WO1999056865A1

WO1999056865A1 - Device and method for producing a system of chain molecules on a support material

Info

Publication number: WO1999056865A1
Application number: PCT/EP1999/002958
Authority: WO
Inventors: Stephan Burkhardt; Jonathan Hall
Original assignee: Novartis Ag; Novartis-Erfindungen Verwaltungsgesellschaft Mbh
Priority date: 1998-05-02
Filing date: 1999-04-30
Publication date: 1999-11-11
Also published as: DE19819735A1; EP1079920A1; JP2002513549A; AU3825899A

Abstract

The invention relates to a method, a device and the use of a device for producing a support material having chain molecules synthesized in synthesis fields, especially oligonucleotides which are complementary to segments of carriers of genetic information. According to the invention, after a synthesis run for synthesizing chain molecules (21) in a sequence of synthesis fields (1 to 16), at least one additional synthesis run is carried out, during which chain elements (n4 to n15) of one base-type sequence are linked to the chain molecules (21) synthesized during the preceding synthesis run or during each preceding synthesis run and comprising other chain elements (N1 to N12; N9 to N17) of another base-type sequence. The delivery of chain elements (N1 to N17; n4 to n15) to the synthesis fields (1 to 16) in the synthesis runs corresponds to the predetermined succession of chain elements (N1 to N17; n4 to n15) in the chain molecules (21) to be synthesized. The groups of synthesis fields (27) assigned to synthesis regions (1 to 16) are during each synthesis cycle displaced by one synthesis field (1 to 16), always moving in the same direction. In this way it is possible efficiently to synthesize, for example, chain molecules (21) which are complementary to at least parts of segments (20) of carriers of genetic information, with groups of chain elements (N1 to N17; n4 to n15) so as to obtain different base-type sequences.

Description

- 1 -

Device and method for producing an arrangement of chain molecules on a carrier material

The invention relates to a method for producing a carrier material with chain molecules synthesized in synthesis fields with a predetermined sequence of chain building blocks, in particular oligonucleotides complementary to at least parts of sections of genetic information carriers, in which at least one reaction cell is relative in one synthesis cycle in a sequence of synthesis cycles in one cycle direction positioned to the carrier material with partially overlapping synthesis areas and chain building blocks of a basic type series intended for chain extension are passed through the reaction cell, so that chain molecules with sequences of the same sequences of chain building blocks are synthesized in several synthesis cycles in synthesis fields repeatedly covered by synthesis areas of a reaction cell.

The invention further relates to a device, in particular for producing a carrier material with chain molecules synthesized in synthesis fields with a predetermined sequence of chain building blocks, in particular oligonucleotides complementary to at least parts of sections of genetic information carriers, with a carrier material holder to which the carrier material can be applied, with a storage unit, with at least one reaction cell connected to the supply unit, through which chain components provided for chain extension when the or each reaction cell is placed on the support material in a sealing manner can be passed through in contact with the support material in a synthesis area, with a positioning unit with which the or each reaction cell can be passed through in a synthesis process in a sequence of synthesis cycles, each by a displacement path relative to the carrier material with synthesis areas partially overlapping in synthesis fields is possible, and with a control unit for controlling the supply unit and the positioning unit, so that chain molecules with the same sequences of chain components can be synthesized in several synthesis cycles in synthesis fields repeatedly covered by synthesis areas of a reaction cell.

The invention further relates to the use of a device for producing a carrier material with chain molecules synthesized in synthesis fields with a predetermined sequence of chain building blocks, in particular at least in part of - 2 -

Sections of genetic information carriers of complementary oligonucleotides, in particular for carrying out a method according to one of claims 1 to 4, with a carrier material holder to which the carrier material is applied, with a supply unit, with at least one reaction cell connected to the supply unit, by means of which the or each reaction cell on the support material for chain extension intended chain blocks are passed through in contact with the support material in a synthesis area, with a positioning unit with which the or each reaction cell in a synthesis run in a sequence of synthesis cycles each by a displacement path relative to the support material with partially in Positioned synthesis field overlapping synthesis areas, and with a control unit for controlling the storage unit and the positioning unit, so that in several synthesis cycles in the synthesis area repeatedly In a reaction cell covered by synthesis cells, chain molecules with the same sequences of chain building blocks are synthesized.

Such a method and such a device are known from the article "Arrays of complementary oligonucleotides for analyzing the hybridization behavior of nucleic acids" by E.M. Southern, S.C. Case-Green, J.K. Eider et al., Pages 1368 to 1373, Nucleic Acid Research, 1994, vol. 22, no. 8, known. In the known method and the known device, a reaction cell with a diamond-shaped or circular synthesis area is offset in one synthesis step in a sequence of synthesis cycles in one passage direction relative to a carrier material with partially overlapping synthesis areas, with a reaction solution with one provided for chain extension in each synthesis cycle Chain building block is passed through the reaction cell in contact with the carrier material. Chain molecules with a predetermined sequence of chain building blocks, in particular oligonucleotides complementary to sections of genetic information carriers, can be synthesized by passing a reaction solution with a predetermined chain building block in the synthesis area over the carrier material in each case during the synthesis cycles, so that a reaction cell is repeated from synthesis areas covered synthetic fields, chain molecules with sections of the same sequence of chain building blocks are synthesized.

The chain molecules are preferably designed as oligonucleotides. In the present arrangement on the carrier material (as a so-called "scanning array"), the oligonucleotides serve to analyze the hybridization behavior of nucleic acids. With the previously known method - 3 -

and the known device, a large number of sequence-homologous complementary oligonucleotides with different chain lengths can be synthesized.

When inhibiting the expression of a protein encoded by a target nucleic acid by interaction (hybridization) of relatively short oligonucleotides complementary to the base sequence of the target nucleic acid with the target nucleic acid or a target nucleic acid - protein complex, the target nucleic acid in particular a single-stranded target nucleic acid with a secondary and tertiary structure, in particular a "messenger RNA" (mRNA) (so-called "antisense technology"), there is the problem of inhibiting translation due to the complicated secondary and tertiary structure of the mRNA to identify available binding sites for a specific mRNA for an oligonucleotide, the localization of which is difficult to predict. A carrier material on which the above-mentioned arrangement of oligonucleotides is located can be used in particular in an assay with which it is determined whether or how strongly a target nucleic acid, such as such an mRNA molecule, is able to hybridize one or more of the oligonucleotides on the support material. In such an assay, the hybridization conditions are chosen as far as possible so that the secondary or tertiary structure of the target nucleic acid is largely preserved and matches the corresponding in vivo structure of the RNA. The strength of the hybridization can serve as a measure of the accessibility of a specific region of the target nucleic acid by the oligonucleotides. A single oligonucleotide, identified in this way and capable of hybridizing with the target nucleic acid, can then be produced by known methods and used in antisense technology for medical-therapeutic or diagnostic purposes.

It is known from WO 95/21265 to synthesize oligonucleotides that can be hybridized to an mRNA on a solid support as so-called “antisense” to identify binding sites that can be used for inhibition, the oligonucleotides each consisting entirely of a single defined type of chain building block, for example of the deoxyribonucleotide -Type, of the deoxyribophosphorothioate nucleotide type or of the 2'-O-methyl-ribonucleotide type (ie in each case from chain building blocks of a single basic type series). - 4 -

The known methods have the disadvantage that they can only be used to synthesize and identify oligonucleotides on the support which are composed entirely of chain molecules of a single specific basic type series. Such oligonucleotides often do not have the multiple properties desired in antisense technology, such as, for example, high binding affinity for the target nucleic acid and or high resistance to endogenous nucleases combined with the ability to effectively inhibit expression of the target protein at the level of the target nucleic acid.

