DE10117857A1 - Detecting specific interactions between molecular targets and probes, useful for determining genotypic and physiological status of cells, by attaching an markable adapter to the target - Google Patents

Abstract

Method for detecting specific interaction between target sequence (T), especially nucleic acid, and a probe on an array, in which adapter molecules (AM) are fixed to T, forming a continuous sequence, and labeling of T, independent of T, is mediated by interaction of AM with a label (L).

Description

The invention relates to a modular, sensitive system for qualitative and quantitative Detection of certain molecular targets with the help of probe arrays.

Biomedical tests are often based on determining the interaction between a molecule that is present in a known amount and position (the molecular probe) and the molecule or molecules to be detected (the molecular Target). Modern tests are usually done with a sample in parallel on some probes performed (D. J. Lockhart, E. A. Winzeler; Genomics, gene expression and DNA arrays;  Nature 2000, 405, 827-836). The probes are usually of the specified type and manner on a suitable matrix, for example described in WO 00/12575 immobilized (see e.g. US 5412087, WO 98/36827) or synthetically produced (see e.g. US 5143854, US 5658734, WO 90/03382).

Such an interaction is usually demonstrated as follows: The The probe or probes are attached to a specific matrix in a predetermined manner fixed. The targets are brought into contact with the probes in a solution and under incubated defined conditions. As a result of the incubation takes place between probe and target a specific interaction takes place. The resulting bond is much more stable than that Binding of molecules for which the probe is unspecific. Then the system washed with appropriate solutions so that the molecules that are not specifically bound are removed.

A variety of methods are used today to demonstrate the interaction between target and probe, some of which are described below:
Nature Biotechnology 1998, 16, 725-727 describes the detection of the complex of target and probe by mass spectrometry. In addition, mass-sensitive methods such as surface plasmon resonance are used (JM Brockman et al., A multistep chemical modification procedure to create DNA Arrays on Gold surfaces for the study of Protein-DNA Interactions with surface plasmon resonance imaging, J. Am. Chem. Soc. 1999, 121, 8044-8051). US Patent 5605662 discloses a method for direct electrical detection of the interaction. DE 195 43 232 describes the marking of the target with detection spheres, the presence of which is optically detected after the interaction of the target with the probes.

When using arrays with large dimensions, the target often becomes radioactive marked. The interaction is by incubation with an x-ray film or Phosphoimager detected. Furthermore, the target can be marked with a dye and its presence can be verified using a photometer.

DE 100 33 334 discloses a method in which the interaction of targets with specific probes on probe arrays can be visualized via a reaction, where an insoluble product is formed and deposited at the point of interaction. The Process implements signal amplification and is characterized by an extreme simple detector design.

Analyzes based on highly integrated probe arrays are u. a. because of high Local resolution and less effort compared to other conventional methods usually read by fluorescence optics (A. Marshall, J. Hodgson, DNA Chips: An array of possibilities, Nature Biotechnology 1998, 16, 27-31; G. Ramsay, DNA Chips: State of the Art, Nature Biotechnology 1998, 16, 40-44). For this, the targets are before, after or in Course of the specific interaction with the probe array with fluorescent markers Mistake.

Various methods are available for the effective labeling of targets with fluorophores known. On the one hand, conjugates from a fluorophore or an anchor group for fluorescent molecules and a reactive group used to target chemical To mark ways. Various are especially for labeling nucleic acids commercial products; available, such as B. BiotinChemLink from Roche Biochemicals, Mannheim, Germany; Ulyssis from Molecular Probes, Eugene, Oregon, USA; psoralen Biotin from Ambion Inc., Austin, Texas, USA. Targets are created as a product of the process, which are modified internally with substituents. Since the latter the hybridization behavior of the Influencing targets in a way that is difficult to predict is optimally stringent  Hybridization conditions such as those used for the detection of point mutations are necessarily more difficult to define.

On the other hand, enzymatic activities are used to target molecules with fluorophores to mark. For example, nucleic acid targets are copied using polymerases, where in the copy fluorescent monomers or with anchor groups such as Biotin, digoxigenin or the like coupled monomers are incorporated. In a Further development of this cDNA protocol it is possible to use quantitative array Experiments to mark two different samples with different dyes, and hybridize them simultaneously with the array (comparative hybridization), the signals generated by one sample (sample to be examined) with those of the other (Standard) are compared and each array element is thus internally calibrated.

All marking methods based on copying the target have an effect disadvantageous that the individual target sequences copied with different efficiency and in extreme cases, e.g. B. by forming stable secondary structures, for certain Targets a loss of information can occur. In addition, here too arise as with the above chemical methods internally labeled copies of the target with the disadvantages described.

This problem can be avoided by marking outside of the detecting region, for example at the terminus of the molecule to be detected. For this purpose, template-independent polymerases like the terminals Deoxynucleotidyl transferase or the poly (A) polymerase used the successive bases link with the 3 'terminus of DNA or RNA (G. Martin, W. Keller, Tailing and 3' end labeling of RNA with yeast poly (A) polymerase and various nucleotides. RNA 1998, 4, 226-230, V. Rosenmeyer, A. Laubrock, R. Seibl, Nonradioactive 3 'end labeling of RNA molecules of different length by Terminal Deoxynucleotidyl Transferase. Analytical  Biochemistry 1995, 224, 446-449, D. Figeys, A. Renborg, N.J. Dovichi, Labeling of double stranded DNA by ROX-dideoxycytosine triphosphate using Terminal Deoxynucleotidyl Transferase and separation by capillary electrophoresis. Anal. Chem. 1994, 66, 4382-4383).

A general disadvantage of the enzymatic incorporation of bases with fluorophores or Anchor molecules are modified, is that these are usually poor substrates for Represent polymerases and are therefore incorporated inefficiently. This situation is with the above-mentioned template-independent polymerases particularly pronounced, so that the desired high specific marking is not achieved in this way. A efficient labeling is only possible with selected combinations of polymerase and to achieve marked base.

