EP2217364A2

EP2217364A2 - Flexible extraction method for the production of sequence-specific molecule libraries

Info

Publication number: EP2217364A2
Application number: EP08851271A
Authority: EP
Inventors: Markus Beier; Stephan Bau; Daniel Summerer; Mark Matzas; Peer STÄHLER
Original assignee: Febit Holding GmbH
Current assignee: Febit Holding GmbH
Priority date: 2007-11-23
Filing date: 2008-11-24
Publication date: 2010-08-18
Also published as: US20100256012A1; WO2009065620A2; WO2009065620A3; DE102007056398A1

Abstract

The invention relates to an extraction method for isolating target molecules from a sample with the help of a molecule library.

Description

Flexible extraction procedure for the preparation of sequence-specific

molecule libraries

s description

The invention relates to an extraction method for isolating target molecules from a sample using a library of molecules. o The selective extraction of molecules is a central and important process in many areas of biochemistry, biology and medicine. These areas include the extraction and purification of nucleic acids, proteins, sugars and other biochemical functional molecules. 5

Also in genetics biochemical methods for the extraction of certain molecules, compounds or classes of substances play an important role. Particular importance is attached to the purification of nucleic acids, usually DNA and RNA. Essential steps of recombination techniques are to isolate and purify certain nucleic acids, e.g. of plasmid DNA or genomic DNA. Also, the construction of messenger RNA (mRNA) gene libraries, which is of central importance in genetic engineering, relies on the isolation of a desired RNA population. From such libraries, genes or gene fragments are "fished" for further investigation or manipulation.

Biochemical, biological and medical analysis methods can be enormously increased in their efficiency and significance by miniaturization and parallelization. Such miniaturization relates, for example, to the analysis of genetic material by means of hybridization experiments on DNA microarrays. By developing microarrays of suitable DNA probes as receptors, whole genomes and Analyze transcriptomes. In addition to the rather widespread DNA microarrays, methods have also been miniaturized and parallelized, which serve to screen for molecules with special properties, eg, ribozymes. Other examples of receptors on microarrays are proteins and those molecules that are not found in nature, such as peptide nucleic acids (PNA). Many such assay formats are grouped under the Biochips heading. All of these assay formats and biochips are potentially useful for isolation and purification of sample material and sample preparation.

Microreaction techniques can be coupled with such microarrays to provide fast and efficient systems both in sample preparation and in the fabrication of the actual array. This also includes the use of microfluidic methods.

Numerous methods are available for the biochemical isolation of nucleic acids. Although the methods allow the isolation and possibly also the purification of RNA or DNA via their biophysical properties, they are completely unspecific with regard to the sequence of the nucleic acid strand.

Thus, from total RNA containing different sorts of RNA, mRNA molecules can be isolated relatively specifically by growing on a solid phase, e.g. Latex beads, magnetic beads, controlled pore glass beads or the matrix of a column immobilized poly-thymidine strands (poly-T strands). These poly-T strands, upon addition of total RNA, hybridize with the poly-adenine (poly-A) tail of mRNA molecules and allow separation of the unbound RNA molecules. The mRNA molecules are then isolated by appropriately changing the buffer so that hybridization in favor of single strands is abolished. The liberated mRNA molecules can then be eluted.

The information content of such an isolation matrix is comparatively small, since only two categories of target molecules can be distinguished, namely with or without poly-A tail.

In the case of isolation methods for proteins, it is usually similar. Thus, immunoglobulins are often isolated by an isolation matrix in an immobilized Protein A (from Staphylococcus aureus) column (Brown et al., Biochem. Soc. Transactions (England) 26 (1998), 249). In other versions of the method, antibodies to one or a few target molecules are bound to the isolation matrix.

Such isolation methods are commonly referred to as affinity chromatography. A disadvantage of these methods is that no parallel selective isolation of different target molecules is possible.

U.S. Patent 6,013,440 describes a method of making an affinity matrix wherein a set of different nucleic acid probes are immobilized on a solid support to thereby enrich target nucleic acids of a still unknown sequence from a sample. Among other drawbacks, it should be emphasized that the proposed method uses sheet carriers as the isolation matrix, allowing only unfavorable elution. Even with this method, the known from the prior art disadvantages can not be eliminated.

