EP1565569A2 - Method for the detection of mutations - Google Patents

Abstract

A method for the detection of mutations in nucleic acids is disclosed, comprising a) a number of nucleic acid strands for analysis are brought into contact with at least two types of oligonucleotides, receptor oligos and probe oligos, under conditions which bring about hybridisation of the oligos with the DNA for analysis to give a sample complex and a reference complex in such a manner that a double-sided linkage of sample complex and reference complex is produced which is or may be immobilised, b) the linkage is then optionally immobilised, c) a tensile force is applied to the linkage until one of the complexes separates and d) whichever of the complexes has separated is determined.

Description

Procedure for the detection of mutations

Description

The invention relates to a method for the detection of mutations in a nucleic acid.

The study of variations or mutations in a genomic nucleic acid sequence is becoming increasingly important and interesting as the changes allow conclusions to be drawn that can be used for different areas.

There are different types of variations and mutations, all of which are summarized under the generic term polymorphism. A polymorphism is a variation, a genomic nucleic acid sequence that can often be found in the same place. This can involve the exchange of individual bases, a so-called point mutation, as well as small deletions, insertions and inversions, but also the reorganization of large sequence sections. In any case, the sequence is changed in one or more places compared to the wild type.

Findings about polymorphisms in a genome are helpful in various areas. In genetics, for example, polymorphisms are used as markers to determine the degree of relationship between two organisms by tracking the inheritance of the polymorphisms. The most important genetic markers include single base mutations in which one of the four nucleotides is exchanged for another and which are also called SNP (single nucleotide polymorphism).

In medicine, polymorphisms serve, on the one hand, to detect certain diseases in which genes are changed by single-base mutations, thereby triggering diseases. Another important field of application is the so-called HLA typing, which is used to compare the similarity of immune systems of two individuals based on the state of many loci on the HLA genes. HLA typing is primarily used to determine whether a graft will be compatible with transplants. Another area in which the detection of polymorphisms is important is pharmacogenomics, in which, by detecting certain polymorphisms, drug tolerance should be predictable when treating a particular patient.

For the detection of polymorphisms, in particular Einzelbasenpoiymorphismen, several methods have been developed ^0. A good overview is provided by Nollau et al. in Clinical Chemistry 43, No. 7 (1997), pages 1115-1128. Methods for the identification of new SNPs as well as methods for the detection of SNPs at known loci were presented. Known polymorphisms can be detected by hybridization, the sample usually being amplified by PCR, by sequencing, by primer extension, or by an oligonucleotide ligation assay. In all of these processes, complex steps are usually necessary, for example electrophoretic separations, which take a long time.

All these methods have in common that a homoduplex (both strands have the same origin) is cleaved and at least one of the strands hybridizes with a "foreign" strand (a probe) and forms a heteroduplex (strands of different origins). The heteroduplex can then open different ways can be used for detection.

One can differentiate screening methods which serve to identify unknown SNPs from other methods which are suitable for the detection of polymorphisms at known loci. DNA sequencing, on the other hand, can be used universally, but is very expensive for routine diagnostic tests.

The pure screening methods can either be traced back to the principle of the different migration behavior of wild-type or mutant heteroduplexes in electrophoresis gels, or to the enzymatic digestion of heteroduplexes.

In the pure detection methods, too, a heteroduplex is formed from sample DNA and an oligonucleotide probe. The discrimination between wild type and mutant can then be achieved either by using stringent hybridization conditions or by enzymatic reactions.

In the case of discrimination by stringent hybridization, oligonucleotides are used which hybridize to the wild type under perfect buffer and temperature conditions for this sequence (perfect match) but not to the mutant (mismatch). The problem with this procedure is that when such tests are carried out in parallel in one approach, no conditions have to be set which are equally stringent for all analytes.