Further restrictions result from the detection technology. The currently The most sensitive fluorescence readers use the narrow ones to excite the fluorophore spectral lines from laser sources, so that for sensitive detection only dyes can be used that can be excited by available lasers. A disadvantage of Fluorescence-based detection on probe arrays consists in comparison to radioactive labeling about 100 times lower sensitivity (F. Bertucci et al., Sensitivity issues in DNA-array based expression measurements and performance of nylon microarrays for small samples. Human Molecular Genetics 1999, 8 (9), 1715-1722). The detection of one Targets are therefore often only after their amplification or after signal amplification possible. U.S. Patent 4,683,202 discloses amplification by PCR for qualitative evidence. Quantitative assays on probe arrays require a procedure with linear amplification kinetics. One such method is from J. Phillips, J.H. Eberwine (Antisense RNA amplification: A linear amplification method for analyzing the mRNA population from single living cell. Methods 1996, 10 (3), 283-288) and by Wang et al., (High fidelity mRNA amplification for gene profiling., Nat. Biotechnol. 2000, 18 (4), 457- 459).  

WO 92/01813 discloses a method according to which, with linear kinetics Variety of copies of a circular single strand template can be made. In WO 2000/04193 uses this as RCA (Rolling Circle Amplification) designated mechanism for the detection of molecular interactions on probe arrays described. After specific interaction between that at the 3 'end on the array immobilized probe and the target becomes an adapter oligonucleotide with the target hybridized. This consists of a sequence complementary to the target and one Sequence that is complementary to a circular single-stranded DNA molecule, together. The two modules are linked by a 5'-5 'link. To Addition of the circular single-stranded DNA molecule, a polymerase and the The corresponding partially marked building blocks are carried out on the probes on which one Interaction has taken place, DNA synthesis according to the RCA mechanism, whereby through the installation of a large number of marked blocks, a marking and Signal amplification takes place. This method is characterized by a high sensitivity and due to the double specific hybridization between probe and target as well between target and adapter oligonucleotide by a high specificity. However, it is very complex and not suitable for a variety of different interactions to be detected in parallel on a probe array.

Methods for the detection of molecular interactions are also known, in which direct labeling of the target nucleic acid can be dispensed with (A. R. Dunn, J. A. Hassel, A novel method to map transcripts: evidence for homology between an adenovirus mRNA and discrete multiple regions of the viral genome. Cell 1977, 12, 23-36; Ranki, M. et al., Sandwich hybridization as a convenient method for the detection of nucleic acids in crude samples. Gene 1983, 21 (1-2), 77-85). In this as a sandwich The technique referred to as hybridization uses two probes bind different, non-overlapping regions of the target nucleic acid. One of the  both probes act as a so-called capture probe, with the help of which the target Nucleic acid can be bound specifically to a surface. The second, target-specific probe carries a detectable unit, so that the target nucleic acid Hybridization to this second probe is marked. Such sandwich hybridizations therefore perform two functions, namely increasing the specificity of the detection by double interaction with two specific probes and the label hybridized target nucleic acids by hybridization with the detection probe.

An overview of variations of the sandwich hybridization method is in F. Lottsspeich and H. Zorbas (ed.), Bioanalytik, Spektrum, Akad. Verl., Heidelberg, Berlin, 1998. In US 4,486,539 the use of sandwich hybridizations for Detection of microbial nucleic acids described. In US 5,288,609 a sandwich Method disclosed, which the immobilization, hybridization and detection of Allows target nucleic acid in one step. In US 5,354,657 a sandwich Hybridization method described, in which the detection on the interaction of Digoxin or digoxigenin based with specific detectable antibodies. In U.S. 4,751,177 discloses a sandwich hybridization method in which a bispecific capture probe is used, which has a specificity for hybridization with the target DNA and its other specificity for hybridization with a surface. In EP 0198662 and EP 0192168 are methods based on sandwich hybridization described in which the immobilization of the resulting complex hybridization upstream of the capture and detection probe with the target nucleic acid in solution.

US Pat. No. 5,641,630 discloses a method in which a complex of target and Capture probe is formed, which is then immobilized and then by Hybridization with the detection probe is detected. In US 5,695,926 is a Method described in which the capture probes have a length of 11 to 19 bases and are not covalently immobilized on a hydrophobic substrate. From US 4,882,269  known a method based on sandwich hybridization, the one Signal amplification guaranteed.

The above methods have the common disadvantage that for Detection of each target, both a specific capture probe and one specific detection probe is necessary. For the parallel proof of a number different targets, for example on probe arrays, is an equally large number specific detection probes necessary, whereby the above-mentioned methods only can be used up to a low degree of parallelization.

In order to solve this problem, methods have been described in the prior art in which Provide all targets of a target mixture with a uniform adapter sequence by copying the target mixture starting from a primer attached to its 5 'end contains the adapter sequence. The homogeneous and efficient introduction of However, an adapter sequence via an adapter primer is disadvantageous for those targets that are not have the same sequence modules to which the primer can bind, so that this Process has only a limited scope.

A sensitive sandwich that requires the introduction of a uniform adapter sequence Hybridization method for the sensitive detection of interactions on probe arrays based on the use of multi-labeled dendrimers (see US 5,487,973, US 5,484,904, US 5,175,270, commercially available from Genisphere Inc., Montvale, NJ, UNITED STATES). This method uses copies of the target provided with an adapter sequence incubated with a probe array, with a specific hybridization taking place. The The specific hybridization is verified in a second step Hybridization of a multi-labeled dendrimer, which is the complement of Carries adapter sequence and thus binds to the adapter sequence. Because of the restrictions when introducing the adapter sequence via an adapter primer is also the applicability  this method on targets with uniform sequence modules such as limited polyadenylated eukaryotic RNA.

So far, no methods are known with which a multitude of different ones Interactions between targets and probes on a probe array are efficient, i. H. especially with high sensitivity and specificity; homogeneous, d. H. with the same efficiency for different targets; and can be detected in parallel.