WO 03/031965 describes a method and an apparatus in which a microfluidic carrier is used as the isolation matrix, which allows a selective isolation of certain biochemical functional molecules (target molecules), in particular a sequence-specific parallel isolation of multiple species of target molecules from a mixture.

The present invention relates to a method of isolating target molecules from a sample using a library of molecules comprising free capture receptors that are specific - A -

Have interactions with the target molecules. The method preferably comprises parallel isolation of multiple target molecules using a library of several different capture receptors. The synthesis of the molecule library takes place on a solid support, in particular on a microfluidic support. Before being brought into contact with the sample, the catcher receptors forming the molecule library are detached from the carrier, in particular by chemical or / and physical methods, or copied off or copied off, in particular by enzymatic methods. The capture receptors are tagged with functional groups and then contacted with the sample so that binding of the target molecules to targeting receptor specific receptors can occur and complexes of target molecules and capture receptors are formed. The complexes formed, e.g. consist of a capture receptor and a target molecule are then separated from remaining sample components, preferably by immobilization to a solid phase, e.g. about the functional group. Subsequently, the target molecules bound to the solid phase can be eluted.

The functionality of a target molecule is defined by its ability to selectively bind to a targeting molecule-specific capture receptor, preferably through bioaffinity interactions such as hybridization, receptor-ligand binding, antigen-antibody binding, saccharide-lectin binding, etc.

The method according to the invention comprises the provision of a carrier with a molecule library, ie an array of selectively binding receptors or capture probes which are suitable for isolating the target molecules. In a preferred embodiment, an in situ synthesis of the receptors takes place on or in the carrier, preferably a microfluidic reaction carrier. This procedure allows for the use of appropriate methods for in situ synthesis a very high information content of the molecule library. In the case of nucleic acids as Target molecules can make a suitable system for the in situ synthesis of the corresponding capture probes thousands of defined sequences of a molecule library. Thus, the invention provides a way to selectively isolate hundreds to thousands or even millions of individual DNA or RNA molecules from a mixture.

A variation of the method described above is to use the sample containing several different target molecules, e.g. Nucleic acids, to contact with a carrier having reversibly bound different freely selectable capture molecules. After removal of unbound or insufficiently bound components of the sample, the complexes of target molecules and capture receptors are detached from the carrier. Subsequently, a separation of the target molecules from the capture receptors, e.g. via functional groups of capture receptors that allow biophysical separation.

The method of the invention may e.g. They are used in academic research, basic research, industrial research, quality control, pharmaceutical research, biotechnology, clinical research, clinical diagnostics, screening, patient-specific diagnostics, and clinical trials , in forensics, for genetic testing, such as parenting, in animal and plant breeding or in environmental monitoring.

An object of the invention is thus a method for isolating target molecules from a sample, comprising the steps:

(a) providing a carrier having an array of a plurality of different freely selectable capture molecules which are immobilized at respectively different positions on or in the carrier,

(b) detaching the catcher molecules from the carrier,

(c) labeling the capture molecules, wherein marking occurs before or after Detachment of the capture molecules according to step (b) can take place,

(d) contacting the labeled capture molecules with a probe containing target molecules to be isolated under conditions in which target molecules can specifically bind to the labeled capture molecules,

(e) separating material not bound to catcher molecules from the sample and

(f) isolating the target molecules.

Another object of the invention is a method for the isolation of target molecules from a sample, comprising the steps: (a) providing a support with an array of several different optional catcher molecule matrices immobilized at respectively different positions on or in the support (b) copying the capture molecule templates to obtain capture molecules in free form,

(c) marking the catcher molecules, wherein the marking can take place during or after the copying according to step (b),

(D) contacting the labeled capture molecules with a sample containing target molecules to be isolated under conditions in which

Target molecules can specifically bind to the labeled capture molecules,

(e) separating material not bound to catcher molecules from the sample; and (f) isolating the target molecules.