Discrimination by enzyme occurs either through DNA polymerase or DNA ligase. DNA polymerase is used in PCR with sequence-specific primers (SSP) and in "mini-sequencing". The principle of the SSP is based on the fact that a PCR primer, which is a strand of a heteroduplex, only then through the polymerase reaction if it is a perfect match, the polymerase reaction does not take place in the event of a mismatch, which is why no PCR product can be detected.

Mini-sequencing is a modification of SSP-PCR. In this case a heteroduplex is formed which ends at the 3 'end exactly one nucleotide in front of the SNP locus. This primer can be extended by the DNA polymerase by incorporating dideoxynucleotide triphosphates (ddATP, ddTTP, ddGTP and ddCTP), which leads to a strand termination immediately after the incorporation of one of these bases. An individual color marking of the four dideoxynucleotides can now be used to determine which base has been incorporated or which of the corresponding complementary bases is present on the sample molecule.

A similar strategy is used in the so-called oligonucleotide ligation assay (OLA). Two oligos are tailored in such a way that they hybridize directly next to each other at the SNP site with the sample molecule. The 5 'end of the first oligo is provided with a phosphate and the 3' end of the other oligo Wundt. If there is a perfect match, the oligos are now connected by the DNA ligase. This reaction does not occur in the event of a mismatch. The identity of the base at the SNP position can finally be deduced from the detection of the ligation product.

The object of the present invention was to provide a method with which mutations, in particular polymorphisms, can be detected without the need for complex separation steps, such as electrophoresis. Furthermore, it was an object of the invention to provide a method which offers the possibility of parallelizing tests.

This object is achieved with a method for the detection of mutations in a ^' nucleic acid, which comprises that a) a plurality of strands of nucleic acid to be analyzed are brought into contact with at least two types of oligonucleotides, receptor oligos and probe oligos, under such conditions that the Oligos hybridize with the DNA to be analyzed to form a sample complex and a reference complex, so that there is a chain of sample complex and reference complex immobilized or immobilizable on both sides, b) the chain is subsequently immobilized if necessary, c) a tensile force is applied to the chain until it pulls one of the complexes is separated and d) it is determined which of the complexes has been separated.

According to one embodiment of the method according to the invention, to detect mutations in analyte nucleic acids, the analyte nucleic acid is brought into contact with two types of oligonucleotides, receptor oligos and probe oligos, under conditions such that the oligos hybridize with the analyte nucleic acid, the oligonucleotides being immobilized or immobilizable, and where appropriate, the oligonucleotides are immobilized after the hybridization, so that a mutually immobilized or immobilizable chain of receptor oligo - analyte nucleic acid - probe oligo is present; a tensile force is applied to the linkage so that one of the bonds is released and it is determined which of the bonds has been released, the receptor oligonucleotides having a sequence which is essentially complementary to a sequence section of the analyte nucleic acid in which a mutation or Variation is suspected.

The principle of the differential force test is used for the method of the present invention. The general principle of the differential force test is described in PCT / EP01 / 09206. It has been found that the use of the differential force test allows the detection of polymorphisms reliably and to a large extent in parallel and in a miniaturized manner, whereby point mutations (SNP) as well as other changes in the sample sequence can be detected, provided that they lead to a change in the separating force of the duplex formed when hybridizing with the strand complementary to the Wiid type.

To summarize briefly, a nucleic acid molecule in a sample, which is also referred to below as analyte nucleic acid, is to be examined for the presence of mutations or variations. A sample complex is formed from a first and a second binding partner. One of the two binding partners - the analyte nucleic acid - contains a defined sequence section (sample sequence) of an analyte molecule. The other of the two binding partners contains a defined sequence section (receptor sequence) of a receptor oligo, which is essentially complementary to the section of the analyte sequence of interest. The hybridization of the first binding partner with the second binding partner leads to the formation of a sample complex.