It is also known that pharmaceutically particularly efficient oligonucleotides are those which combine several properties, for example a high binding affinity for the target nucleic acid / or resistance to degradation by endonucleases combined with the property that the target nucleic acid is cleaved efficiently by the endogenous one To cause RNAse H. It has been possible to combine a large number of such properties in one and the same molecule in oligonucleotides, in that such an oligonucleotide is constructed in sections from chain building blocks of a specific basic type series, each section being responsible for a specific property or function of the oligonucleotide. Such oligonucleotides are often referred to as "chimeric" oligonucleotides. Chimeric oligonucleotides consist of chain building blocks of at least two different basic type series, which are synthesized in at least two successive or adjacent sections, often also in three or more such sections (see, for example, ST Crooke et al., J. of Pharmacology and Experimental Therapeutics 277 (1996) , Pp. 923-937).

The invention has for its object to provide a method and a device of the type mentioned, in which on the carrier material an efficient synthesis of an arrangement of chain molecules with the same sequence of sections of chain building blocks of different basic type series, in particular oligonucleotides with a chimeric structure, but otherwise largely the same characteristic characteristics such as base recognition characteristics can be carried out on oligonucleotides.

The invention is based in particular on the object of providing a method and a device of the type mentioned at the beginning for chain molecules which are at least partially complementary to oligonucleotides of genetic information carriers, the oligonucleotides having a chimeric structure, as a result of which, for example, a high binding affinity and or a high resistance against such oligonucleotides - 5 -

acting natural degradation enzymes associated with the ability to efficiently inhibit translation, or more generally expression, of a target nucleic acid.

These objects are achieved with a method of the type mentioned at the outset in that after a sequence of synthesis cycles in a synthesis run, at least one further synthesis run is carried out with chain building blocks of a basic type series which is different from the previous synthesis run, in that one or each further synthesis run involves synthesis cycles of a synthesis run the reaction cell used in this synthesis run is positioned in the same direction as in the previous synthesis run in such a way that the associated synthesis region is overlaid in sections with a synthesis field hitherto uncovered in this synthesis run, in which chain molecules with a maximum chain length synthesized in the or each previous synthesis run are present, and that provided at this chain position chain building blocks of the chain molecule to be synthesized use in this synthesis run th basic type series are passed through the reaction cell.

These objects are achieved in a device of the type mentioned at the outset that chain components of at least two different basic type series are stored in the supply unit, that the or each reaction cell can be positioned with the control unit in at least two synthesis cycles in the same direction in such a way that synthesis cycles of a synthesis cycle the associated synthesis area is overlaid in sections with a synthesis field hitherto uncovered in this synthesis run, in which chain molecules with a maximum chain length synthesized in the or each previous synthesis run are present, and that, controlled by the control unit, chain building blocks of different basic type series can be passed through the or each reaction cell in two successive synthesis runs .

As an alternative to such a device, the supply unit of which stores chain components of at least two different basic type series, a device according to the invention can comprise a corresponding supply unit, which in each case contains chain components of a single basic type series, and in which the supply unit is designed to be interchangeable, so that after each synthesis cycle the supply unit is replaced by a another supply unit with chain components of another basic type series can be exchanged. - 6 -

When using a device of the type described, these objects are achieved in that chain components of at least two different basic type series are stored in the supply unit, that with the control unit the or each reaction cell can be positioned in at least two synthesis cycles in the same direction in such a way that one during synthesis cycles The associated synthesis area is partially sectioned with a synthesis field hitherto uncovered in this synthesis process, in which chain molecules with a maximum chain length synthesized in the or each previous synthesis process are present, and that, controlled by the control unit, chain building blocks of different basic type series are passed through the or each reaction cell in two successive synthesis processes become.

The present invention thus furthermore relates to the use of a device for producing a carrier material comprising an arrangement of chain molecules synthesized in synthesis fields with a predetermined sequence of chain building blocks, in particular of oligonucleotides complementary to at least parts of sections (20) of genetic information carriers, adjacent synthesis fields Chain molecules with sections of the same sequence of chain building blocks of different basic type series include. Such an arrangement thus comprises chain molecules that comprise at least two sections, each with different basic type series of chain building blocks.

Another object of the invention is a carrier material, comprising an arrangement of chain molecules synthesized in synthesis fields with a predetermined sequence of chain building blocks, in particular of oligonucleotides complementary to at least parts of sections (20) of genetic information carriers, adjacent synthesis fields being chain molecules with the same sequence of chain building blocks of different basic type series include. Such an arrangement thus comprises chain molecules that comprise at least two sections, each with different basic type series of chain building blocks.

In a further aspect, the present invention is directed to a carrier material which can be produced by a method according to the present invention. - 7 -

By carrying out at least two superimposed synthesis runs according to the method according to the invention, the device according to the invention and the use of a device according to the invention, chain molecules with blocks of chain building blocks of different basic type series, but otherwise the same characteristics as base recognition characteristics, can be synthesized, whereby, for example, different sequences of basic type series and / or differently designed synthesis areas of reaction cells used in the various synthesis runs can achieve high varieties in the synthesized chain molecules with high efficiency.

The synthesis areas of each reaction cell are expediently rectangular in shape, each having the same transverse sides, the length of each long side of a reaction cell corresponding to an integral multiple of a displacement path in the longitudinal direction. The reaction cells are shifted from one synthesis cycle to the next by the displacement distance corresponding to the size of a synthesis field in the longitudinal direction. In the case of synthesis areas of different lengths, the reaction cells are to be positioned in a first synthesis cycle of each synthesis run in such a way that the synthesis areas of the reaction cells are flush with one another in the passage direction.

In the context of the present invention, as already mentioned, the chain molecules are preferably oligonucleotides. Such oligonucleotides are capable of pairing with a complementary nucleic acid strand, in particular an mRNA, and consist of individual chain building blocks, namely nucleosides or nucleoside analogues, which as a rule carry a nucleic acid base or a base analog, which mediate the properties of the oligonucleotides for base pairing . A chain building block also includes an intemucleosidic bridge bond to the next building block, for example of the phosphodiester, phosphorothioate or amide type, as described, for example, in A. de Mesmaeker et al., Acc. Chem. Res. 28 (1995), pp. 366-374.

In the context of the present invention, a basic type series of the chain molecules is in each case chain building blocks of the same basic structure, members of a first basic type series being members of a second basic type series in particular by the properties to be achieved, for example those by the part of the chain molecule under consideration - 8th -

caused or mediated physiological or pharmacological properties. In the case of oligonucleotides, such properties of the chain molecules are, for example, the affinity for a target nucleic acid, the nuclease resistance, the sensitivity to RNAse H or the toxic properties. An advantageous oligonucleotide, which is used as a drug in the context of antisense technology, combines several advantageous properties.