There is therefore a strong need for further methods for marking targets before, after or during the specific interaction with the probe array that the Not have disadvantages of the prior art.

The present invention is therefore based on the object of a flexible method for Provide detection of molecular targets with the help of probe arrays, an efficient, homogeneous and parallel marking of targets before, during or after ensures their interaction with the probe arrays.

Furthermore, it is an object of the present invention to provide a method which the Allows labeling of DNA and RNA alike.

These and other objects of the present invention are achieved by providing the solved in the claims characterized embodiments.

Surprisingly, it has now been found that different interactions between molecular targets, especially nucleic acids, and probes on probe arrays highly specific and highly sensitive can be detected by using adapter molecules the targets to form a continuous sequence consisting of adapter molecule and  Target, are added and the target is marked independently of the target Interaction of the adapter molecules with a detectable marker is mediated.

In particular, in the method according to the invention, adapter sequences are sent to the Targets to form a continuous sequence consisting of adapter sequence and Target added and the target is marked independently of the target sequence by hybridizing the adapter sequence with a detectable adapter sequence complementary marker sequence.

An essential feature of the method according to the invention is that the adapter sequences the target sequences are added and the labeling with the probes interacting targets regardless of the target sequence through the adapter sequence is conveyed. In contrast to the previously known methods of sandwich Hybridization, the adapter sequence is linked to the target without copies of the Targets are generated using a primer containing the adapter sequence. While the Efficiency of inserting an adapter molecule by copying the target from that When the primer is attached to the respective target sequence, the is added Adapters in the method according to the invention regardless of the sequence of the particular Targets. That is, the method according to the invention comes for the labeling of nucleic acids without copying target sequences. This offers the advantage that the efficiency and Homogeneity of the labeling of hybridized targets regardless of the target sequence is guaranteed.

The expression "attach" the adapter sequence to or "link" the adapter sequence with the target sequence means in the context of the present invention that a continuous Strand from target sequence and adapter sequence is formed. Procedures in which a Adapter oligonucleotide hybridized to the target are not within the scope of present invention.  

Within the scope of the present invention, a molecule becomes a molecular probe understood that for the detection of other molecules by a certain, characteristic Binding behavior or a certain reactivity is used.

The adapter molecule or the adapter sequence can be any molecule, in particular one be any nucleic acid molecule that, in particular, with the target to be detected a nucleic acid can be linked and is able to label the targets convey. The adapter sequence is preferably a homopolymer of 5 to 10,000 bases Length, preferably from 10-5000 bases in length, particularly preferably from 15 to 1000 bases Length. The homopolymer preferably consists of poly (A).

The marker or the marker sequence can be any molecule, especially one be any nucleic acid molecule, with the proviso that it interacts with the adapter sequence is capable of, in particular that it is one to the adapter sequence includes complementary nucleic acid sequence. The marker sequence also includes a Fluorophore, an anchor group, or other detectable entity.

Within the scope of the present invention, an arrangement of understood molecular probes on a surface, the position of each probe is determined separately.

Within the scope of the present invention, an array element is used for Deposition of a molecular probe understood certain area on a surface, where the sum of all occupied array elements is the probe array.

In the context of the present invention, a target is a molecular target Probe molecule to be understood.  

In the context of the present invention, a sample is a complex mixture understood that contains a variety of targets.

The term "marked" denotes the presence in the context of the present invention a fluorophore, an anchor group, or other detectable entity. A preferred embodiment of the method according to the invention is a nucleic acid in the target to be detected. The method according to the invention however, analogous to the detection of protein / probe interactions, Antibody / probe interactions, etc. can be used by the detected Protein or the antibody to be detected is linked to the adapter sequence.

The essential features of the method according to the invention are the addition of Adapter sequences on targets, their specific interaction with probes on probe Arrays should be detected, and the mediation of the labeling of the probes interacting targets regardless of the target sequence through the adapter sequence.

In a preferred embodiment of the present invention, the method comprises the following steps:

• a) optionally fragmentation of the targets;
• b) adding adapter sequences to the targets;
• c) specific hybridization of the targets with arranged on a probe array probes;
• d) Marking the targets by hybridizing the adapter sequence with a detectable marker sequence complementary to the adapter sequence; and
• e) detection of the signals

The fact that the labeling of the hybridized targets in the invention Procedures are achieved without a copy process, those with the on the copying of the Disadvantages associated with target-based marking methods of the prior art, especially the copying of individual target sequences with different efficiency, the Loss of information for certain target sequences through training more stable Secondary sequences and the difficulty of setting optimally more stringent Hybridization conditions when incorporating internally with substituents for labeling modified targets, overcome.

Furthermore, the inventive method has the advantage that the labeling of DNA and RNA is equally possible. Further advantages of the method according to the invention are that the method provides an interface for signal amplification and comparative Hybridizations are possible, as explained in detail below. After all it is advantageous that in the process according to the invention comparatively small amounts of labeled substances, for example in the range from 10 μM to 10 pM, preferably from 1 µM to 100 pM final concentration of detectable units can be used, which minimizes unspecific signals.

The marking of the hybridized targets is done via the attached to the targets Mediated adapter sequence. The marking is preferably carried out outside of the detecting region, d. H. the actual target sequence, at the terminus of the Target sequence and the molecule comprising the attached adapter sequence. This offers the Advantage that the efficiency of the labeling of hybridized targets only on the Sequence of the adapter that is the same for all hybridized targets and not on the sequence of the target, so that an efficient, homogeneous and parallel marking of all hybridized targets regardless of the respective target sequence is possible.

Preferred methods of marking are:

• - Sandwich hybridization with a labeled complementary to the adapter sequence Oligonucleotide.
• - Sandwich hybridization of those hybridizing in chain form with the adapter sequence labeled oligonucleotides: in chain form with the adapter sequence hybridizing labeled oligonucleotides is within the scope of the present Invention understood a set of labeled oligonucleotides, at least of which a complementarity to the adapter sequence as well as to another Has oligonucleotide. The other oligonucleotides are self-complementary or mutually complementary to each other, so that a chain during hybridization of labeled oligonucleotides bound to the adapter sequence is formed.
• - Sandwich hybridization with one complementary to the adapter sequence Oligonucleotide with a multi-labeled structure, for example one Dendrimer, described e.g. B. in WO 99/10362.