A third and a fourth binding partner together form a reference complex. Sample complex and reference complex are linked together in series. Two of the participating binding partners are immobilized or immobilizable so that a tensile force can be exerted after the reaction to the chain formed. A tensile force is applied to the ends of the chain until one of the two complexes is separated, namely either the first from the second binding partner, ie the sample complex, or the third from the fourth binding partner, ie the reference complex. Thus, either the sample complex is dissolved if its separating force under the test conditions is less than that of the reference complex or the reference complex is dissolved if its separating force is less than that of the sample complex under the test conditions.

If this experiment is carried out very often, preferably in the form of many concatenations on one spot and simultaneously, and the number of separated sample _{complexes is} summed to n _Pro and the number of divided reference _complexes to n _Ref- , and these are compared, then obtained the quotient Q ^* , which correlates with the ratio of the mean separation forces (F ^* ) of the complexes: Q ^* = n _Ref - / n _Pro correlates with F ^* _Pra / F ^* _Ref

If one starts from a certain sequence, which is referred to as wild type (WT), all changes in this sequence represent a mutation (M) Procedures are demonstrated. In this case, a comparative experiment shows:

F ^* _Ref / F ^* _P rα (WT) ≠ F ^* _Ref / F ^* _P ro (M) or Q ^* (WT) ≠ Q ^* (M)

Since mutations in the sample sequence generally lead to a reduction in F ^* _Pro , the following also usually applies:

F ^* _Ref / F ^* _P ro (wild type) <F ^* _Ref / F ^* _Pra (mutant) or Q ^* (WT)> Q ^* (M)

The discrimination of mutations compared to the wild type by a force comparison is based on the fact that the ratio F ^* _Re f / F ^* _{Pra also} changes due to the change in F ^* _Pr o. The reference complex is usually also a nucleic acid duplex, the fourth binding partner corresponding to the probe sequence and being called probe oligo. Binding partner 3 is essentially complementary to or corresponds to the reference sequence and is preferably part of the sample molecule to be analyzed.

The method according to the invention is described for determining a mutation, but it can also be used for determining variations in a nucleic acid. Therefore, when the term "mutations" is used below, variations should also be included.

According to the invention, an analyte nucleic acid, on which mutations are to be detected, is brought into contact with different oligonucleotides in such a way that at least two nucleic acid duplexes are formed which are linked to one another in such a way that a chain is formed on the two ends of which force can be exerted. Furthermore, this linkage is designed such that, when a force is exerted on this linkage, one of the two hybridized duplexes or complexes separates, depending on which duplex or complex has the lower binding force under the given conditions. In the case of nucleic acid strands which are not hybridized in the Unzip mode, this depends primarily on the number of paired bases, the type of bases, in particular the GC content, and the number of mismatches.

There are various embodiments for the method according to the invention, which are explained in more detail below and are briefly described below. The essence of the invention is that the binding force of two duplexes is compared with one another, the analyte nucleic acid being involved in at least one of the two duplexes. By determining which of the two duplexes has come loose, conclusions can be drawn as to which of the two duplexes had the stronger binding force. From this it can again be concluded whether the duplex formed by the analyte nucleic acid had formed mismatches.

When one speaks of "an analyte nucleic acid", this naturally only ever means the description of one representative from a large number, so that when it is carried out tion of the method according to the invention normally always a large number of nucleic acids react. For the sake of simplicity, the description refers only to a selected representative.

In order to be able to exert a force on the chain formed by hybridization of the individual partners, the chain must be immobilized on both sides on a carrier. Therefore strands are used for the duplexes, which are either already immobilized or immobilizable. In some variants of the method according to the invention, immobilized nucleotides are already used for the hybridization, while in other embodiments the hybridizations are carried out first with mobile nucleotides and the immobilization takes place in a subsequent step. In the latter case, a binding possibility must of course be provided for the immobilization on the strands to be immobilized.

In the following, for the sake of simplicity, the term "stamp" and "base" will be used, without the intention to determine the spatial position, nature or function, since the stamp can also serve as a base and vice versa. The same statements also apply if the immobilization on beads or particles takes place instead of macroscopic surfaces.