Within a chain molecule, such different properties are often linked to the presence of building blocks of certain basic structures or basic types. Examples of various basic type series of chain building blocks of oligonucleotides are known to the person skilled in the art. For example, the basic types of building blocks are natural nucleosides, such as 2'-deoxyribonucleosides or ribonucleosides; or by 2'-0-alkyl-modified ribonucleosides, the 2'-O-alkyl group, which is preferably 2'-OC ₁ -C ₅ alkyl, in particular 2'-O-methyl or 2'-O-ethyl, unsubstituted or, in the case of 2'-O-ethyl at the β-position, can be substituted with -OH, -F or alkoxy, preferably with dC ₅ -alkoxy, in particular methoxy, where 2'-O- (2-methoxyethyl ) -modified ribonucleosides are very particularly preferred; to building blocks of nucleotides or nucleotide analogs which lead to oligonucleotides with an intemucleosidic bridging of the phosphodiester or phosphorothioate type, or to oligonucleotides with a modified backbone (intemucleosidic bridging), such as PNA building blocks, amide building blocks or 3-methylenephosphonate building blocks ; or base-modified nucleoside building blocks, such as 5-propynyl nucleoside building blocks, where the individual basic types have corresponding nucleic acid bases or base analogues depending on the base sequence of the target nucleic acid (see also A. De Mesmaeker et al., as mentioned above; E Uhlmann et al., Chem. Rev. 90 (1990), pp. 543-584). Combinations of chemical modifications lead to building blocks that belong to different basic type series. For example, 2'-O- (2-methoxyethyl) ribonucleotides with a phosphodiester group as building blocks of a basic type series, and 2'-O- (2-methoxyethyl) ribonucleotides with a phosphorothioate group as building blocks of another basic type series are considered in the context of the present invention. According to the invention, building blocks of a basic type series are synthesized in one section in an oligonucleotide, an oligonucleotide consisting of at least 2, for example 2 or 3 sections. - 9 -

Chimeric oligonucleotides which consist of two sections are preferred as chain molecules, one section consisting of 2'-O- (2-methoxyethyl) ribonucleosides which are linked to one another via phosphodiester bridges or phosphorothioate bridges, and the other section 2'-deoxyribonucleosides, which are linked together via phosphorothioate bridges.

Further preferred chain molecules are chimeric oligonucleotides which consist of three sections, the first section consisting of 2'-O- (2-methoxyethyl) ribonucleosides, viewed from 5'- in 3'-direction, via phosphodiester bridges or phosphorothioate bridges are linked together, the adjoining second section consists of 2'-deoxyribonucleosides which are linked together via phosphorothioate bridges, and the adjoining third section in turn consists of 2'-O- (2-methoxyethyl) ribonucleosides , which are linked together via phosphodiester bridges or phosphorothioate bridges.

In such chimeric oligonucleotides which contain sections with 2'-0- (2-methoxyethyl) ribonucleosides, two individual sections are preferably linked to one another via a phosphorothioate bridge if the nucleoside at the 3 'end of a section is one 2'-deoxyribonucleoside; or alternatively, two individual sections are linked to one another via a phosphodiester bridge or via a phosphorothioate bridge if the nucleoside at the 3 'end of a section is a 2'-O- (2-methoxyethyl) ribonucleoside.

Suitable, preferred chimeric oligonucleotides have a total length of about 15 to about 25, for example 20, nucleoside building blocks, the section consisting of 2'-deoxyribonucleoside building blocks comprising at least about 5 such building blocks.

Further expedient refinements and advantages of the invention are the subject of the subclaims and the following description of exemplary embodiments with reference to the figures of the drawing. Show it:

1 shows a schematic representation in one exemplary embodiment of a carrier material strip with a reaction cell which has been placed on and has a synthesis area covering four synthesis fields, after a first synthesis - 10 -

thesis cycle of a first synthesis run with graphic representation of the synthesized chain building blocks of a basic type series,

2 shows the arrangement according to FIG. 1 after the last synthesis cycle of the first synthesis run has been carried out,

3 shows the arrangement according to FIG. 2 after carrying out a first synthesis cycle of a further second synthesis run with chaining of a chain building block of a different basic type series used in relation to or in the first synthesis run,

4 shows the arrangement according to FIG. 3 after the last synthesis cycle of the second synthesis run has been carried out,

Fig. 5 in a schematic representation in a further embodiment

Carrier material strips with a reaction cell having a synthesis area covering three synthesis fields after carrying out a tenth synthesis cycle of a first synthesis run with representation of the chain building blocks of a basic type series which were linked up to that point,

FIG. 6 shows the arrangement according to FIG. 5 with a reaction cell placed on top of it, which has a synthesis area covering five synthesis fields, after a second synthesis cycle of a further second synthesis run, FIG.

FIG. 7 shows the arrangement according to FIG. 6 after the last synthesis cycle of the second synthesis run has been carried out and

Fig. 8 shows the arrangement of FIG. 7 with a covering two synthesis fields

Reaction cell having synthesis area after carrying out the last synthesis cycle of a further third synthesis pass.

9 shows a device for producing a carrier material with chain molecules synthesized in synthesis fields according to the invention in a schematic perspective view. - 11 -

Fig. 10 shows the lifting unit of the device schematically in a perspective enlarged view.

11 shows the lifting unit according to FIG. 10 in a top view.

Fig. 12 shows a cross section through the support beam of the lifting unit with a

Support bars attached reaction cell in a side view, partially cut open:

13 is a plan view of a reaction cell

1 shows, in a schematic representation in an exemplary embodiment of a method and a device according to the invention, a support material strip 17 which can be occupied with a total of 16 synthesis fields 1 to 16 and which is made of polypropylene (PP), for example chemically treated for the synthesis of oligonucleotides, as the support material. The carrier material strip 17 is placed on a carrier material holder, not shown in FIG. 1, for example in the form of a metal plate provided with an elastic layer. In the illustration according to FIG. 1, a displaceable reaction cell 18, shown in its outline, is placed on the carrier material strip 17 and is connected to a storage unit, not shown in FIG. 1. In further developments, a plurality of reaction cells 18 are provided which, for example for carrying out a parallel synthesis, are arranged next to one another on a reaction cell carrier and can be moved together. In the storage unit connected to a control unit (also not shown in FIG. 1), reaction fluids, for example reaction solutions, which are provided for a synthesis of chain molecules with chain extension with a predetermined, specific sequence of chain components, are stored or are successively stored, which are controlled by the control unit or can be fed to each reaction cell 18.

The reaction cell 18 can be placed on the carrier material strips 17 with a seal. In the exemplary embodiment according to FIG. 1, the reaction cell 18 has a recess with a rectangle-shaped border which is open on one side and delimits a synthesis region 19, the dimensions of which in the illustrated exemplary embodiment correspond to the size of four adjacent synthesis fields 1 to 16. 1 is the means of a - 12 -

Positioning unit, not shown in FIG. 1, controlled by the control unit in the direction of synthesis fields 1 to 16, reaction cell 18 which can be displaced while being lifted, shown positioned overlapping the first four synthesis fields 1 to 4 counted from the left.

In the following it will be explained how a number of oligonucleotides complementary to at least part of a section 20 of a genetic information carrier are synthesized with the above-described device in a method. The individual bases of section 20 carrying the genetic information are symbolically identified in FIG. 1 with B1 to B17 and each stand for one of the bases A, C, G and U or T according to the Watson-Crick model.

1 shows the structure of oligonucleotides 21 each assigned to a synthesis field 1 to 16 as chain molecules. Each oligonucleotide 21 is attached to a residual group R of the substrate strip 17 prepared for binding.

1, the reaction cell 18 is arranged in the position of a first synthesis cycle of a first synthesis run in which a reaction fluid intended for chain extension with a natural nucleotide N1 complementary to the base B1 of the section 20 has been passed through the reaction cell 18 is. The reaction fluid has washed over the synthesis area 19, so that the nucleotide N1 has been added to the residual groups R of the first four synthesis fields 1 to 4 on the left in the illustration in FIG. 1.