Yet another subject of the invention is a method for isolating target molecules from a sample comprising the steps:

(a) providing a support with an array of several different optional capture molecule templates which are labeled and immobilized at respectively different positions on or in the support,

(b) contacting the labeled immobilized capture molecules with a sample containing target molecules to be isolated under conditions in which target molecules can specifically bind to the labeled immobilized capture molecules,

(c) separating material not bound to catcher molecules from the sample,

(d) detaching the capture molecules and target molecules bound thereto from the support and

(e) isolating the target molecules.

The target molecules isolated by the method of the invention are preferably selected from biological polymers such as nucleic acids, e.g. double-stranded or single-stranded DNA molecules, e.g. genomic DNA molecules or cDNA molecules, or RNA molecules, polypeptides such as proteins, glycoproteins, lipoproteins, nucleoproteins, etc., peptides and saccharides.

Preferably, the target molecules are nucleic acids and the capture molecules are selected from hybridization probes complementary thereto. The hybridization probes can likewise be nucleic acids, in particular DNA molecules, but also nucleic acid analogs, such as peptide nucleic acids (PNA) ₁ Locked nucleic acids (LNA), etc. The hybridization probes preferably have a length corresponding to 10-100 nucleotides and do not necessarily consist of building blocks with bases, ie they may also contain, for example, abasic building blocks, linkers, spacers, etc. The hybridization probes or their complements may be attached to the support at the 3'-end, 5'-end or therebetween or at several positions.

Another preferred example of capture molecules are peptides. Furthermore, of course, low molecular weight substance libraries can be used as receptors. Preferably, the capture molecules or capture molecule matrices immobilized on the support, for example the hybridization probes, are generated on the support by in situ synthesis, for example by stepwise synthesis from synthesis building blocks, and are thus freely selectable. The building blocks for this in situ synthesis may be monomers of the relevant substance class, ie, in the case of nucleic acids, for example nucleotide building blocks. However, it may also be more complex components, such as oligonucleotides or oligopeptides of several, for example, 2, 3, or 4, monomer units.

The sample used for the method according to the invention is preferably a complex sample, i. the target molecules must be selectively isolated from a variety of similar molecular species. The sample may be a biological sample, e.g. a sample from a biological organism, e.g. from a body fluid, a sample from a cell or microorganism culture, etc. Furthermore, the sample may also be made from synthetic sources, e.g. from a synthesizer, or be a mixture of biological and synthetic material. The use of prefabricated molecular libraries in which specific target molecules may be present is also possible.

The sample may optionally be processed prior to contacting the receptors, e.g. by enzymatic reaction such as amplification, restriction cleavage, labeling, transcription, translation, fractionation, pre-purification etc. The target molecules to be isolated can thus be present in labeled or unlabeled form.

The array containing the capture molecules or capture molecule matrices in immobilized form is preferably provided on a structured support, more preferably on a support with channels, for example with closed channels. The channels are, for example, microchannels with a cross section of 10-10,000 microns. Examples of suitable carriers with channels are described in WO 00/13017 and WO 00/13018. Preferably, a carrier is used which is at least partially in the range the positions with the immobilized capture molecules or capture molecule matrices is optically transparent and / or electrically conductive. Alternatively, however, other types of supports, for example planar supports such as microscope slides, can be used, which allow the preparation of defined molecule libraries of capture molecules.

The support used for the method of the invention contains an array of several different species of immobilized capture molecules or capture molecule templates, e.g. with at least 10, preferably at least 100, particularly preferably at least 1000 different species of catcher molecules or catcher molecule matrices. The individual immobilized species differ in that they have a different structure, e.g. Nucleic acid sequence, and optionally - in the case of catcher molecules - bind different target molecules and - in the case of catcher molecule matrices - result in the write off different catcher molecules. The capture molecules or capture molecule matrices are preferably built up in situ stepwise by local or / and time-specific coupling of synthesis building blocks at the respectively predetermined positions on or in the support.

