supplying substances that can solve the interactions or for applying electrical or magnetic fields, etc., which for Support the loosening of these connections are suitable. Furthermore, with the Application device is preferably constructed and maintained by an interphase which the molecules can migrate more easily to the target during transfer and there in the manufactured product are protected against environmental influences. In addition, a Spacer may be provided.  

Further advantages of the present invention are shown in the drawings and are explained. Here show:

Figure 1 is a schematic representation of an embodiment of a method according to the invention in three sub-steps I to III.

Fig. 2 (a) - (f) is a schematic representation of the steps of another method of the invention in seven sub-steps 2 (a) to 2 (f); and

Fig. 3 (a) - (b) is a schematic representation of an embodiment of an inventive device for producing selected molecule libraries in two partial views in Fig. 3 (a) and in Fig. 3 (b).

Fig. 1 shows a schematic representation of an embodiment of a method according to the invention in three sub-steps I to III. An oligonucleotide library 15 , which consists of individual oligonucleotides 15.1 to 15.4, is coupled to the loading chip 10 . These are clearly defined on the loading chip. To simplify this schematic representation, only four oligonucleotides 15 .i are shown in one dimension. Usually, more than 900 positions per square centimeter are provided in a two-dimensional arrangement on a loading chip. Spatial, ie three-dimensional, structures are also conceivable. These oligonucleotides 15 .i have complementary regions or anchor segments 16.1 to 16.4 .

Furthermore, a molecule library 25 is provided which contains individual molecules 25 .i (here: 25.1 to 25.3). These molecules 25 .i have oligonucleotide sections 26 .i which are complementary to the regions 16 .i of the oligonucleotides 15 .i.

In step I, the oligonucleotides are now brought the .i 15 arranged on the loading chip oligonucleotide library 15 with the molecules 25 .i the molecular library in interaction. The molecules 25 .i now couple to the oligonucleotides 15 .i, more precisely, stable interactions form between the oligonucleotide sections 26 .i and the anchor segments or complementary regions 16 .i.

In step II, the loading chip is separated from the molecular library and positioned over the target 50 . Hybrids 35 .i have now formed on the loading chip, which together form a hybrid library 35 . For example, molecules 25.1 bound to 15.1, 25.2 to 15.2 and 25.3 to 15.3. Since there was no molecule 25.4 in the molecule library that could have coupled to the oligonucleotide 15.4 , this location on the loading chip remains unloaded - no hybrid 35.4 is formed here. The "hybrid" 35.4 thus corresponds exactly to the oligonucleotide 15.4 . The hybrid library 35 is now positioned exactly above the target 50 .

In step III, the hybrid library is then split up again by separating the connections between the molecules 25 .i and the oligonucleotides 15 .i by an electric field. The molecules 25 .i now migrate to the target and are positioned there exactly as they were spatially arranged on the loading chip. This creates the selected molecular library 55 . Position 55.4 remains free, since here no molecule 25.4 was coupled to the loading chip. This leaves a targeted vacancy.

This selected molecule library 55 consists of the molecules of the molecule library 25 and additionally obeys the order which is predetermined by the structure of the loading chip and the anchor segments or oligonucleotide sections 16 .i provided there. Through the selection (a) of the type of segments 16 .i, (b) the positioning of these segments 16 .i on the loading chip 10 on the one hand and the selection (c) of the molecules 25 .i and (d) of the molecules offered in the molecule library Sections 26 .i offered there, on the other hand, are given four degrees of freedom for realizing the architecture of the gene chip ultimately planned on the target 50 with the selected molecular library 55 .

After step III, the loading chip 10 could now be used again in step I, for example to select a special molecule 25.4 'with another molecule library 25 ' and thus, analogously to steps II and III, this molecule 25.4 'onto the target 50 at position 55.4 to position. Finally, the segments 56 .i can be separated, so that only the pure molecules 55 .i remain in the selected molecular library. This molecule library is immobilized on the target due to the fixed arrangement. If necessary, the immobilization can be increased again by adding chemical substances or applying light or electrical or magnetic fields. The selected, high-density, spatially resolved, solid-phase-immobilized, selected molecular library can then be detached from the target, preferably used together with any interphase and for carrying out experiments at the molecular biological / biochemical level. It is also conceivable that the selected molecular library 55 with the target is used together for detection or analysis.