1, the further synthesis cycles of the first synthesis run following the first synthesis cycle according to FIG. 1 are now, with the stepwise displacement of the reaction cell 18 by means of the positioning unit in a passage direction, in each case by a displacement path corresponding to the size of a neighboring synthesis field 5 to 16, the synthesis fields covered by the synthesis area 19 1 from left to right in the illustration according to FIG. 1 with natural reaction fluids containing nucleotides N2 to N13 of a basic type series complementary to the bases B1 to B13 of section 20.

FIG. 2 shows the arrangement according to FIG. 1 with the reaction cell 18 in one of the four synthesis fields 13 to 16 located on the left in the selected illustration and in the passage direction. - 13 -

covering arrangement after carrying out the last synthesis cycle of the first synthesis run with a reaction fluid which has the natural nucleotide N13 complementary to the base B13. From FIG. 2 it can be seen that, apart from the first three synthesis fields 1 to 3 and the last three synthesis fields 14 to 16 counted from the left in the illustration according to FIG. 2, in the central synthesis fields 4 to 13 sequence homologues, each four natural nucleotides N1 to N13-containing oligonucleotides 21 have been synthesized, each oligonucleotide 21 synthesized in one of the synthesis fields 4 to 13, as represented by the same sequence of atomic numbers, being complementary to a coherent part of section 20 of bases B1 to B13 of the genetic information carrier.

FIG. 3 shows the arrangement according to FIG. 2 with the reaction cell 18 having the synthesis area 19 covering the four synthesis fields 1 to 16 in the positioning of a first synthesis cycle of a further second synthesis run. In the first synthesis cycle of the second synthesis run, the reaction cell 18 is brought into the same position as in the first synthesis cycle of the first synthesis run, covering the first four synthesis fields 1 to 4 on the left in the illustration in FIG. 3. A reaction fluid with a now modified nucleotide n5 as a chain building block of a different basic type series compared to the one used in the first synthesis run is then flushed out, the base of the modified nucleotide n5 being complementary to the base B5 of section 20 of the genetic information carrier. In the first synthesis cycle of the second synthesis run, an oligonucleotide 21 composed of five chain building blocks in the sequence N1-N2-N3-N4-n5 is synthesized in the fourth synthesis field 4 counted from the left, which oligonucleotide 21 forms the first five bases B1 to B5 of section 20 of the genetic information carrier is complementary and has natural nucleotides N1 to N4 of the basic type series used in the first synthesis run and the modified nucleotide n5 of the further basic type series used in the second synthesis run. In the edge-side synthesis fields 1 to 3, oligonucleotides 21 that are not or not completely complementary to the beginning of section 20 of the genetic information carrier have been synthesized.

In the further synthesis cycles of the second synthesis run following the first synthesis cycle, reaction fluids with modified nucleotides n6 to n17 are then passed through after shifting the reaction cell 18 by a displacement path corresponding to the size of an adjacent synthesis field 5 to 16, whereby the sequence follows - 14 -

the bases B6 to B17 of section 20 of the genetic information carrier the sequence of the modified nucleotides n6 to n17 complementary to the bases B6 to B17 is predetermined.

FIG. 4 shows in the arrangement according to FIG. 3 the reaction cell 18 in the position in the last synthesis cycle of the second synthesis run after flushing with a reaction fluid containing the modified nucleotide n17 complementary to the last base B17 of section 20 of the genetic information carrier. From Fig. 4 it can be seen that now except for the three edge-side synthesis fields 1, 2, 3, 14, 15, 16 in the middle synthesis fields 4 to 13 sequence homologues, each with a part of section 20 of the genetic information carrier complementary oligonucleotides 21 with a Length of eight nucleotides have been synthesized. Each oligonucleotide 21 has four natural nucleotides N1 to N13 in blocks and then four modified nucleotides n5 to n17, each following the natural nucleotides N4 to N13 added last in the first synthesis run in one of the sequences of bases B1 to B17 of section 20 of the Genetic information carrier corresponding order.

The carrier material strip is now, in particular with regard to the oligonucleotides 21 synthesized in the middle synthesis fields 4 to 13, as chain molecules with eight chain building blocks arranged in two blocks or sections with different basic type series, for use in an assay for examining parts of section 20 of the genetic information carrier with regard to accessible Binding sites for so-called "antisense 'oligonucleotides are available, as mentioned above.

5 shows schematically similar to FIGS. 1 to 4 in a further exemplary embodiment of a method and a device according to the invention the carrier material strip 17 according to the aforementioned exemplary embodiment. In the exemplary embodiment explained below, oligonucleotides 21 which are complementary to connected parts of section 20 of the genetic information carrier and have a chain length which is greater than the exemplary embodiment explained with reference to FIGS. 1 to 4 using natural and modified nucleotides in three differently long blocks of pairs of different basic type series are intended be synthesized.

5, a reaction cell 22 with a synthesis area 23 covering three synthesis fields 1 to 16 is arranged in the arrangement with a tenth synthesis cycle - 15 -

first synthesis run shown. In the first synthesis cycle of the first synthesis run, the reaction cell 22 was arranged in such a way that the synthesis area 23 covered the third to fifth synthesis fields 3, 4, 5 counted from the left in the illustration in FIG. 5. Then, in the subsequent synthesis cycles of the first synthesis run, the reaction cell 22 was placed in each case on the next neighboring synthesis field 5 to 14 and flushed with a reaction fluid containing a complementary natural nucleotide N2 to N10 corresponding to the sequence of bases B2 to B10, so that up to the in 5, the tenth synthesis cycle shown have built up the complementary oligonucleotides 21 shown.

FIG. 6 shows the arrangement according to FIG. 5 after a second synthesis cycle of a further second synthesis run. In the second synthesis step, a reaction cell 24 is used, with the synthesis area 25 of which five synthesis fields 1 to 16 can be covered. In a first synthesis cycle of the second synthesis run, the reaction cell 24 is arranged such that the only synthesis field 5 to 12 with a chain of three natural nucleotides N1 to N12 is the fifth synthesis field 5 counted from the left in the illustration in FIG. 6 together with the left side outer four synthesis fields 1 to 4 has been covered for the first time by the synthesis area 25. In the first synthesis cycle of the second synthesis run, the first two synthesis fields 1, 2 located on the left outside in the illustration in FIG. 6 were first flushed with a reaction fluid.

In the first synthesis cycle of the second synthesis run, a reaction fluid was used which contains the modified nucleotide n4 which is complementary to the fourth base B4 of section 20, while in the second synthesis cycle of the second synthesis run shown in FIG. 6 that to the fifth base B5 of section 20 of the genetic information carrier complementary, modified nucleotide n5 was contained in the reaction fluid.

In the further synthesis cycles of the second synthesis run, the reaction cell 24 is in each case displaced by a displacement path corresponding to the size of a synthesis field 1 to 16, and reaction fluids with complementary modified nucleotides n6 to n15 in the corresponding sequence of the bases B6 to B15 of section 20 of the genetic information carrier are passed through . - 16 -

FIG. 7 shows in the illustration in FIG. 6 the positioning of the reaction cell 24 used in the second synthesis run after the last synthesis cycle of the second synthesis run has been carried out. From Fig. 7 it can be seen that apart from the four synthesis fields 1 to 4, 13 to 16 located on the right and left edges in the middle synthesis fields 5 to 12 oligonucleotides with eight chain components N1 to N10, n4 to n15 have been synthesized, the in the first synthesis step, three nucleotides N1 to N10 synthesized block by block of a natural basic type series and the five nucleotides n4 to n15 linked in the second synthesis cycle are assigned in blocks to a modified basic type series.