At a predetermined position, only a single capture molecule species or capture molecule template species or a mixture of several different capture molecule species or capture molecule template species may be immobilized or synthesized. For example, one may use mixtures of species specific for the same target molecule to be isolated, e.g. a set of different hybridization probes for the isolation of a single specific target nucleic acid. Alternatively, the capture molecules can also be synthesized externally and then immobilized on the support, e.g. by spotting.

In a preferred embodiment, the carrier is integrated in a device comprising a programmable light source matrix, a Detector matrix, a preferably arranged between the light source and detector matrix carrier and means for supplying fluids into the carrier and for discharging fluids from the carrier. The programmable light source or exposure matrix may be a reflection matrix, a light valve matrix, eg, an LCD matrix or a self-emitting exposure matrix. Such light matrices are disclosed in WO00 / 13017 and WO00 / 13018. If appropriate, the detector matrix can be integrated in the carrier body.

The in situ buildup of capture molecules on the support can include fluid chemical steps, photochemical steps, electrochemical steps, or combinations of two or more of these steps. An example of an electrochemical synthesis of catcher molecules on a carrier is described in DE 101 20 633.1. An example of a hybrid process comprising the combination of fluid chemical steps and photochemical steps is described in DE 101 22 357.9.

The inventive method is also suitable for the production of multiple libraries of capture molecules on a single carrier. Thus, after the replacement of catcher molecules or after copying catcher molecule matrices another synthesis Ablösungsbzw. Connect write-down cycle. In this way, very different molecular species, such as mRNA molecules or DNA sequences, can be isolated with a carrier. In addition, a switch to new target molecules with a single carrier is possible. The method is easy to automate and can be combined with further subsequent processing steps, such as an amplification reaction, e.g. using a PCR thermocycler, detection or in vitro translation.

In a first embodiment of the method according to the invention, the capture molecules which can specifically bind to the target molecules are provided directly on the support. In this embodiment of the 51

- 11 -

Invention, the capture molecules are coupled via a cleavable bond to the carrier, so that a detachment of the capture molecules from the carrier can follow, for example, by photochemical and / or fluid chemical steps. For this purpose, preferably photolabile and / or chemically labile, e.g. provided by acids, bases and / or reduction cleavable bonds between the capture molecule and the carrier. Before or after detachment from the carrier, the capture molecules are marked. This is preferably done by introducing one or more functional groups. These functional groups can be introduced, for example, during synthesis in the form of functionalized synthesis building blocks and / or after synthesis (on the support or after release) by reaction of the capture molecule with suitable functional groups.

In a further embodiment of the invention, a carrier with immobilized capture molecule matrices is provided. On this support, by transcribing the templates, preferably by enzymatic methods, the capture molecules are prepared in free form, for example by enzymatic elongation of primer molecules which bind to partial sequences of the templates, e.g. hybridize, or by other methods, such as described in WO 2005/051970, the disclosure of which is hereby made the subject of the present application. In this embodiment, the capture molecule matrices are preferably nucleic acids and the capture molecules thereof complementary nucleic acid molecules. The introduction of tag groups into the capture molecules may occur during preparation, e.g. during the enzymatic synthesis, for example by using primer molecules derivatized with functional groups and / or synthesis building blocks, or after the capture molecules have been prepared by derivatization with suitable reactive functional groups.

After elution of the capture molecules from the carrier, amplification of the molecule library consisting of the capture receptors can also be carried out. Preferably, this is a distortion-free Amplification method used in which the original composition of the molecule library is preserved. An example of this is the emulsion PCR. In this embodiment, the functional groups intended for labeling may be introduced, for example, by using labeled amplification primers or labeled nucleoside triphosphate derivatives.

In yet a further modified embodiment of the invention, the catcher molecules are provided directly on the carrier, wherein - as described above - they can be coupled to the carrier via a cleavable bond. The immobilized capture molecules are labeled as described above by introduction of one or more functional groups.