In a further preferred embodiment of the present invention, a Signal amplification by amplifying parts of the adapter sequence with simultaneous Incorporation of labeled bases, particularly preferably using an RCA mechanism Use a circular single-stranded template that is complementary to Has adapter sequence.

The fragmentation of the target has the task of 3'OH termini for the following synthesis to provide the adapter sequence if 3'OH terms are not yet available, as well as the length of the targets and thus their tendency to form the hybridization to reduce disabling secondary structures. The fragmentation can be based on chemical, physical or enzymatic way. Chemical fragmentation of RNA  takes place, for example, by incubation in a suitable, preferably basic Buffer system. Physical fragmentation can be done by ultrasound. This Methods randomly fragment the nucleic acids. The 3 'ends generated in the process must however, e.g. T. be converted into hydroxyl groups. This is done enzymatically, for example by alkaline phosphatase.

The fragmentation in the method according to the invention is preferably carried out enzymatically by using nucleases, especially by using endonucleases. Find particularly preferred when fragmenting RNA or single-stranded DNA single-strand endonucleases such as S1 nuclease and mung bean nuclease Use that are used in a concentration and under conditions that are used Development of average fragment sizes of 5-1000 bases in length, preferably 10- 500 bases in length, particularly preferably 40-200 bases in length.

Particularly preferred when fragmenting double-stranded DNA Restriction endonucleases use; most preferably single or a combination of restriction endonucleases that break the target DNA into fragments of 10-1000, preferably cut 20-700, more preferably 40-500 bases in length while doing so generate overhanging 3 'ends.

The fragmentation of the target is completely eliminated for special tasks of the inventive method, for example if the native structure of nucleic acids to be investigated by hybridization against probe arrays.

The attachment of adapter sequences to the target sequences can on one of the following described ways. The adapter sequences are preferably carried out by Template-independent polymerases to the target sequences, starting from the 3'OH Termini, synthesized. You can also link the adapter sequence with the  Target sequence a telomerase are used or an IDA reaction is carried out as described in WO 99/15698. Another alternative to linking the adapter sequence with the target sequence is a ligation with a suitable ligase, for example a T4 RNA ligase (commercially available from New England Biolabs, Schwalbach, Germany).

If RNA molecules are present as targets, a poly (A) polymerase, particularly preferably the yeast poly (A) polymerase (see US 5525497, commercial) available from USB, Cleveland, Ohio, USA). If DNA molecules as targets a terminal deoxynucleotide transferase is preferred, especially preferred terminal deoxynucleotidyl transferase from calf thymus (commercially available u. a. at New England Biolabs, Schwalbach, Germany).

In a further preferred embodiment, in the case of double-stranded DNA the fragmentation by means of restriction endonucleases and the synthesis of the adapter catalyzed by the terminal deoxynucleotidyl transferase from calf thymus at the same time in a reaction approach.

In another embodiment, the adapter sequence is specific Hybridization of the target to the probes is generated by extending the target in the hybrid.

The hybridization of the targets with the probes arranged on a probe array takes place according to one of the known standard protocols (see, inter alia, Lottspeicher and Zorbas, 1998).

If detection by hybridization of a marked sequence complementary to the adapter sequence is desired, there are several variants for the method according to the invention:
In one embodiment, hybridization takes place with a labeled oligonucleotide complementary to the adapter sequence, or with an oligonucleotide complementary to the adapter sequence, which is coupled to a structure which has been labeled many times, or with a set of labeled oligonucleotides hybridizing in chain form with the adapter sequence simultaneously with the specific hybridization of the targets to the probes on the probe array instead.

In an alternative embodiment, the hybridization takes place with a Labeled sequence complementary oligonucleotide or with a Adapter sequence complementary oligonucleotide that has a multi-labeled structure is coupled or with a set of in chain form with the adapter sequence hybridizing labeled oligonucleotides during a second, the specific Hybridization of the targets to the probes downstream on the probe array Incubation takes place in which the hybridized probe array is labeled with the adapter complementary structure in a suitable buffer under suitable conditions interacts. The two hybridization steps can be carried out by washing steps be separated.

In a further alternative embodiment, the hybridization takes place with a Labeled sequence complementary oligonucleotide or with a Adapter sequence complementary oligonucleotide that has a multi-labeled structure is coupled, or with a set of in chain form with the adapter sequence hybridizing labeled oligonucleotides in one of the specific hybridization of the Targets to the probes are placed on the probe array prior to incubation the marked structure complementary to the adapter in a suitable buffer suitable conditions binds to the adapter sequence.  

Of course, in all embodiments of the present invention it is possible to that the detection takes place via an oligonucleotide complementary to the adapter sequence is coupled to a detection sphere.

After hybridization of the adapter sequence and that complementary to the adapter sequence labeled oligonucleotide or the oligonucleotide complementary to the adapter sequence, which is coupled with a multiple marked structure, or the set of in chain form the oligonucleotides hybridizing the adapter sequence can be carried out by the hybrids covalent binding, for example via psoralen intercalation and subsequent "cross Linking ", or as described in US 4,599,303, by noncovalent binding, for example by binding intercalators.

Following the hybridization of the targets with those on a probe array Arranged probes or the marking of the hybridized targets is usually done a washing step with which non-specific and therefore weakly bound components be removed.

If the target hybridized specifically to the probes on the probe array at the inventive method by hybridization of a complementary to the adapter Structure proven, in a preferred embodiment, the adapter sequence is around many times longer than the sequence complementary to the adapter. In this case it results even when using a simply marked, complementary to the adapter Oligonucleotides a proportional signal amplification, as per hybridized target molecule bind several labeled oligonucleotides complementary to the adapter.