Fig. 2 shows a schematic representation of the steps of another method of the invention in seven sub-steps 2 (a) to 2 (f).

In sub-step a according to FIG. 2.a, a loading chip 10 is provided with a peptide pool 15 , which consists of individual peptides with sections 16.1 to 16.5 . Furthermore, a molecule library 25 is provided, which consists of individual molecules 25 .i. The individual molecules 25 .i have anchor segments 26 .i, which are each shown on the left side of the molecules 25 .i. These anchor segments 26 .i serve to couple the molecules 25 .i to the peptides 15 .i. On the right side of the molecules 25 .i, link molecules are shown as LINK. These serve to later couple the molecules 25 .i onto the target.

An electrical voltage is now applied in order to accelerate the hybridization between the peptides 15 .i and the molecules 25 .i via the anchor segments 26 .i and the sections 16 .i. As a result, hybrids are formed which consist of the molecules 25 .i and the peptides 15 .i.

In sub-step b according to FIG. 2.b, these hybrids 35 are already formed from the molecules 25 .i and the peptides 15 . The complementary anchor segments 26 .i have been coupled to the sections 16 .i. Now an inverse lower electrical voltage is applied to remove the hybrids that do not exactly match. While the hybrids 35.1 to 35.4 could be formed, since the matching partners 25.1 to 25.4 for the peptides 15.1 to 15.4 were found in the offered molecular library 25 , the non-matching molecule 25.5 is separated by the inverse electrical voltage. The place 15.5 remains free.

This cleaned loading chip 10 with hybrid library 35 is shown in Fig. 2.c. This is a loading chip 10 with peptides immobilized with Watson-Crick.

The next sub-step is shown in FIG. 2.d, in which the loading chip 10 with the selected hybrid library 35 is positioned on a target 50 . The hybrids 35 .i couple via the LINK molecule shown on the right to corresponding CATCH molecules which are provided on the target 50 . As a result, covalent immobilization takes place on the contact print. The hybrids are now connected to the loading chip 10 and the target 50 .

In the following sub-step according to FIG. 2.e, the hybridization is dissolved and the hybrids 35 .i are again separated into the molecules 25 .i and the oligonucleotides 15 .i. The now ordered, ie selected, molecular library 55 remains on the target 50 . The solid-phase-immobilized, selected molecular library is thus produced.

In a further optional preferred sub-step, according to FIG. 2.f, the anchor segments 26 .i are split off from the molecules 55 .i along the arrow A. These anchor segments have the transport of the molecules 25 .i on the target and the formation of the selected molecule library 55 served - they may no longer be required for the function of the Bio / Gen chip.

The remaining result is shown in Fig. 2.g. Only the desired molecules 55 .i are arranged in the desired order on the target 50 . This peptide chip can now be used.

3a and FIG. B is a schematic illustration of an exemplary embodiment of an apparatus according to the invention for producing selected molecule libraries in two partial views. Fig. 3a shows an apparatus for the preparation of selective molecular libraries in schematic plan view. Here, a feed device 61 in the form of a conveyor belt is provided, as well as a holding device 62 and an interaction device 63 as a carousel loading station. On the feeding device 61 individual trays are provided, on which the individual targets are .i 50th These targets 50 .i are conveyed further at the level of the holding device 62 and stopped there in a predetermined position. Individual loading chips 10 .i are now selected by means of the interaction device 63 and are positioned above the retention device 62 by rotating the interaction device 63 . The targets 50 .i are loaded there according to the method according to the invention. The interaction device 63 lowers the individual loading chip 10 into the holding device, so that the desired hybrid library can form on the loading chip due to the oligonucleotide library present on the loading chip and the molecular library present in the holding device. The carousel is then rotated 180 ° further along the arrow A via the interaction device 63 , so that the chip 10 .i now loaded comes to lie over the targets 50 .i.