The immobilized labeled capture molecules are now brought into contact with the sample to be examined, wherein the target molecules contained in the sample bind to the immobilized capture molecules. The remaining sample components may be, e.g. by rinsing the carrier, to be separated. Subsequently, complexes of the capture molecules and the target sequences are detached from the carrier, from which the target molecules can be isolated. In this embodiment, contacting the capture molecules immobilized on the support with the sample may be under conditions of variable stringency, eg stringent or less stringent hybridization conditions, such that target molecules having weaker interactions with the immobilized capture molecules (eg nucleic acid sequences having one or more mismatches) , depending on the setting of the stringency bind to the capture molecules or not. Accordingly, the separation of undesired sample components can be controlled by setting more or less stringent washing conditions. The stringency can be controlled by temperature and / or buffer composition.

The functional groups with which the catcher molecules in the are labeled according to the invention preferably solid phase binding groups, ie groups which can be bound to a suitable solid phase, for example to another carrier as indicated above, a microtiter plate, a column or particulate solid phases such as spheres. The functional group used is preferably a first member of a bioaffinity binding pair which can react with high affinity and specificity with the complementary second partner of the binding pair. Examples of suitable pairs of binding partners are antigen or hapten / antibody, biotin / streptavidin, sugar / lectin, ligand / receptor, etc. In a preferred embodiment, the functional group is biotin and the complementary group of the solid phase is streptavidin or avidin. Of course, it is also possible to use biotin derivatives which are capable of binding with streptavidin or avidin instead of biotin.

By immobilization of functionalized capture molecules, the target molecules bound to the capture molecules can be separated from other sample components, for example components of the sample that can not be bound with the capture molecules, in the method according to the invention. The immobilization of the capture molecules on the solid phase can be location-specific, but also - when using different functionalized groups - also site-specific, e.g. on different particles or in different spatial areas of a common solid phase.

In one embodiment of the invention, several sub-fractions of the molecule library of capture molecules used to isolate the target molecules are prepared. These subfractions, which are preferably capable of binding with different species or groups of target molecules, can be prepared on a support spatially separated from other subfractions and spatially or / and temporally detached from / / copied from / to other subfractions from the support. Alternatively, sub-fractions of the molecule library can also be prepared separately on multiple supports. If necessary, different Subfractions of the molecule library can also be labeled with different functional groups. For example, a first subfraction of capture molecules can be labeled with biotin and bind to streptavidin or avidin-coated solid phase. Another subfraction may in turn be labeled with an antigen or hapten and bind to a solid phase coated with the appropriate antibody directed against the antigen or hapten.

Isolation of the target molecules preferably comprises elution of the target molecules from the solid phase under conditions in which the binding of the target molecule to the capture molecule is resolved, but the capture molecule remains bound to the solid phase.

The elution of the target molecules immobilized on the solid phase can take place in a single step. On the other hand, however, a location- or / and time-specific elution can be carried out, wherein individual target molecules or individual groups of target molecules are eluted first in a first step of the solid phase and the elution of other target molecules or

Groups of target molecules in one or more subsequent steps takes place.

In a particularly preferred embodiment of the invention, the elution is carried out by means of a temperature change, e.g. a temperature increase that leads to a denaturation of nucleic acid double strands.

The method according to the invention can also be used for the isolation of proteins and other molecules, in particular DNA-binding molecules, if capture probes suitable as catcher molecules are selected. In a further preferred embodiment, DNA-binding proteins can therefore be isolated with the aid of DNA capture probes, which can be in the form of a double or single strand. In yet another preferred embodiment, peptide capture probes may also be used for the isolation of proteins or DNA molecules be used. In yet another preferred embodiment, the capture probes used are nucleic acids having specific binding properties, for example aptamers or ribozymes.

The isolated target molecules can be used directly or indirectly for diagnostic or therapeutic purposes. Furthermore, the extracted material can be used in follow-up reactions. Thus, from extracted nucleic acids, proteins or peptides, e.g. by transfer into suitable vectors (cloning), or into suitable target cells (transformation or transfection) or by in vitro translation, in particular isolation of mRNA target molecules. Further examples of follow-up reactions to which the isolated target molecules can be subjected are, in particular with nucleic acid target molecules, sequencing reactions or microarray analyzes.