In the simplest variants, three strands react: the analyte nucleic acid, a receptor oligonucleotide and a probe oligonucleotide. In this embodiment, receptor and probe oligonucleotides must be immobilized or immobilizable. The three components are brought into contact with one another in such a way that they can hybridize with one another. For this purpose, for example, the receptor oligo can be immobilized in a manner known per se on a surface mentioned for the sake of simplicity, for example by covalent bonding via functional groups located on the support. The immobilization should take place in such a way that a duplex is formed in the hybridization, which is not zipped open when a tensile force is applied. The pad with the bound receptor oligos is then brought into contact with the analyte nucleic acid so that hybridization can take place, a sample complex being formed. The probe oligonucleotide can then either simultaneously with the analyte nucleic acid or subsequently added or immobilized on a second surface called a stamp and then brought into contact with the nucleic acids bound on the support in such a way that hybridization occurs between probe oligo and analyte nucleic acid, a reference complex being formed. In this way, a chain can be formed, which consists of immobilized reference oligo, analyte nucleic acid and immobilized probe oligo. A tensile force can be exerted on this concatenation via the surfaces or supports on which the oligonucleotides are immobilized. When a tensile force is applied, the weaker of the two bonds breaks, ie either the bond between receptor oligo and analyte nucleic acid or between probe oligo and nucleic acid. The sequence is only described here as an example. Other sequences of chaining are also possible. It is then proven which of the two bonds has broken.

In another variant of the method according to the invention, a receptor oligonucleotide is immobilized and brought into contact with the sample which contains the nucleic acid to be analyzed, so that the analyte nucleic acid can hybridize with the receptor oligo, the sequence of the working steps being arbitrary. A stamp is prepared on which the probe oligonucleotide is immobilized. Furthermore, a reference nucleotide is hybridized with this probe oligonucleotide, which has a base sequence which is outside of the range in which mutations are suspected to a sequence of the analyte nucleic acid. Here, too, the order of the work steps is arbitrary. The number of base pairs hybridizing with one another between this reference strand and the analyte nucleic acid is far higher than that of the reference complex and sample complex to be formed and only serves to reliably connect the two complexes to be checked. This additional hybridization strand should definitely be as many

J base pairs that bind to each other have that their separating force is far above the separating force of the two complexes, so that it remains in any case. As with the previous variant, a tensile force is then subsequently exerted on the chain formed and it is then proven which of the two complexes has dissolved. In a further variant (in which the sequence of the work steps can be freely selected, as far as is meaningful), the analyte nucleic acid is immobilized on a base. The base with immobilized analyte nucleic acid is brought into contact with the receptor oligonucleotide under conditions such that hybridization can take place. A probe oligonucleotide is immobilized on a stamp and hybridized with a reference strand, as explained in the previous variant. Both the receptor oligonucleotide and the reference strand have mutually complementary bases. The base and stamp are then brought into contact with one another in such a way that the complementary bases of the reference strand and receptor oligonucleotide can hybridize with one another. The number of base pairs hybridizing with one another between this reference strand and the receptor oligonucleotide is far higher than that of the reference complex and sample complex to be formed and only serves to reliably connect the two complexes to be checked. This additional hybridization strand should in any case have so many base pairs that bind to one another that its separating force is far above the separating force of the two complexes, so that it remains in any case. After the hybridization, a tensile force is then again exerted on the linkage formed from analyte nucleic acid, receptor oligonucleotide, reference strand, probe oligonucleotide, which in turn releases the bond which has the lowest separating force.

Further variants of the method variants described are possible and can be easily found by a person skilled in the art. As stated, it is an essential feature that two duplexes can be compared with one another in terms of their binding strength, and conclusions as to the type of hybridization can be drawn from which binding is being released.