Thereafter, as shown in Fig via the positioning means 64 can,. 3b shown in section, the hybrid library, which has been positioned by the interaction means 63 over the target 50 .i dissolved and the molecules to be transferred. For this purpose, a voltage is applied across the voltage device as a transfer device 65 , which then, as described above, opens the hybrid library and transfers the molecules to the target 50 .i. accomplished. In addition, the interaction device 63 can preferably be moved along the double arrow B in order to achieve the exact position of the loading chip 10 .i above the target 50 .i. In this way, the molecular transfer is effected with the positioning device 64 and the transfer device 65 .

In Fig. 3a, the point C at which the actual experiment takes place is shown schematically with the arrow C after loading. Here the sample is placed on the chip and the result is evaluated, for example using a scanner.

In Fig. 3a is also shown that the targets are .i further transported after the experiment 50, and for example, a cleaning device 66 are fed, on which they are washed and regenerated.

The experiment comes after loading; only then the regeneration !!

In this way, a method and an apparatus have been provided with which Bio / Gen chips manufactured faster and with greater flexibility in architecture can be.  

LIST OF REFERENCE NUMBERS

10

loading chip

15

Oligonucleotide
15.i oligonucleotide from the oligonucleotide library (

15

)
16.i (complementary) area of a representative (

15

.i) the oligonucleotide library (

15

) to an oligonucleotide section (

26

.i) a representative (

25

.i) the Molecular library (

25

)

17

oligonucleotide probe

25

molecule library
25.i molecule of the molecule library (

25

)
26.i oligonucleotide section of a representative (

25

.i) the molecular library (

25

) / Anchor segment
27.i a magnetic bead from a library

27

, one each

27

.i containing one

25

.i

30

Loading chip (

10

) with hybrid library (

35

)

35

hybrid library
35.i hybrid of the hybrid library (

35

)

40

interphase

50

target

55

selected molecule library (immobilized in a high density spatially resolved manner)

60

Device for the production of selective molecular libraries

61

feeding

62

Vorhalteeinrichtung

63

Interaction device

64

positioning device

65

transmission equipment

66

cleaning device

Claims (34)

1. A method for producing selected molecular libraries ( 55 ), in particular gene or bio chips, comprising the steps:

• a) production of a loading chip ( 10 ) with a defined oligonucleotide library ( 15 );
• b) Provision of a molecular library ( 25 ), in which at least one molecule (25.i) of the molecular library ( 25 ) has an oligonucleotide section (26.i) which extends to at least one region (16.i) of an oligonucleotide (15.i) the oligonucleotide library ( 15 ) contained on the loading chip ( 10 ) is complementary to the formation of stable interactions between the molecule (25.i) of the molecule library ( 25 ) and the oligonucleotide (15.i) of the oligonucleotide library ( 15 ), in particular that Enable formation of a stable hybrid (35.i);
• c) the molecule library ( 25 ) with the oligonucleotide library ( 15 ) contained on the loading chip ( 10 ) to form stable interactions between oligonucleotides (15.i) of the oligonucleotide library ( 15 ) and molecules (25.i) of the molecule library ( 25 ) in Brought interaction, resulting in a hybrid library ( 35 );
• d) the hybrid library ( 35 ) is positioned adjacent to a target ( 50 );
• e) the hybrid library ( 35 ) is dissolved by solving the stable interaction between the molecules (25.i) of the molecular library ( 25 ) and the oligonucleotides (15.i) of the oligonucleotide library ( 15 ) and those previously bound to the oligonucleotide library ( 15 ) Molecules (25.i) of the molecular library ( 25 ) transferred spatially ordered to the target ( 50 ) and immobilized there as a selected molecular library ( 55 ).

2. The method according to claim 1, characterized in that the method further comprises the step:

• a) the loading chip ( 10 ) is regenerated and used again in step (c).