The term multiple is not an integer in the context of the present invention Multiples limited, but also includes all multiples from the set of positive ones rational numbers.  

In an alternative embodiment, the signal can be obtained using in Chain-shaped labeled oligonucleotides hybridizing with the adapter sequence, for example, by a mechanism as described in US 5,124,246 the average chain length.

In a preferred embodiment of the present invention, the Signal amplification by hybridization with one complementary to the adapter sequence Oligonucleotide with a multi-labeled structure, such as a dendrimer (see WO 99/10362, commercially available from Genisphere Montvale, NJ, USA). A particularly strong signal amplification is achieved in this way.

In a further preferred embodiment, the specific hybridization of the Targets to the probe on the probe array by enzymatic extension of the Adapter sequence, for example by primer extension or rolling circle amplification (P.M. Lizardi et al., Mutation detection and single molecule counting using isothermal rolling circle amplification. Nature Genetics 1998, 19, 225-232), with simultaneous installation of labeled bases detected.

The linear amplification is particularly preferably carried out using an RCA mechanism Use a circular single-stranded template that is complementary to Has adapter sequence. The sequence of the adapter is used as a primer and extended several times by copies of at least parts of the adapter sequence. This method allows the parallel implementation of all targets and hybridized on the probe array ensures a high proportionality of the signal to the amount of each array element hybridized targets because (i) all targets have the same adapter sequence; (ii) the original length of the adapter sequence does not affect the number of in the course of Amplification has incorporated labels; and (iii) in the course of the amplification  all targets have the same marked sequence regardless of the sequence of the target, namely copies of the circular single-stranded template.

The RCA reaction can start in a separate step after hybridization of the targets the specific probes of the probe array take place. Alternatively, the amplification can also hybridization.

The method according to the invention enables different marking of different nucleic acids and their subsequent comparative hybridization and Detection on a probe array. For example, a sample of chromosomal DNA an organism according to the inventive method with a poly (C) adapter sequence be provided, while the RNA from the same organism with a poly (A) - Adapter is provided. The detection is carried out either by sandwich hybridization two complementarily marked to complement each adapter Oligonucleotides (e.g. poly (G) or poly (T)) or by sandwich Hybridization of two sets of in chain form, each with one of the two Labeled oligonucleotides hybridizing adapter sequences or by sandwich Hybridization with two differently labeled to one of the two Adapter sequences complementary oligonucleotides that are labeled with a multiple Structure are coupled. Alternatively, the detection can be carried out by amplifying parts of the Adapter sequences occur with simultaneous installation of differently marked bases, for example via an RCA mechanism using two different ones circular single-stranded templates that complement one of the two Have adapter sequences.

The method according to the invention proves with regard to the possibilities of detection compared to the prior art methods as extremely flexible. That's how it is inventive method with a variety of physical, chemical or  biochemical detection method compatible. The only requirement is that the detecting structure directly coupled to an oligonucleotide or via a with the Oligonucleotide linkable anchor group can be linked. So that allows The method also uses non-fluorescence-based detection.

The detection is preferably based on the use of fluorophore-labeled Structures or building blocks. In conjunction with fluorescence detection, the method allows labeling with any oligonucleotides during or after their synthesis couplable dye, for example cy dyes (Amersham Pharmacia Biotech, Uppsala, Sweden), Alexa dyes, Texas red, fluorescein, rhodamine (Molecular Probes, Eugene, Oregon, USA), lanthanides such as samarium, yterbium and europium (EG Wallac, Freiburg, Germany).

Another advantage of the method according to the invention is that the bleaching of Fluorescent dyes is minimized, as this is only at a very late point in time inventive method are added.

In an alternative embodiment of the method according to the invention, the Detection on the use of structures or building blocks with anchor groups are coupled. Preferred anchor groups are biotin, digoxigenin and like. The anchor groups become specific in a subsequent reaction binding components, for example streptavidin conjugates or antibody Conjugates implemented, which are detectable themselves, or trigger a detectable reaction. When anchor groups are used, the anchor groups are converted into detectable ones Units after step d) of the method described above.

In a further alternative embodiment of the method according to the invention Detection methods are used which result in an adduct with a certain  Provide solubility product that results in precipitation. That is, become a marker Substrates used, which are converted into a sparingly soluble, usually colored product can be. For example, enzymes can be used in this labeling reaction that catalyze the conversion of a substrate into a poorly soluble product.

In a further alternative embodiment of the method according to the invention used as marker detection beads, in particular made of gold. The signal at the Detection can preferably be enhanced by silver deposition on the detection beads become. So the above-mentioned detection method can be done by means of poorly soluble Products based on metal markings are carried out. In doing so for example colloidal gold or defined gold clusters directly or via certain ones Mediator molecules such as streptavidin coupled to the structure complementary to the adapter, brought to the target by sandwich hybridization and by a subsequent one Reaction with less noble metals such as silver intensified. Evidence of through the specific reaction amplified areas is done using a very simple optical Structure in transmitted light (contrast due to shadowing) or incident light (contrast due to Reflection).

Another alternative embodiment in the detection is based on the use of radioactively marked structures or building blocks.

The following examples serve to illustrate the invention, without this in restrict in any way.  

example 1 Detection of hybridization of genomic DNA from Corynebacterium glutamicum against an array of 328 probes

DNA arrays are often used to determine the genotypic state of cells measure up. For this, DNA is isolated from the corresponding cells with a suitable one Method marked and hybridized against an array with complementary probes (G. Ramsay, DNA chip state of the art, Nature Biotechnol. 1998, 16, 40-44). In this example it was the method according to the invention for the detection of cellular genomic DNA Corynebacterium glutamicum used.