For the method according to the invention, a nucleic acid to be examined is brought into contact with two different oligonucleotides, of which one oligonucleotide has a defined sequence section which is complementary to the region of the nucleic acid to be analyzed in which the variation or mutation is suspected. This oligonucleotide is called receptor oligo. The second type of oligonucleotide, with which the nucleic acid to be analyzed is brought into contact with has a defined sequence section which is complementary to another section. This oligonucleotide is called probe oligo. The sequence of the probe oligo should not hybridize to the same section as the receptor oligo to avoid cross reactions. In addition, the single strands should not interfere with each other in the hybridization. In a preferred embodiment, however, the probe oligo can have the reverse sequence to the sequence of the receptor oligo. This has several advantages. It simplifies the production of the oligonucleotides, prevents cross reactions and creates complexes that are comparable in terms of binding power.

In order to be able to prove which bonds have broken, at least one of the nucleic acids used has a label. Two of the nucleic acids used are preferably provided with a label. The label can be inherent to the molecules or duplexes or can be introduced.

In a preferred embodiment, the nucleic acid to be analyzed is provided with a label. There are various ways of doing this. The type of labeling depends, among other things, on how the nucleic acid to be examined was obtained. In this way, the label can be bound directly to the nucleic acid to be examined. In a preferred embodiment, the labeling is introduced by generating the nucleic acid to be examined using PCR, the primer to be used for this purpose having a label, the pair of primers used for the PCR having a sequence which flanks the site, which contains the possible variation, or wherein the incorporated nucleotides have a label.

Any analytically detectable group or substance can be used as a marker for the method according to the invention. In the context of the invention, labeling is understood to mean a property by which some binding partners differ from others and which is detectable. Physically detectable parameters are preferably used. The label may be inherent to the molecule or complex, or may be introduced or bound. In particular, radioactive atoms or groups that change the optical or electrical effect properties. All reporter groups known to the person skilled in the art are suitable here. Examples are radioactive markers such as ³ H, ¹⁴ C, ³² P, ³⁵ S, fluorescent, luminescent, chromophoric groups or dyes, semiconductor particles, metals or conductive groups, but also substrates of enzymes or reporter enzymes. Fluorescent or chromophoric groups are preferably used as the label. At least one of the oligonucleotides has a label.

The label is preferably a fluorescent dye, for example fluorescein isothiocyanate (FITC), fluorescein, rhodamine, tetramethylrhodamine 5- (and-6) -isothiocyanate (TRITC), Texas Red, cyanine dyes (CY3 or CY5) etc. fluorescent dyes are advantageous because they can be detected in very small amounts.

In a preferred embodiment, two markings are used per chain, which are preferably different from one another. When using different fluorescent markings, those can be used which are each excited by light of the same wavelength or by radiation of different wavelengths.

The marking serves to demonstrate where the probe oligo or reference oligo is located and, where appropriate, where the receptor oligonucleotide or the analyte which can be hybridized with the analyte nucleic acid is after applying a tensile force (examples can be found in FIG. 4). This information can then be used to derive which strands have connected and separated from one another. From this it can be concluded whether the analyte nucleic acid shows variations or mutations compared to the wild type. The double marking ensures that only those chains that were linked are evaluated.

The nucleic acid to be examined can be genomic DNA, RNA, PNA or modifications. It can also be c-DNA. For the investigation, the nucleic acid can be obtained directly from cells and, if enough copies of the sequence are present in the cell, this sequence can be used directly for the test.

In a preferred embodiment, however, the nucleic acid to be examined is amplified in a first step with a PCR and the amplified product is then used for the method according to the invention. If a PCR is used for the amplification, the amplification is preferably carried out with a labeled primer in order to obtain labeled nucleic acid sequences in this way. The primers are selected in a manner known per se so that they flank the sequence of interest. The primers can each have a length of up to 30 nucleotides, preferably 15 to 20 nucleotides.

The length of the nucleic acid used depends, among other things, on whether a mutation or variation or several variations or mutations are to be detected and in which region these are then located. The method according to the invention is particularly well suited for nucleic acid sequences with up to 100 nucleotides, in particular for those sequences which have 30 to 50 nucleotides. Even shorter nucleic acid sequences can be used to detect known single base mutations.