In the following, the schematic sequence of a preferred embodiment of the method is explained using the example of nucleic acids as target molecules:

1. Determination of data on the sequences of the target molecules to be extracted from the sample, e.g. Genes from a model organism under investigation;

2. Identification of suitable capture probes which are complementary to regions in the selected genes, preferably with the aid of a computer;

3. Preparation of the sample to be examined, e.g. by isolating the nucleic acid molecules from an organism;

4. providing a device for in situ synthesis of corresponding capture probes in or on a microfluidic reaction support or other suitable support;

5. feeding the detected capture probe sequence into a synthesis control unit that may be integrated with the support;

6. Synthesize the selected capture probe sequences into or on the carrier (see Figure 1A);

7. detachment of the capture probes synthesized on the microfluidic support and elution from the support (see FIG. 1B). Alternatively, the recovery of the molecule library may also be via a copy reaction of capture probe templates. This can e.g. enzymatically over one

Polymerase reaction take place. This results in the complement of the carrier-bound molecule library;

8. Amplification and labeling of capture probes, synthesis of double-stranded labeled molecules (see Figure 1C); 9. mixing the labeled capture probes and the sample (for example, fragmented genomic DNA) so that binding between target molecules and capture probes (hybridization) can occur under suitable buffer conditions and temperature (see Figures 1 D and E); 10. Immobilizing the capture probes / target molecule complexes via the capture molecule (e.g., biotin) to a specific support material (such as coated magnetic beads, coated microtiter plates, or a suitable columnar material, e.g., strepavidin-coated) (see Figure 1F); 11. removing unbound or unspecifically bound components via suitable buffer conditions and suitable temperature; 12. detaching and capturing the nucleic acid fragments to be isolated / extracted from the support via suitable reagents and suitable temperatures (see FIG. 1G); 13. Further use of the selectively extracted nucleic acids, e.g. in a sequencing, PCR, cloning, microarray experiments.

Optionally, the sample to be tested may be pretreated to selectively remove certain components from a sample containing a target molecule or a target molecule mixture. Disturbing components may be removed, eg, repetitive elements or telomeric sequences upon analysis of gene fragments; Filtering out certain genes (eg housekeeping genes) in transcriptional analyzes; Protein or Protein classes that interfere with the proteome analysis of (rare) proteins. Thus, known components can be trapped by binding to specific ("negative") catcher molecules specific for these interfering complements and only desired, eg known or unknown, target molecules eluted, resulting in a concentration of desired nucleic acids or proteins or other desired biomolecules for example, by a method comprising the steps of:

(i) providing another support with an array of several different capture molecules, each on different ones

Positions are immobilized on or in the support, and (ii) passing a sample containing target molecules to be isolated through or over the support under conditions where interfering components from a sample can specifically bind to the capture molecule immobilized on the support.

By doing so, interfering components are depleted so that subsequent analysis of the desired molecules can be performed with higher precision. Furthermore, a concentration of the desired target molecules of the sample to be examined is achieved and interfering molecules, e.g. several species in parallel, can be selectively removed from the molecule mixture.

Finally, the present invention also encompasses an embodiment relating to a method for isolating target molecules from a sample, wherein at least one process cycle in which the target molecules bind to immobilized capture molecules on a support, as in

WO 03/031965 and at least one process cycle in which the target molecules bind to free labeled capture molecules are combined.

Claims

claims

A method of isolating target molecules from a sample comprising the steps of:

(a) providing a support having an array of a plurality of different freely selectable capture molecules which are immobilized at respectively different positions on or in the support, (b) detaching the capture molecules from the support,

(c) marking the catcher molecules, wherein the marking can take place before or after the detachment of the catcher molecules according to step (b),

(e) separating material not bound to catcher molecules from the sample and

(f) isolating the target molecules.