Preparation of the total DNA from Corynebacterium glutamicum

Total DNA from Corynebacterium glutamicum was prepared by standard methods (phenol extraction; J. Sambrook et al (1989): Molecular Cloning: A Laboratory Manual, 2 ^nd Edition, Cold Spring Harbor Laboratory Press.) Recovered. 8 µg of this DNA were cut in an appropriate buffer using the restriction endonucleases HhaI and NlaIII (enzymes and buffers from MBI Fermentas, Vilnius, Lithuania) and at the same time with the enzyme terminal transferase (MBI Fermentas, Vilnius, Lithuania) with a poly-A adapter provided at the 3 'end. The reaction was incubated at 37 ° C. for 1 hour, stopped with EDTA and, after phenol treatment and ethanol precipitation (Sambrook et al., Supra), purified by the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.

Hybridization of the poly-A adapted DNA

The DNA processed in this way was taken up in 100 μl hybridization buffer (0.25 M NaPO ₄ , 4.5% SDS, 1 mM EDTA in 1 × SSC) and denatured for 3 minutes at 95 ° C. The area of the slide covered with DNA was capped with a hybridization chamber (Hybrislip, Sigma, Deisenhofen). The slide was preheated to 50 ° C on a thermal shaker with a microtiter plate attachment (Eppendorf, Hamburg, Germany).

The denatured hybridization solution was then introduced and the Hybridization chamber closed according to manufacturer's instructions. The incubation was for 60 min continued at 50 ° C. The hybridization solution was then removed, the Hybridization chamber removed and the slides with shaking at 30 ° C for 10 min 2 × SSC + 0.2% SDS and 10 minutes each at room temperature in 2 × SSC and 0.2 × SSC (Sambrook et al., Supra) washed and blown dry with compressed air.

Evidence of hybridization

Two different variants of the applied method according to the invention. On the one hand, as described above 20 bp long oligo-dT with a Texas red dye (MWG Biotech AG, Ebersberg, Germany) again against the Poly-A adapter for 30 min at 30 ° C without previous Denaturation hybridizes and then an image under one Fluorescence microscope (Zeiss, Jena, Germany) with an appropriate filter setting performed.

On the other hand, the slide was also used after hybridization of the poly-A-adapted DNA a reaction solution (1-fold Klenow reaction buffer and 8 µl Klenow exo from MBI Fermentas, 160 µM circular DNA, 25 µM dATP, dCTP and dGTP, 12.5 µM dTTP, 25 µM Tetramethylrhodamine-4-dUTP (Amersham Pharmacia Biotech, Uppsala, Sweden)) 30 min wetted in a humid chamber at 37 ° C. This allowed rolling circle amplification starting from the poly-A adapter, whereby tetra-methylrhodamine-4-dUTP was installed and linear on-chip labeling was thus carried out. The corresponding matching circular DNA (Circle) was synthesized by the AUA company in Jena and had the sequence TTTTTTTTTTTTTTTTTTTTATATATATAT.  

Example 2 Detection of chromosomally encoded resistance genes in Staphylococcus aureus isolates by hybridizing genomic, in vitro polyadenylated DNA against a cDNA array

DNA arrays are often used to determine the genotypic state of cells determine. For this, DNA is isolated from the corresponding cells with a suitable one Method marked and hybridized against an array with complementary probes. In this The method according to the invention for the detection of resistance genes was used in example Genomic DNA from Staphylococcus aureus used.

Preparation of the total DNA from Staphylococcus aureus

Total DNA from various clinical Staphylococcus aureus isolates was analyzed using the DNeasy tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. 40 µg of this DNA were in an appropriate buffer using the restriction end nucleases HhaI and NlaIII (New England Biolabs, Schwalbach, Germany) cut and simultaneously with the enzyme terminal transferase (New England Biolabs, Schwalbach, Germany) with a poly A adapter at the 3 'end.

The approach had the following composition:
40 µg chromosomal DNA
8 µl 10 × buffer NEB4 (New England Biolabs)
8 µl 2.5 mM CoCl ₂
40 units of terminal transferase
40 units of restriction endonuclease NlaIII
80 units of restriction endonuclease HhaI
4 µl 100 mM dATP
made up to 80 µl total volume with deionized water.

The reaction was incubated at 37 ° C for 1 hour, stopped with EDTA and after Phenol treatment and ethanol precipitation (Maniatis et al., 1989) using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) cleaned according to the manufacturer's instructions. The elution was done with water. The eluate in water was then concentrated to a few µl.

Manufacture of the array

On epoxidized slides (Quantifoil, Jena, Germany) oligonucleotides that were on the 5'- Terminus were modified with an amino link (MWG, Ebersberg, Germany) by Spotting immobilized using a Microgrid II spotter (Biorobotics, Great Britain). A total of 52 oligonucleotides and one marker substance were in 3 subarrays per slide applied. The individual subarrays had a format of 11 × 10 spots. Table 1 gives one Assignment of the sample identification numbers to the positions on the subarray:

Table 1

Table 2 gives an assignment of the probe identification numbers to the sequences.

Table 2

Hybridization of the polyadenylated DNA

The polyadenylated DNA was mixed with 100 μl hybridization buffer (0.25 M NaPO ₄ , 4.5% SDS, 1 mM EDTA in 1 × SSC) and denatured for 5 minutes at 95 ° C. The surface of the slide covered with DNA was capped with a hybridization chamber (Hybriwell, Sigma, Deisenhofen, Germany). The slide was preheated to 50 ° C on a thermal shaker with a microtiter plate attachment (Eppendorf, Hamburg, Germany). The denatured hybridization solution was then filled in and the hybridization chamber was closed according to the manufacturer's instructions. Incubation was continued at 50 ° C for 60 min. The hybridization solution was then removed, the hybridization chamber was removed and the slides were shaken for 10 min at 20 ° C. in 2 × SSC + 0.2% SDS and for 10 minutes each at 20 ° C. in 2 × SSC and 0.2 × SSC (Maniatis et al., 1989) washed and dried with compressed air.