To carry out the method according to the invention, the labeled nucleic acid to be analyzed is brought into contact with a receptor oligo and a probe oligo. The receptor oligo is a nucleic acid that is complementary to one end of the nucleic acid to be analyzed, while the probe oligo is complementary to the other end or a reference strand. The receptor oligo has a nucleotide sequence that either corresponds to the section of interest of the wild-type sequence of the nucleic acid to be examined, or has a variation or mutation. The sequence corresponding to the wild-type nucleic acid is referred to as receptor _wτ- oligos, while those receptor oligos which contain variations and mutations are referred to as receptor _M- oligos. The mutations and variations can be the exchange of one or more bases, preferably a base, or the change of one or more bases, which leads to a change in the binding behavior. Any conceivable change in the chemical structure of the nucleotide is understood as a mutation or variation, which causes a separation force difference in a force comparison compared to an original state.

The probe oligo is used to form a reference complex with another strand, which can be the analyte nucleic acid or a reference strand. The reference complex should be as similar as possible to the test complex in order to enable the differentiation of small separation force differences between two test complexes.

A nucleotide sequence which is complementary to a part of the nucleic acid to be analyzed and in which no mutation or variation is suspected is preferably used as the probe oligo. In addition, the sequence of the probe oligo is preferably selected such that a perfect match occurs here when hybridizing with the reference strand or the reference sequence. In particular embodiments, it can also be preferred to provide a predetermined number of mismatches if, for example, more than one mismatch is suspected or is to be detected in the nucleic acid to be analyzed.

The length of the hybridizing sequence of the probe oligo is preferably selected so that the force necessary to separate the reference complex is very close to the force necessary to separate the sample complex. If a sequence is selected for the reference complex that has the same GC ratio as the sample complex, the number of base pairs is preferably in the same range and preferably differs only by up to 2 base pairs. If the GC ratio differs between the two complexes, the length of the sequence to be hybridized can be selected so that the separation force approaches that of the sample complex. The insertion of one or more mismatches can also be used to set the optimal reference complex. In one embodiment, the nucleic acid to be analyzed is contacted with receptor oligos and probe oligos under conditions such that hybridization can take place. The probe oligo hybridizes with one end of the DNA to be analyzed to form a perfect match, while the other end of the DNA to be examined hybridizes with receptor oligos, resulting in a perfect match or a mismatch, depending on whether the sequence of the receptor corresponds to corresponds to investigating DNA or not.

Forms by bringing them into contact with the two types of oligos. in the method according to the invention in the embodiment described a concatenation of receptor oligo-analyte nucleic acid-probe oligo, as shown for example in FIG. 4. The chain must be or must be immobilized for further investigation of the bonds that the analyte nucleic acid has entered into. According to the invention, the oligonucleotides or the analyte are therefore either already used in immobilized form or else immobilizable oligonucleotides or analytes are used which are immobilized after the hybridization.

Immobilization of the oligonucleotides can be carried out in a manner known per se by binding the nucleotides to a support or a surface via spacers or bridge molecules. In any case, the support or the surface must be designed so that a tensile force can be applied. These can be macroscopic surfaces, but also other solid bodies, such as beads, or molecules. The immobilization takes place particularly preferably in that the receptor oligonucleotides are or are immobilized on a first surface, while the probe oligos are immobilized or are immobilized on a second surface.

If surfaces are used for the immobilization, the material from which the surfaces are made can be the same or different and one or both surfaces can be coated in a suitable manner. Preferably, either one surface is made of a rigid material and the other is made of an elastic material, or both surfaces are made of elastic material, so that the respective surface sections of the first and second surfaces are accurate when brought into contact can adapt to each other and thus an optimal contact of the individual nucleic acid ranks and possibly binding partners is possible. The surfaces can be formed, for example, from glass, silicone, in particular polydimethylsiloxane (PDMS), nylon, polystyrene or other plastics. At least one of the two surfaces is preferably produced from an elastic material, preferably an elastic plastic. At least one surface is particularly preferably formed from a siloxane, in particular polydimethylsiloxane. Various other flexible materials or mixtures thereof are possible. Another possible material is polyacrylamide gel, the elastic properties of which can be adapted to experimental requirements through the molecular weight and the degree of crosslinking. The surface can be constructed from a single material, a mixture of materials or also a system of elements from one or different materials.