3. The method according to claim 2, characterized in that the method between step (c) and step (e) further comprises the step:

• a) Excess and / or incompletely coupled molecules (25.i) of the molecular library ( 25 ) are separated from the hybrid library ( 35 ).

4. The method according to the immediately preceding claim, characterized in that the method further comprises the step:

• a) Excess molecules (25.i) of the molecule library ( 25 ) are used again after their removal in step (d) in step (c) which has been carried out again.

5. The method according to any one of the preceding claims, wherein for the production of the oligonucleotide library ( 15 ) of the loading chip ( 10 ) oligonucleotide probes ( 17 ) with a length of 5 to 30 nucleotides, which are coupled to the loading chip by a covalent chemical bond, are used. 6. The method according to the preceding claim, wherein more than 900 different oligonucleotide probes ( 17 ) are used per square centimeter. 7. The method according to any one of claims 1 to 4, wherein for the production of the oligonucleotide library ( 15 ) of the loading chip ( 10 ) in addition to normal and modified oligonucleotide probes ( 17 ') are used, which allow a more specific interaction with the molecular library ( 25 ). 8. The method according to the immediately preceding claim, wherein oligonucleotide analogs of the following classes are used as oligonucleotide probes ( 17 '):

• - PNA (Peptide Nucleic Acids)
• - LNA (Locked Nucleic Acids)
• - 2'-5'-linked 3'-deoxyribonucleotides
• - p-RNA (pyranosyl DNA or RNA)
• - Compositions of at least two of the aforementioned molecules or of DNA / RNA and at least one of the aforementioned
• - 2'-0-methyl RNA

9. The method according to any one of the preceding claims, wherein the molecule library ( 25 ) is produced by combinatorial chemistry and / or by the "split and pool" method. 10. The method according to any one of the preceding claims, wherein the oligonucleotide library ( 15 ) and / or the molecular library ( 25 ) is produced by the so-called "codon triplet synthon" method. 11. The method according to any one of the preceding claims, wherein
the molecular library ( 25 ) is a chimeric molecule library ( 25 ) which also contains molecules (25.i),
which have an anchor segment (26.i) for interaction with oligonucleotides (15.i) of the oligonucleotide library ( 15 ) of the loading chip ( 10 ) and / or
which have a link molecule (26.i) for interaction with a catch molecule (54.i) of the target ( 50 ). 12. The method according to the immediately preceding claim, wherein the anchor segment (26.i) is strongly interacting with the representatives of the oligonucleotide library ( 15 ) and these molecules are assigned to the following substance classes:

• - peptides
• - "Aptamer oligonuleotides"
• Molecular Beacon oligonucleotides
• - Single strand of DNA
• - Double stranded DNA
• - Single strand RNA
• - Self-complementary DNA / RNA chimera