Evidence of hybridization

To demonstrate the hybridization, a was used in a new hybridization step the 20 base long oligo-dT labeled with the Cy3 dye against the poly-A adapter bound. This hybridization was carried out in the hybridization buffer for 30 min at 30 ° C without prior denaturation. The concentration of the oligo-dT 20mer was 100 nM. Following the second hybridization was the hybridization solution removed, the hybridization chamber removed and the slides with shaking for 10 min  20 ° C in 2 × SSC + 0.2% SDS and 10 minutes each at 20 ° C in 2 × SSC and 0.2 × SSC (Maniatis et al., 1989) washed and dried with compressed air.

The hybridization signals were detected with a laser scanner of the type Scan array 4000 (GSI-Lumonics, USA) ( Fig. 1).

Example 3 Hybridization of a mixture of eight in vitro and polyadenylated RNAs against a probe array

Another area of application for probe arrays is called transcription profiling denotes and consists in the detection and quantification of RNAs in an RNA Mixture by hybridization. This example shows the suitability of the invention Method for the detection of RNA hybridizations on probe arrays.

Polyadenylation of the RNA

Eight in vitro RNAs with the following sequence sections were used:

RNA 1

RNA 2

RNA 3

RNA 4

RNA 5

RNA 6

RNA 7

RNA 8

Fragmentation of the RNA

The RNAs were mixed equimolar, precipitated with ethanol and in 10 ul 1 × Mung Bean Nuclease reaction buffer (Biozym, Hessisch Oldendorf, Germany) added. The Final concentration was 2 µM for each RNA. The solution was heated to 20 ° C then 0.125 units of mung bean nuclease (Biozym, Hessisch Oldendorf, Germany) added and the mixture incubated for 5 minutes at 20 ° C. The reaction  was stopped by adding 0.2 µl of 0.5 M EDTA. The average Fragment size must be approximately 100 bases.

The mixture was analyzed using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) cleaned according to manufacturer's instructions. The elution was carried out with water.

Polyadenylierungsreaktion

The fragmented RNA was polyadenylated with yeast poly (A) polymerase in the following approach:
1 × Poly (A) polymerase reaction buffer (USB, Cleveland, USA)
1 mM ATP (MBI Fermentas, Vilnius, Lithuania)
1000 units poly (A) polymerase (USB, Cleveland, USA)
Total volume 50 µl

The reaction was incubated for 30 minutes at 30 ° C and then with 2 ul 0.5 M EDTA stopped.

Manufacture of the array

On epoxidized slides (Quantifoil, Jena, Germany) oligonucleotides that were on the 5'- Terminus were modified with an amino link (MWG, Ebersberg, Germany) by spot immobilized using a Microgrid II spotter (Biorobotics, Great Britain). A total of 69 oligonucleotides and one marker substance in 3 subarrays per slide applied. The individual subarrays had a format of 12 × 18 spots.

Table 3 gives an assignment of the sample identification numbers to the positions on the Subarray.  

Table 3

Table 4 gives an assignment of the probe identification numbers to the sequences.  

Table 4

Hybridization of the polyadenylated RNA

The polyadenylated RNA was taken up in different concentrations in the range from 40 nM to 40 pM in 100 μl hybridization buffer (0.25 M NaPO ₄ , 4.5% SDS, 1 mM EDTA in 1 × SSC) and for 5 minutes at 80 ° C. denatured. The surface of the slide covered with DNA was capped with a hybridization chamber (Hybriwell, Sigma, Deisenhofen, Germany). The slide was preheated to 50 ° C on a thermal shaker with a microtiter plate attachment (Eppendorf, Hamburg, Germany). The denatured hybridization solution was then poured in and the hybridization chamber was closed according to the manufacturer's instructions. Incubation was continued at 50 ° C for 60 min. The hybridization solution was then removed, the hybridization chamber was removed and the slides were shaken for 10 minutes at 30 ° C. in 2 × SSC + 0.2% SDS and for 10 minutes each at 20 ° C. in 2 × SSC and 0.2 × SSC (Maniatis et al., 1989) washed and dried with compressed air.

Evidence of hybridization

To demonstrate the hybridization, a was used in a new hybridization step the 20 base long oligo-dT labeled with the Cy3 dye against the poly-A adapter bound. This hybridization was carried out in the hybridization buffer mentioned above for 30 min at 30 ° C without prior denaturation. The concentration of the oligo-dT 20mer was 100 nM. Following the second hybridization was the hybridization solution removed, the hybridization chamber removed and the slides with shaking for 10 min 20 ° C in 2 × SSC + 0.2% SDS and 10 minutes each at 20 ° C in 2 × SSC and 0.2 × SSC (Maniatis et al., 1989) washed and dried with compressed air.

The hybridization signals were recorded using a Scan Array 4000 laser scanner (GSI- Lumonics, USA) was detected.

Fig. 2 shows the result of a hybridization with a final concentration of 40 nM of each RNA, Fig. 3 shows the hybridization pattern of a hybridization with a final concentration of 40 pM per RNA.

Example 4 Hybridization of a mixture of 8 in vitro fragmented and polyadenylated in vitro RNAs against a probe array, detection of hybridization by silver staining

In this example, the RNA sequences given in Example 3 and the same Array as used in example 3. The mixing, fragmentation, and polyadenylation of the RNA, the production of the arrays and the hybridization of the polyadenylated RNA was carried out exactly as described in Example 3.

Evidence of hybridization

To demonstrate the hybridization, a was used in a new hybridization step Biotin as an anchor group labeled oligo-dT of 20 bases in length against the poly-A adapter bound. This hybridization took place in the hybridization buffer for 30 min at 30 ° C without prior denaturation. The concentration of the oligo-dT 20mer was 100 nM. Following the second hybridization was the hybridization solution removed, the hybridization chamber removed and the slides with shaking for 10 min 20 ° C in 2 × SSC + 0.2% SDS and 10 min each at 20 ° C in 2 × SSC and 0.2 × SSC (Maniatis et al., 1989) washed and dried with compressed air.