The immobilization of the oligonucleotides or nucleic acids can be carried out in a manner known per se, both covalent and non-covaient bonds and bonds directly to the surface or via bridging molecules can be considered. Immobilization via the partners of a specifically binding couple is also possible. The immobilization should take place in such a way that a duplex is formed in the hybridization, which is not separated like a zipper when a tensile force is applied. In the latter case, one of the two partners of the specific binding pair is permanently immobilized and the other is bound to a nucleotide or the analyte nucleic acid, whereby permanent binding is understood here to mean a bond which remains essentially stable before the surfaces are brought into contact and which is also bonded of the attachment partner to its specific counterpart. A permanent bond can e.g. via functional groups which the binding partner has or which have been introduced into the molecule, to functional groups which are provided by the surface.

A preferred method is to biotinylate a partner and to connect it to the likewise biotinylated surface via a streptavidin molecule. Monoclonal antibodies can be activated chemically by oxidizing certain groups of their glycosylations to aldehyde groups. These aldehyde groups can in turn Bind amino groups or hydrazide groups of a modified surface (see Solomon et al., Journal of Chromatographie, 1990, Vol. 510, 321-329). Another method which is known to the person skilled in the art is the conjugation of amino groups of the antibody with carboxy groups of a surface by using ethyl (dimethylamino) carbodiimide / N-hydroxy-succinimide.

The immobilization of the oligonucleotides can be carried out in a manner known per se via spacers or bridge molecules. The person skilled in the art knows possibilities with which nucleotides can be bound to surfaces. The immobilization should take place in such a way that the sequence complementary to the nucleic acid to be analyzed is accessible for hybridization.

In a further embodiment, oligonucleotides are used which can be immobilized and are only immobilized after the hybridization.

According to the invention, both only one type of oligonucleotide and both types of nucleotides can be immobilized.

The immobilizable nucleotides used are those which have a partner of a specific binding pair at one end, the other partner of the specific binding pair being covalently bound to a surface. When the nucleotides are brought into contact with the surface, the specific binding partners are bound, which results in immobilization. A wide variety of mutually bindable partners can be considered as specifically bindable pairs, the specificity of the bond being able to relate both to a specific partner, e.g. Antigen-related antibody or biotin-avidin / streptavidin, as well as on a group or class of compounds, e.g. Antibody protein A.

In one embodiment, antibodies and a substance with an epitope recognized by it, such as antigens, haptens or anti-idiotype antibodies, are used as the binding pair. In the present description, the term “antibody” also includes antibody fragments or antibody derivatives, as well as functional fragments of Antibodies or derivatives thereof that can recognize and bind the epitope are understood. The antibodies can be polyclonal or monoclonal antibodies, but monoclonal antibodies are preferred. Known fragments and derivatives are Fv, Fab, Fab 'or F (ab') ₂ fragments, "single-chain antibody fragments", bispecific antibodies, chimeric antibodies, humanized antibodies and fragments that contain CDRs (complementarity determining regions). contain an epitope of the sample.

The selection of the specifically binding pair is not critical. It is only important that the separation force required to separate the specifically binding pair is greater than the separation force required to separate the hybridized strands.

Various variants are possible for carrying out the method according to the invention, which can be selected depending on the intended use and the circumstances of the respective test. Some variants of the sequence and type of chaining are given below. In a first variant, the DNA to be analyzed is reacted with receptor oligonucleotide and probe oligonucleotide under conditions which permit hybridization and then immobilization by bringing the hybridized molecules into contact with two surfaces, each of which provides specifically binding partners for the two oligonucleotides brought about.