FIG. 8 shows the arrangement according to FIG. 7 after carrying out the last synthesis cycle of a further third synthesis run, in which, as chain building blocks, a basic type series different from the previous synthesis run in turn complements the natural nucleotides N9 to N17 to the bases B9 to B17 to those in the first two Synthesis runs synthesized oligonucleotides 21 have been linked. In the third synthesis step, a reaction cell 26 is used, with the synthesis region 27 of which two adjacent synthesis fields 1 to 16 can be covered.

In a first synthesis cycle of the third synthesis run, the reaction cell 26 was positioned such that the fifth synthesis field 5 counted from the left in the illustration in FIG. 8 was the only synthesis field covered for the first time with a full number of eight chain building blocks synthesized in the previous two synthesis runs. In this first synthesis cycle, a reaction fluid with a natural nucleotide N9 complementary to the ninth base B9 of section 20 of the genetic information carrier was passed through the reaction cell 26.

In the further synthesis cycles of the third synthesis run, after moving the reaction cell 26, reaction fluids containing complementary natural nucleotides N10 to N17 predetermined by the sequence of the bases B10 to B17 were flushed through the reaction cell 26 to cover for the first time the next neighboring synthesis field 6 to 16. - 17 -

As can be seen from FIG. 8, 5 to 12 sequence homologous oligonucleotides 21 with a length of ten chain building blocks, which are each complementary to part of section 20 of the genetic information carrier and consist of a block of three, are now synthesized in the middle synthesis fields 5 to 12 after completion of the third synthesis run natural nucleotides N1 to N10, a block of five modified nucleotides n4 to n15 and a further block of two natural nucleotides N9 to N17 are constructed. Each block of natural nucleotides N1 to N10 or N9 to N17 and of modified nucleotides n4 to n15 has a length which corresponds to the number of synthesis fields covered by the synthesis areas 23, 25, 27 of the reaction cells 22, 24, 26 used in the corresponding synthesis runs Corresponds to 1 to 16.

It goes without saying that in further embodiments, not shown, longer-chain chain molecules can be synthesized by using reaction cells having larger synthesis areas and by carrying out a plurality of synthesis cycles having a greater number of synthesis cycles than in the exemplary embodiments explained in detail above. By providing reaction cells with differently sized synthesis areas, diverse variations in the block-wise sequence of basic type series can be achieved, it being expedient that the rectangular sides of the synthesis areas have the transverse sides of the reaction cells of the same size and the size of the long sides a full-page multiple of that due to a displacement path in the passage direction correspond to the specified dimension of the synthesis fields and the reaction cells are displaced in the longitudinal direction by the corresponding dimension of the synthesis fields.

It goes without saying that more than two different basic type series can be provided in the different synthesis runs, so that with a corresponding number of synthesis runs a wide variety of variations in the sequence of basic type series can be achieved. - 18 -

Examples:

1. Production of an activated polypropylene carrier material

A cut polypropylene film (PP: Mobil 40MB400, 400 mm x 300 mm) is placed in a cylindrical glass drum (diameter: 170 mm, length: 400 mm) in a solution (180 ml) of 0.9 g chromium (VI) oxide (Fluka), dissolved in a 1: 1 mixture of acetic anhydride: acetic acid (Fluka), immersed. The reaction drum is quickly rotated for 1 hour using a mechanical rolling device. The film is then removed, immersed in a bath of 1 M aqueous acetic acid for 30 minutes and then rinsed with methanol (Fluka) for 30 minutes. The film is dried in a high vacuum, being located between 2 glass plates for protection. The film is then immersed in a corresponding glass container in a 1 M solution of BH ₃ (Aldrich Chemical Co.) in tetrahydrofuran (180 ml), which is diluted to 0.2M with additional tetrahydrofuran. The glass vessel is rotated quickly for 1 hour. The film is removed, immersed in 1 M aqueous acetic acid, rinsed in methanol and dried under vacuum. The film treated in this way is used without further treatment as a carrier material in a process according to the invention.

2. Automated device for the production of a carrier material according to the invention with chain molecules synthesized in synthesis fields

FIG. 9 shows an automated device (so-called "array maker") which is used for the synthesis of a carrier material according to the invention with chain molecules synthesized in synthesis fields. The device comprises an automatic cell positioning device or cell positioning unit 36 and a DNA synthesizer (ABI 394/4) 31. The DNA synthesizer 31 is connected to the automatic cell positioning device 36 by means of hose connections for the reagents (not particularly reaction solutions) which are not shown in the drawing , Purge liquids and purge gases). The inlet tubing connections that are normally used to deliver the reagents from the DNA synthesizer to the 4 reaction columns, which comprise a solid support and are standard for the synthesis of oligonucleotides, are instead with the inlet openings of 4 reaction cells 32, 33, 34, 35 connected. The outlet tube connections that normally connect the DNA synthesizer to the respective outlet openings of the 4 reaction columns mentioned are instead to the outlet openings of the 4 reaction cells - 19 -

32, 33, 34, 35 connected. The cell positioning machine 36 is connected via a trigger line (not shown in the drawing) to an electronic control unit 37, with the aid of which a trigger signal ("advance fraction collector signal") for the respective next cell position of the cell positioning machine 36 from the DNA synthesizer 31 the cell positioning machine 36 is transmitted. The automatic cell positioning device 36 and the DNA synthesizer 31 stand on a stable base 38 below an extractor hood 39 with a rear wall 40. The automatic cell positioning device 36 has its own protective hood 41, which is only shown in parts in the drawing, and which has a vertical sliding door is provided, which are moved by means of a handle 42 and via pulling ropes (not shown in the drawing), which are connected to a counterweight (not shown in the drawing) and are deflected via rollers 43, into an indicated upper position and a likewise indicated lower position can. The rollers 43 are connected via brackets and struts to the profile frame 45, which consists of several profile tubes 44 and is supported on the base 38. The automatic cell positioning device (36) is moved by an electrically operated unit 46, which, controlled by the control unit 37, moves a pressure plate 47 vertically in stages between an illustrated upper position and an indicated lower position.

The PP film 49 is fastened on the pressure plate 47 with the aid of 5 vertical metal holding bars 48 by means of screws located at the upper and lower ends of the holding bars. A thin piece of rubber 90 (see FIG. 11) is located between the PP film 49 and the pressure plate 47. The pressure plate 47 is moved vertically to preprogrammed positions. The reaction cells 32, 33, 34 and 35 are horizontally next to each other on a support beam

50 a lifting unit 51 mounted.

The lifting unit 51 is shown separately and enlarged in FIG. 10. 11 shows the lifting unit

51 in plan view together with the reaction cells 32, 33, 34 and 35 and with the pressure plate 47. The lifting unit 51 has a left-hand guide 52 and a right-hand guide 53, each of which is designed as a hollow cylinder. A left push rod 54 and a right push rod 55 each extend through the guides 52 and 53. Both push rods 54, 55 are prestressed with the aid of pressure springs (not shown in the drawing) arranged in the guides 52, 53 such that the reaction cells 32, 33, 34, 35 are pressed onto the pressure plate 47. To lift the reaction cells 32, 33, 34, 35 away from the pressure plate 47, eccentric disks, not shown in the drawing, are provided, which cooperate with the end faces 56 and 57 of the pressure rods 54, 55, around the - 20 -

Carrier bar 50 and thus the reaction cells 32, 33, 34, 35 to move away from the pressure plate 47. The eccentric discs are adjusted with the aid of an electric drive, which is also connected to the control electronics 37.