13. The method according to any one of claims 11 to 12, wherein the chimeric molecular libraries ( 25 ) are produced by biochemical or molecular biological methods. 14. The method according to any one of the preceding claims, wherein a molecule library ( 25 ) coupled to so-called "magnetic beads" (17.i) is used as the molecule library ( 25 ). 15. The method according to any one of the preceding claims, characterized in that the interactions in step (c) double helices according to Watson-Crick, in particular parallel double helices. 16. The method according to the preceding claim, characterized in that double helices are formed between each a 2'-5'-oligonucleotide (15.i) of the loading chip ( 10 ) and an RNA anchor segment (26.i) of the molecular library ( 25 ) , 17. The method according to any one of the preceding claims, characterized in that in step (c) and / or (d) are used to accelerate the formation of the interaction and / or to increase discrimination against different interactions. 18. The method according to claim 17, characterized in that
the temperature is changed in a controlled manner during the formation of the interactions; and or
an electric field is applied and changed in a controlled manner; and / or when using "magnetic beads" a magnetic field is applied and changed in a controlled manner. 19. The method according to any one of the preceding claims, characterized in that the target ( 50 ) comprises nylon, polyamide, nitrocellulose, cellulose, modified celluloses, glass, polyimide, polyacrylamide, silicon, silicone, noble metals or combinations of these materials. 20. The method according to the preceding claim, characterized in that the target ( 50 ) represents a complex spatial structure and contains further integrated functionalities. 21. The method according to any one of the preceding claims, characterized in that an interphase ( 40 ) is used in step (e) for the formation of a physical contact between the loading chip ( 10 ) and the target ( 50 ). 22. The method according to claim 21, characterized in that
a buffered aqueous solution as interphase ( 40 ); and or
a highly viscous solution; and or
a gel
is used. 23. The method according to any one of claims 21 or 22, wherein the interphase ( 40 ) remains on the target ( 50 ) after step (c). 24. The method according to any one of claims 21 to 23, characterized in that a spacer ( 45 ) is introduced between the loading chip ( 10 ) and the target ( 50 ), the spacer ( 45 ) allowing a spatially high-resolution transfer. 25. The method according to the preceding claim, characterized in that the spacer ( 45 ) is a microchannel system. 26. The method according to any one of claims 24 to 25, characterized in that the spacer ( 45 ) allows focusing or spatial reduction of the transfer process in step (f). 27. The method according to any one of the preceding claims, characterized in that in step (f) to solve the primary interaction thermal and / or electrical and / or magnetic energy or radiation is used.   28. The method according to any one of the preceding claims, characterized in that
in step (f) between the target ( 50 ) and the transferring molecules (25.i) of the molecule library ( 25 )
a stable interaction and / or
a covalent chemical bond is established. 29. The method according to the immediately preceding claim, characterized in that after binding, the oligonucleotide sections no longer required (26.i) be split off hydrolytically and / or enzymatically. 30. The method according to any one of the preceding claims, characterized in that the structure of the target ( 50 ) is carried out in several substeps using a plurality of part libraries ( 25 ). 31. The method according to any one of the preceding claims, characterized in that complex molecule libraries ( 25 ) are transferred to the target ( 50 ) in several substeps. 32. The method according to any one of claims 30 to 31, characterized in that due to the spatial reduction, the transfer from step (f) to the target ( 50 ) takes place in several steps. 33. The method according to any one of the preceding claims, characterized in that the target ( 50 ) is regenerated and used again. 34. Device ( 60 ) for producing selected molecular libraries ( 55 ), in particular gene chips, comprising:

• a) a feed device ( 61 ) for providing a loading chip ( 10 ) with a defined oligonucleotide library ( 15 );
• b) a holding device ( 62 ) for providing a molecule library ( 25 ), in which at least one molecule (25.i) of the molecule library ( 25 ) has an oligonucleotide section (26.i) which has at least one region (16.i) Oligonucleotide (15.i) of the oligonucleotide library ( 15 ) contained on the loading chip ( 10 ) is complementary to the formation of stable interactions between the molecule (25.i) of the molecular library ( 25 ) and the oligonucleotide (15.i) of the oligonucleotide library ( 15 ), in particular to enable the formation of a stable hybrid (35.i);
• c) an interaction device ( 63 ) with which the molecule library ( 25 ) with the oligonucleotide library ( 15 ) contained on the loading chip ( 10 ) to form stable interactions between oligonucleotides (15.i) of the oligonucleotide library ( 15 ) and molecules (25.i) the molecule library ( 25 ) is interacted, whereby a hybrid library ( 35 ) is formed;
• d) a positioning device ( 64 ) with which the hybrid library ( 35 ) is positioned adjacent to a target ( 50 );
• e) a transfer device ( 65 ) with which the hybrid library ( 35 ) is dissolved by loosening the stable interaction between the molecules (25.i) of the molecule library ( 25 ) and the oligonucleotides (15.i) of the oligonucleotide library ( 15 ) and the previously molecules (25.i) of the molecular library ( 25 ) bound to the oligonucleotide library ( 15 ) are spatially arranged on the target ( 50 ) and immobilized there as the selected molecular library ( 55 ).