A streptavidin-gold conjugate (EM. STPS, British BioCell International, Cardiff, GB) in a 1:50 dilution in 6 × SSPE + 0.005% Triton (Maniatis et al., 1989) were added to the slide and incubated for 1 S min at 30 ° C. The slide were under Shake for 10 min each in 2 × SSC + 0.2% SDS, 2 × SSC and 0.2 × SSC and wash with Compressed air dried.

The subsequent reinforcement of the gold particles now immobilized on the slide was done with the LM / EM Silver Enhancing Kit (SEKL15, British BioCell International, Cardiff, GB). According to the manufacturer, 2 drops of the initiator and the  Mixing enhancer solution and pipetting 15 µl of each onto the chip surface. By Silver plating detectable spots were made using a conventional slide scanner recorded.

pictures

Fig. 1 shows the hybridization of genomic, in vitro polyadenylated DNA against a cDNA array for the detection of chromosomally encoded resistance genes in Staphylococcus aureus isolates. The hybridization signals were detected with a laser scanner of the type Scan array 4000 (GSI-Lumonics, USA).

Fig. 2 shows the result of a hybridization of a mixture of eight in vitro fragmented and polyadenylated RNAs with a final concentration of 40 nM each against a probe array.

Fig. 3 shows the hybridization pattern of a hybridization of a mixture of eight in vitro fragmented and polyadenylated RNAs with a final concentration of 40 pM per RNA against a probe array.

Claims (30)

1. A method for detecting the specific interaction between molecular targets, in particular nucleic acids, and probes on probe arrays, characterized in that adapter molecules are added to the targets to form a continuous sequence consisting of adapter molecule and target, and the labeling of the targets is independent is mediated by the target through interaction of the adapter molecules with a detectable marker. 2. The method according to claim 1, characterized in that adapter sequences to the targets to form a consecutive sequence, consisting of adapter sequence and target, and the Labeling of the targets independently of the target sequence by hybridizing the Adapter sequence with a detectable complementary to the adapter sequence Marker sequence is conveyed. 3. The method of claim 2, comprising the following steps:

• a) fragmentation of the targets;
• b) adding adapter sequences to the targets;
• c) specific hybridization of the targets with probes arranged on a probe array;
• d) labeling of the targets by hybridizing the adapter sequence with a detectable marker sequence which is complementary to the adapter sequence; and
• e) detection of the signals.

4. The method according to claim 3, characterized in that the fragmentation is chemical, physical or enzymatic he follows.   5. The method according to claim 4, characterized in that the fragmentation by using nucleases, in particular by using endonucleases. 6. The method according to any one of the preceding claims, characterized in that the attachment of the adapter sequences to the targets via free 3'OH termini of the targets. 7. The method according to any one of the preceding claims, characterized in that the adapter sequences to the targets by means of a template independent polymerase can be added. 8. The method according to any one of the preceding claims, characterized in that the adapter sequences to the targets by means of a telomerase, an IDA reaction or a ligase. 9. The method according to any one of the preceding claims, characterized in that in the case of RNA molecules as targets the attachment of Adapter sequences to the targets with a poly (A) polymerase. 10. The method according to any one of the preceding claims, characterized in that in the case of DNA molecules as targets the attachment of Adapter sequences to the targets with a terminal deoxynucleotide transferase. 11. The method according to any one of the preceding claims, characterized in that the adapter sequences after the specific hybridization of the target to the probes in the hybrid are attached to the target.   12. The method according to any one of the preceding claims, characterized in that the labeling of the hybridized targets outside of the detecting region at the terminus of the target sequence and the adapter sequence comprehensive molecule takes place. 13. The method according to any one of the preceding claims, characterized in that the labeling of the hybridized targets by sandwich Hybridization with a labeled oligonucleotide complementary to the adapter sequence he follows. 14. The method according to any one of claims 1 to 12, characterized in that the labeling of the hybridized targets by sandwich Hybridization of labeled in chain form hybridizing with the adapter sequence Oligonucleotides. 15. The method according to any one of claims 1 to 12, characterized in that the labeling of the hybridized targets by sandwich Hybridization takes place with an oligonucleotide complementary to the adapter sequence is coupled with a structure which is marked many times, for example a dendrimer. 16. The method according to any one of claims 1 to 12, characterized in that the labeling of the hybridized targets by Amplification of parts of the adapter sequence with simultaneous incorporation of marked Bases done. 17. The method according to claim 16, characterized in that the labeling of the hybridized targets via an RCA Mechanism using a circular single-stranded template, the  Has complementarity to the adapter sequence, with the simultaneous incorporation of marked Bases done. 18. The method according to any one of the preceding claims, characterized in that the adapter sequence is many times longer than that for Adapters complementary labeled oligonucleotide. 19. The method according to any one of the preceding claims, characterized in that the labeling of the hybridized targets simultaneously with the specific hybridization of the targets to the probes takes place on the probe array. 20. The method according to any one of claims 1 to 18, characterized in that the labeling of the hybridized targets during a second, the specific hybridization of the targets to the probes on the probe array downstream incubation takes place. 21. The method according to any one of claims 1 to 18, characterized in that the labeling of the hybridized targets during a second, the specific hybridization of the targets to the probes on the probe array upstream incubation takes place. 22. The method according to any one of the preceding claims, characterized in that the hybrid of adapter sequence and labeled Oligonucleotide can be stabilized by covalent or non-covalent binding. 23. The method according to any one of the preceding claims, characterized in that fluorophores are used as markers.   24. The method according to any one of claims 1 to 22, characterized in that anchor groups are used as markers. 25. The method according to any one of claims 1 to 22, characterized in that the marking takes place by means of reactions which lead to a poorly soluble product. 26. The method according to any one of claims 1 to 22, characterized in that, as a marker, detection beads, in particular made of gold, be used. 27. The method according to claim 26, characterized in that the detection signal by silver deposition on the Detection beads is amplified. 28. The method according to any one of claims 1 to 22, characterized in that radioactive markers are used. 29. Use of the method according to one of claims 1 to 28 for Investigation of the genotypic state of cells. 30. Use of the method according to one of claims 1 to 28 for Investigation of the physiological state of cells.