The push rods 53, 54 are detachably connected to the support beam 50 by means of knurled nuts 58. In this way, the reaction cells 32, 33, 34, 35 can be removed together with the support bar 50. After dismantling the support bar 50, it is possible to detach each individual reaction cell 32, 33, 34, 35 from the support bar 50 and, if necessary, to replace it with another reaction cell 32, 33, 34, 35.

The connection of the reaction cell 32 is shown in FIG. 12, for example. The reaction cell 32 is fastened to a pin 59 which extends through a bore 60 in the support beam 50. On the side of the support beam 50 pointing away from the reaction cell 32, the pin 59 is provided with a thread onto which a knurled nut 61 is screwed. The pin 59 with the screwed-on knurled nut 61 is prestressed with the aid of a compression spring 62, so that the knurled nut 61 is pressed against the back of the support beam 50. The compression spring 62 is designed to be significantly weaker than the compression springs provided in the guides 52, 53, so that when the reaction cells 32, 33, 34, 35 are placed on the plastic film 49 and the pressure plate 47, the compression spring 52 is compressed and the pressure force of the reaction cell 32, 33, 34, 35. The optimal pressure force can be set quickly and easily by changing the pressure spring 62. A pressure spring 62 with 42N is used for the PP film 40MB400 used as carrier material in this example. In addition, the compression spring 62 ensures a uniform pressure of the reaction cells 32, 33, 34, 35 against the plastic film 49 (see FIG. 11), which either directly on the pressure plate 47 or with the interposition of an intermediate carrier film 90 (see FIG. 11) elastic material is attached to the pressure plate 47 with the aid of the holding webs 48. The intermediate carrier film 90, which is preferably up to 1 mm thick (see FIG. 11), consists, for example, of a polymer film made of Neoprene 60 ° Shore A or natural rubber (for example Paragummi 40 ° Shore A), both from Richterich + Zeller AG, Basel, Switzerland are distributed.

The structure of the reaction cells 32, 33, 34, 35, which are open on one side, is explained in more detail below with reference to FIGS. 12 and 13. - 21 -

As can be seen in FIGS. 12 and 13, the reaction cells 32, 33, 34, 35 consist of a block with a rectangular cross section made of a deformable material or a plastic. In this case, PTFE is used. The rear side 63 facing the support beam 50 and the outer sides 64, 65, 66, 67 and 68 are each flat. On the upper side 69 pointing away from the support beam 50, a peripheral edge strip 70 is provided, which encloses a flat space 71 (corresponding to 19, 23, 25, 27 in FIGS. 1 to 8). The height of this flat space 71 and the height of the edge strip 70 are one millimeter. The four edge strip sections circumscribing a rectangle are each 40 mm x 10 mm in size. In the area of the corners 72, 73, 74 and 75, the edge strip sections run in an arc shape, the inner radius being approximately 0.5 millimeters.

Between the corners 72 and 75 or 73 and 74 there are bores 76 and 77 running at right angles to the upper side, which in the exemplary embodiment shown likewise have a radius of 0.5 millimeters. The bore 76 opens into an outlet channel 78. The lower bore 77 opens into an inlet channel 79. Each inlet channel 79 and each outlet channel 78 of the reaction cells 32, 33, 34, 35 is connected to the synthesizer 1 via a hose connection, not shown in the drawing.

3. Operation of the synthesis device

During operation of the device, the support bar 50 is moved horizontally in order to press the reaction cells 32-35 against the PP film. When an electrical signal is received from the advance fraction collector, the control unit 37 sends a signal to the cell positioner 36. The support bar 50 is then moved horizontally to remove the reaction cells 32-35 from the surface of the PP film . The pressure plate 47 is then moved vertically into a new position. Finally, the carrier bar 50 is again moved horizontally, and the reaction cells 32-35 are pressed again onto the surface of the PP film.

The preparation of an arrangement of chimeric oligonucleotides on the solid support material comprises the following steps: - 22 -

1. A PP film 49 preactivated as in Example 1 is fastened in the cell positioning device 36 using a rubber piece 90 in order to guarantee a good seal.

2. The vertical support bars 48 are attached in place to ensure that the film 49 holds well.

3. The selected reaction cells (between 1 and 4) 32, 33, 34, 35, with corresponding compression springs 62 are attached to the support bar 50, which is installed on the cell positioning machine.

4. The selected reaction cells 32, 33, 34, 35 are connected to the inlets and outlets of the DNA synthesizer via hose connections.

5. The coordinates of the starting position and the length of the displacement path are programmed into the control unit 37, the pressure plate 47 is moved into the starting position, and the reaction cells 32, 33, 34, 35 are brought into contact with the PP film 49.

6. The DNA synthesizer is programmed to synthesize the desired oligonucleotide sequences in the desired order and the synthesis begins.

7. As soon as the so-called "advance fraction collector" signal is generated according to the synthesis protocol (see below), the lifting unit 51 lifts the reaction cells away from the surface of the PP film, the pressure plate moves vertically upwards by the selected displacement length, and moves the lifting unit 51 forwards the support beam 50, whereby the contact between the reaction cells and the film is established. A reaction cycle is then carried out by means of the DNA synthesizer in order to synthesize a first building block of an oligonucleotide on the surface of the film or in order to carry out a chain extension reaction of an oligonucleotide.

8. Step 7 is repeated as many times as necessary for a synthesis run.

4. Production of an arrangement of oligonucleotides on a support (so-called "scanning arrav"), which comprises chimeric oligonucleotides with a length of 16 nucleotide building blocks

The PP film 49 is pretreated as described herein. A sheet of natural rubber (Paragummi 40 ° Shore, Richterich & Zeller, Basel, Switzerland, 0.5 mm thick, 400 x 300 mm) 90 is used as described. As described above, 4 rectangular reaction cells 32, 33, 34, 35 made of Teflon are used, each reaction cell being held by two compression springs 62 (42N). Each reaction cell is connected to the inlet and outlet tubing connections of an ABI 394/4 DNA synthesizer (Perkin Elmer Corp., Foster City, CA, USA). The - 23 -

The starting position of the beam is set to 5 mm below the top edge of the PP film. 5 mm are programmed as the length of the displacement path of the reaction cells after each synthesis cycle. Due to the length of the reaction cells of 40 mm, an arrangement of oligonucleotides is generated in one synthesis run, which have a maximum length of 8 building blocks.

The reaction sequences described below result in an arrangement of chimeric oligonucleotides (total length of a chimeric oligonucleotide: 16 nucleotide building blocks, of which (i) building blocks 1 to 8: deoxyribonucleosides, which are linked to one another via phosphorothioate bridges, and (ii) building blocks 9 to 16 : 2'-0- (2-methoxyethyl) ribonucleosides which are linked to one another via phosphodiester bridges). In analogy to the procedure described in general form above (FIGS. 1 to 3; chimeric oligonucleotide with a total length of 8 building blocks, of which the first 4 are called N1 to N4 and the last 4 are called N5 to n8), the nucleotides of the in a part of the desired oligonucleotides comprising nucleotides N1 to N8 produced in a first synthesis step and comprising blocks 1 to 8, while the nucleotides of the part of the desired oligonucleotides comprising blocks 9 to 16 produced in the second synthesis step would be referred to analogously as n10 to n16.