2. A method of isolating target molecules from a sample comprising the steps of:

(a) providing a support having an array of a plurality of different optional capture molecule templates which are immobilized at respectively different positions on or in the support,

(b) transcribing the capture molecule templates to obtain capture molecules in free form,

(c) marking the catcher molecules, wherein the marking can take place during or after copying the catcher molecules according to step (b),

(d) contacting the labeled capture molecules with a sample containing target molecules to be isolated under conditions which target molecules can bind specifically to the labeled capture molecules, (e) separating material not bound to capture molecules from the sample, and s (f) isolating the target molecules.

3. The method according to claim 1 or 2, characterized in that the target molecules are selected from nucleic acids, o polypeptides, peptides and saccharides.

4. The method according to any one of claims 1 to 3, characterized in that the target molecules of nucleic acids, in particular DNA and / or RNA molecules are selected.

5. The method according to claim 4, characterized in that are used as catcher molecules hybridization probes.

6. The method according to claim 5, characterized in that one uses as hybridization probes nucleic acids or nucleic acid analogs.

7. The method according to claim 5 or 6, characterized in that the hybridization probes have a length corresponding to 10-100 nucleotides.

8. The method according to any one of the preceding claims, characterized in that a sample of biological or / and synthetic origin used.

9. The method according to any one of the preceding claims, characterized in that one uses a sample, the one or more

Pretreatment steps has been subjected.

10. The method according to any one of the preceding claims, characterized in that one uses a microfluidic carrier with closed channels, in particular with microchannels having a diameter of 10-1000 microns.

11. The method according to any one of the preceding claims, characterized in that the array on the support contains at least 10, preferably at least 100 positions with different capture molecules or capture molecule matrices.

12. The method according to any one of the preceding claims, characterized in that the capture molecules or capture molecule matrices at the individual positions contain individual sequences and / or sequence mixtures.

13. The method according to any one of the preceding claims, characterized in that the capture molecules or capture molecule matrices of the array are built in situ step by step and / or time-specific immobilization of synthesis blocks at the respective predetermined positions on or in the carrier.

14. The method according to claim 13, characterized in that one uses the carrier for one or more integrated synthesis analysis cycles.

15. The method according to claim 13 or 14, characterized in that one uses the carrier together with a programmable light source matrix and a detection matrix.

16. The method according to any one of claims 1 or 3 to 15, characterized in that the detachment of the capture molecules from the carrier by photochemical and / or fluid chemical steps takes place.

17. The method according to any one of claims 2 or 3 to 15, characterized in that the copying of the capture molecule matrices comprises an enzymatic polymerase reaction.

18. The method according to any one of the preceding claims, characterized in that the marking comprises the introduction of one or more functional groups, in particular solid phase binding groups in the capture molecules.

19. The method according to claim 18, characterized in that the separation of non-bound to catcher molecules material binding to a solid phase and the separation of not to the

Solid phase bound material from the sample comprises.

20. The method according to claim 19, characterized in that isolating the target molecules comprises eluting the target molecules from the solid phase.

21. The method according to claim 20, characterized in that the elution takes place without detachment of the capture molecules of the solid phase.

Method according to one of the preceding claims, characterized in that the isolated target molecules of a follow-up reaction, e.g. sequencing and / or microarray analysis.

23. The method according to any one of the preceding claims, characterized in that one uses the isolated target molecules directly or indirectly for diagnostic or therapeutic purposes.

24. A method for isolating target molecules from a sample, comprising at least one process cycle according to one of claims 1 to 23, wherein the sample is subjected to a pretreatment step, comprising the steps: (i) providing a further support with an array of several different capture molecules, on different ones

Positions are immobilized on or in the carrier, and

(ii) passing a sample containing target molecules to be isolated through or over the support under conditions in which interfering components from a sample are specifically immobilized to the support immobilized on the support

Capture catcher molecules can bind.

25. A method of isolating target molecules from a sample comprising the steps

(b) contacting the labeled immobilized capture molecules with a probe containing target molecules to be isolated under conditions in which target molecules can specifically bind to the labeled immobilized capture molecules,

(c) separating material not bound to catcher molecules from the sample,

(d) detaching the capture molecules and target molecules bound thereto from the support and (e) isolating the target molecules.

26. The method of claim 25, comprising the features of one or more of claims 3 to 24.