The supply unit comprises, among other things, 8 bottles with nucleoside building blocks, 4 bottles each containing one of the four deoxyribonucleotides (corresponds to chain building blocks of a first basic type series), and the 4 remaining bottles each to one of the four 2'-0- (2-methoxyethyl) -ribonucleosides (corresponding to Chain components of a second basic type series) included. In the following, the corresponding bottles with the building blocks of the first basic type series are activated in the synthesis of the 1st to 8th building blocks of the oligonucleotides, and the corresponding bottles with the building blocks of the second basic type series are activated for the synthesis of the 9th to 16th building blocks.

(i) Synthesis of the 1st to 8th building blocks of an arrangement of oligonucleotides on the PP film (DNA building blocks linked via phosphorothioate bridges)

The synthesis of the DNA phosphorothioate portion of the oligonucleotides is accomplished by programming the DNA synthesizer to use one of the four possible reaction cells, for example cell 32, to provide the desired arrangement of oligonucleotides - 24 -

produce. The three remaining reaction cells, for example 33, 34 and 35, are freely available in order to simultaneously synthesize adjacent arrays of oligonucleotides on the same PP film which correspond to other target sequences. Commercially available phosphoramidites are used as nucleotide building blocks (Cruachem, A: 208120-21; G: 208190-2; C: 208130-21; T: 208100-21). The synthesis machine dilutes 2 g of the phosphoramidites with 50 ml of acetonitrile (MWG-Biotech). In the synthesis, tetrazole (Cruachem. 17112044) as used by the manufacturer is used; Trichloroacetic acid in 1, 1, 1-trichloroethane (2.5% w / v) is used for detritylation; Phenylacetyl disulfide (prepared according to page 329 of the article by HCPF Roden et al., Recl. Trav. Chim. Pays-Bas 110 (1991), pages 325-331) is used as a 0.5 M solution in a mixture of acetonitrile / 3 -Picolin (4: 1) used for oxidation. The first base (from the 3 'end) is coupled to the PP film at the start position, and subsequent bases are added by the reaction cell moving vertically downwards in accordance with the appropriately programmed control unit.

An arrangement of oligonucleotides is generated which is based on the following sequence:

5'-GGC CTG AAG GTG CTG GAC AGT GTC TGA AAG TAG GGC ACT AGC CAA GTT GCA GCA GAA GGC-3 '(SEQ ID NO 1).

In the form shown, this sequence is complementary to the sequence of the target nucleic acid. This sequence is programmed into the synthesizer, the synthesis taking place from 3 'to 5'.

The synthesis protocol reproduced below for coupling a nucleotide building block to the film or to a nucleotide chain (ie a synthesis cycle) is a protocol of the DNA synthesizer ABI 394/4, which is based on the specific circumstances of the production of an arrangement of oligonucleotides has been adapted on a PP film. The protocol is reproduced in English, since the machine must be programmed in English: -25-

Step function duration

1. begin

2. 18 to waste 3s

3. 18 to column 8s

4. reverse f flush 6s

5. block f lush 3s

6. phos prep 4s

7. column 1 on

8. block vent. 2s

9. B + tet to column 4s

10. column 1 off

1 1. column 2 on

12. 18 to waste 4s

13. block flush 3s

14. block vent 2s

15. B + tet to column 4s

16. column 2 off

17. column 3 on

18. 18 to waste 4s

19. block flush 3s

20. block vent 2s

21. B + tet to column 4s

22. column 3 off

23. column 4 on

24. 18 to waste 4s

25. block flush 3s

26. block vent 2s

27. B + tet to column 4s

28. column 4 off

29. wait 20 s

30. 18 to waste 3s

31. block flush 3s

32. 18 to column 8s - 26 -

33. reverse flush 4 s

34. 10 to column 6 s

35. wait 15 s

36. 10 to column 1 s

37. wait 10 s

38. start detrityi

39. 18 to waste 3 s

40. 18 to column 6 s

41. reverse flush 6 s

42. 18 to column 6 s

43. reverse flush 6 s

44. 14 to waste 3 s

45. 14 to column 9 s

46. wait 15 s

47. 18 to column 4 s

48. reverse flush 4 s

49. 18 to column 4 s

50. reverse flush 15 s

51. advance fraction collector

52. wait 2 s

53 end

Bottle 18 contains acteonitrile, bottle 14 the detritylation solution, bottle 10 the phenylacetyl disulfide reagent and bottle B the respective phosphoramidite solution.

To couple the other building blocks, the described protocol is repeated correspondingly often until the desired arrangement of the first part of chimeric oligonucleotides has been completed, the first part, based on a single oligonucleotide, each comprising a maximum of the first 8 building blocks, and the arrangement as a whole includes the sequence given above.

(ii) Synthesis of the 9th to 16th building blocks of an arrangement of oligonucleotides on the PP film (2'-0- (2-methoxyethyl) ribonucleosides linked via phosphodiester bridges) - 27 -

The reaction cell is reset to the original starting position. The DNA synthesizer is programmed so that 2'-0- (2-methoxyethyl) ribonucleoside phosphoramidite building blocks (2g) are used for the 9th to 16th building blocks, which according to P. Martin, Helv. Chim. Acta 78 (1995), pp. 486-504 and dissolved in 50 ml of acetonitrile. The tetrazole (Cruachem. No. 17112044) used in the further course of the synthesis is used as obtained from the manufacturer. Trichloroacetic acid in 1, 1, 1 trichloroethane (2.5% w / v) is used for demetilation. A mixture of iodine / lutidine water / acetonitrile (0.65 g (Fluka) / 50 ml (Fluka) / 2.5 ml / 450 ml) is used for the oxidation. The synthesis protocol is modified according to the use of the modified phosphoramidite building blocks. The first base (3 'end) in each sequence is coupled to the PP film at the starting position of the reaction cell. The starting position corresponds to the starting position at the beginning of the first synthesis run (5mm below the top edge of the PP film). Subsequent bases are added while the reaction cell is moved vertically along the carrier film (displacement length 5 mm). The synthesis sequence is based on the following base sequence:

5'-ccc acc gag gcc tga agg tgc tgg aca gtg tct gaa agt agg gca cta gcc aag ttg cag-3 '(SEQ ID NO 2).

This sequence is programmed into the synthesizer; the synthesis again takes place in the 3'-5 'direction. The first base "g" at the 3 'end of SEQ ID No 2 corresponds to the 9th base "G" counted from the 3' end of SEQ ID NO 1, since the maximum length of the oligonucleotides synthesized in the 1st synthesis step is 8 chain building blocks .

Synthesis protocol of the synthesis machine ABI 394/4 (modified with regard to the production of the oligonucleotide arrangement ("array"); the "advance fraction collector" signal is used to shift the reaction cell). The protocol is reproduced in English, since the machine must be programmed in English:

Step function duration

1. begin

2. 18 to waste 3 s

3. 18 to column 8 s -28-

4. reverse flush 6s

5. block flush 3s

6. phos prep 4s

7. column 1 on

8. block vent. 2s

9. B + tet to column 4s

10. column 1 off

11. column 2 on

12. 18 to waste 4s

13. block flush 3s

14. block vent 2s

15. B + tet to column 4s

16. column 2 off

17. column 3 on

18. 18 to waste 4s

19. block flush 3s

20. block vent 2s

21. B + tet to column 4s

22. column 3 off

23. column 4 on

24. 18 to waste 4s

25. block flush 3s

26. block vent 2s

27. B + tet to column 4s

28. column 4 off

29. wait 50 s

30. 18 to waste 3s

31. block flush 3s

32. 18 to column 8s

33. reverse flush 4s

34. 15 to column 8s

35. wait 20 s

36. 15 to column 2s - 29 -

37. wait 10 s

38th start detrityl

39. 18 to waste 3 s

40. 18 to column 6 s

41. reverse flush 6 s

42. 18 to column 6 s

43. reverse flush 6 s

44. 14 to waste 3 s

45. 14 to column 9 s

46. wait 15 s

47. 18 to column 4 s

48. reverse flush 4 s

49. 18 to column 4 s

50. reverse flush 15 s

51. advance fraction collector

52. wait 2 s

53 end

Bottle 18 contains acteonitrile, bottle 14 contains the detritylation solution, bottle 15 contains the oxidation reagent (l ₂ / lutidine / H ₂ 0) and bottle B contains the respective phosphoramidite solution.

In this way, the desired scanning array of chimeric oligonucleotides on the support is finally obtained.

5. Deprotection of the oligonucleotides

The PP film is removed from the device and rinsed in isopropanol. The film is then washed in 32% ammonia at room temperature in a reaction drum (see Example 1). The chamber is rotated rapidly for 16 hours to ensure good reagent mixing. The film is removed from the chamber, rinsed with water and methanol and briefly dried under vacuum. The film with the desired arrangement of chimeric oligonucleotides can now be used in a standard hybridization assay, as mentioned above.

Claims

- 30 -PATENT CLAIMS

1. Process for producing a carrier material with synthesized in synthetic fields

Chain molecules with a predetermined sequence of chain building blocks, in particular of oligonucleotides complementary to at least parts of sections (20) of genetic information carriers, in which in one synthesis step in a sequence of synthesis cycles in one passage direction at least one reaction cell relative to the carrier material (17) with partially overlapping synthesis areas positioned and chain components of a basic type series intended for chain extension are passed through the reaction cell, so that in the case of several synthesis cycles in synthesis fields repeatedly covered by synthesis areas of a reaction cell, chain molecules with the same sequence of chain components in sections are synthesized, characterized in that after a sequence of synthesis cycles in one synthesis cycle at least one further synthesis run with chain building blocks of a different basic type compared to the previous synthesis run h it is carried out that in the or each further synthesis run in synthesis cycles of a synthesis run, a reaction cell (18, 22, 24, 26) used in this synthesis run is positioned in the same passage direction as in the previous synthesis run in such a way that the associated synthesis region (19, 23, 25, 27) in sections with a synthesis field (1 to 16) hitherto uncovered in this synthesis run, in which chain molecules (21) with a maximum chain length synthesized in the or each previous synthesis run are present, and that chain building blocks provided at this chain position of the synthesizing chain molecules of the basic type series used in this synthesis run through the reaction cell (18, 22, 24, 26).

2. The method according to claim 1, characterized in that in a first synthesis cycle of the or each further synthesis run, the or each reaction cell used in this synthesis run (18, 22, 24, 26) is positioned so that a single synthesis field (1 to 16) with chain molecules (21) of a maximum chain length synthesized in the or each previous synthesis step is overlaid by their synthesis region (19, 23, 25, 27), and that in each further synthesis cycle a further adjacent synthesis field (3 to 16) with one in the or everyone - 31 -

The synthesis step (19, 23, 25, 27) is superimposed for the first time.

3. The method according to claim 1 or 2, characterized in that in each

Synthesis pass the or each reaction cell (18, 22, 24, 26) is displaced by the same displacement path in each synthesis cycle, so that the synthesis fields (1 to 16) are of the same size.

4. The method according to claim 3, characterized in that at least two

The synthesis areas (23, 25, 27) of the reaction cells (22, 24, 26) used in each case are of different sizes, with each synthesis area (23, 25, 27) in one direction being a multiple of the dimensions of the synthesis fields (1 to 16) in corresponds to this direction.

5. Device in particular for producing a carrier material with chain molecules synthesized in synthesis fields with a predetermined sequence of chain building blocks, in particular oligonucleotides complementary to at least parts of sections (20) of genetic information carriers, in particular for carrying out a method according to one of claims 1 to 4, with a Carrier material holder to which the carrier material (17) can be applied, with a storage unit, with at least one reaction cell connected to the storage unit, by means of the chain components provided for chain extension when the or each reaction cell is placed on the carrier material (17) in sealing contact with the carrier material (17) can be passed through in a synthesis area, with a positioning unit with which the or each reaction cell in a synthesis run in a sequence of synthesis cycles each by a displacement path relative to the carrier material (17) mi t can be positioned partially in synthesis areas overlapping synthesis areas, and with a control unit for controlling the storage unit and the positioning unit, so that chain molecules with sequences of identical sequences of chain building blocks can be synthesized in several synthesis cycles in synthesis fields repeatedly covered by synthesis areas of a reaction cell, characterized in that in the Supply unit chain building blocks (N1 to N17, n4 to n15) of at least two different basic type series are stocked so that with the control unit the or each reaction cell (18, 22, 24, 26) is used in at least two synthesis runs in the same way - 32 -

Passage directions can be positioned so that during synthesis cycles of a synthesis run, the associated synthesis area (19, 23, 25, 27) is overlaid in sections with a synthesis field (1 to 16) previously uncovered in this synthesis run, in which chain molecules (21) with one in or each previous synthesis pass synthesized maximum chain length, and that the control unit can be passed through the or each reaction cell (18, 22, 24, 26) in two successive synthesis passes chain building blocks (N1 to N17, n4 to 15) of different basic type series.

6. The device according to claim 5, characterized in that the or each reaction cell (18,

22, 24, 26) has a rectangular synthesis area (19, 23, 25, 27).

7. The device according to claim 6, characterized in that the longitudinal dimensions of each

Synthesis area (19, 23, 25, 27) of a reaction cell (18, 22, 24, 26) an integer multiple of the size of the synthesis fields (1 to 16) in the longitudinal direction of the synthesis areas (19, 23,) determined by the same displacement paths in all synthesis runs. 25, 27) corresponds.

8. Device according to one of claims 5 to 7, characterized in that reaction cells used in different synthesis runs (22, 24, 26) have differently sized synthesis areas (23, 25, 27).

9. Use of a device defined in claim 5 for producing a

Carrier material comprising an arrangement of chain molecules synthesized in synthesis fields with a predetermined sequence of chain building blocks, in particular of oligonucleotides which are complementary to at least parts of sections (20) of genetic information carriers, adjacent synthesis fields comprising chain molecules with the same sequence of chain building blocks of different basic type series.

10. Use of a device according to claim 9, characterized in that the or each reaction cell (18, 22, 24, 26) has a rectangular synthesis area (19, 23, 25, 27). - 33 -

11. Use of a device according to claim 10, characterized in that the

Longitudinal dimensions of each synthesis area (19, 23, 25, 27) of a reaction cell (18, 22, 24, 26) an integer multiple of the size of the synthesis fields (1 to 16) in the longitudinal direction of the synthesis areas (19, 16), which are determined by the same displacement paths in all synthesis runs. 23, 25, 27).

12. Use of a device according to one of claims 9 to 11, characterized in that reaction cells used in different synthesis runs (22, 24, 26) have differently sized synthesis areas (23, 25, 27).

13. Carrier material, comprising an arrangement of chain molecules synthesized in synthesis fields with a predetermined sequence of chain building blocks, in particular oligonucleotides complementary to at least parts of sections (20) of genetic information carriers, adjacent synthesis fields comprising chain molecules with the same sequence of chain building blocks of different basic type series.

14. Support material, comprising an arrangement of synthesized in synthetic fields

Chain molecules with a predetermined sequence of chain building blocks, in particular of oligonucleotides complementary to at least parts of sections (20) of genetic information carriers, can be produced by a method according to one of claims 1 to 4.