In a second embodiment, one of the two oligonucleotides, either receptor oligonucleotide or probe oligonucleotide, is immobilized on a surface, then brought into contact with the other type of oligonucleotide and the nucleic acid to be analyzed so that it binds to receptor oligonucleotide - analyte nucleic acid - probe oligonucleotide one surface, and then the second surface is brought into contact with this concatenation so that a binding partner of a specific binding pair bound to the second surface can bind to the nucleotide which has not yet been immobilized and provided with a binding partner.

In a further variant of the method according to the invention, receptor oligonucleotides are immobilized on a first surface, while probe oligonucleotides are immobilized on one second surface can be immobilized. Either the first surface or the second surface is then brought into contact with the analyte nucleic acid under conditions such that hybridization takes place and then the other surface is brought into contact with the analyte nucleic acid now bound to an oligonucleotide so that further hybridization can take place. so that a chain is formed again.

In all variants, when the linkage has formed and the immobilization of the two types of oligonucleotides has taken place, the two surfaces are separated so that one of the bonds is released.

Then it is then determined which of the bonds has largely broken, from which conclusions can then be drawn as to whether the nucleic acid to be analyzed corresponds to the wild type or has a mutation or variation. This is done by checking where the mark is located. If the label is on the surface on which the probe oligonucleotide is bound, the binding between probe oligo and analyte nucleic acid in the embodiment described above must be stronger than the bond between receptor oligo and analyte nucleic acid. Since the hybridization of probe oligonucleotide with analyte nucleic acid is usually a perfect match, this is the case if there is at least one mismatch in the hybridization between receptor oligonucleotide and the DNA to be analyzed or if a nucleotide is changed so that the binding is weaker than a "normal" nucleotide.

Significant results can be obtained if the linkages were separated simultaneously on many complexes under the same conditions. If one then relates the number of separated sample complexes and the number of separated reference complexes, one obtains a ratio which allows conclusions to be drawn about the type of hybridization, as already explained above.

By selecting the appropriate probe oligonucleotide for any desired analyte nucleic acid, a sensitive detection method for variations and Perform mutations by contacting the analyte DNA with receptor oligonucleotides, some of which correspond to the wild type and others have at least one change in sequence, so that a comparison between wild type and change is possible.

In a preferred embodiment, four different receptor oligos can be used for the detection of an SNP, for example, which differ in the base at the location suspected for the SNP, so that each base occurs once at this location. If the process according to the invention is then carried out simultaneously with the four receptor oligos and the frequency with which complexes with receptor oligos have separated is compared, it can be expected that the result for the three oligos in which a mismatch is to be expected differs from the one oligo that created a perfect match. This embodiment therefore leads to particularly meaningful results.

According to the invention, a method is thus made available with which mutations or variations of a nucleic acid sequence can be detected simply and quickly. The essence of the invention is that many hybridizations can be carried out at the same time and the separating force of the hybridized strands on many molecules can be demonstrated simultaneously.

In a further embodiment, the method becomes even more meaningful by using a second marking. In this embodiment, both the nucleic acid to be analyzed is labeled and the probe oligonucleotides. This is an embodiment in which the probe oligonucleotides can be immobilized, but are not yet immobilized in the reaction with the DNA to be detected. The probe oligonucleotides are provided with a label which differs from the label with which the DNA to be analyzed is provided. After carrying out the method according to the invention, in which the analyte nucleic acid is again brought into contact with receptor oligonucleotides and probe oligonucleotides, hybridization and then immobilization is brought about and then a tensile force is applied to the chaining on both sides in order to bind one of the To solve chaining. A surface is then analyzed to determine how much of the probe oligonucleotide has bound to the surface and how much of the DNA to be analyzed has bound. By determining the quotient of bound probe oligonucleotides to bound analyte nucleic acids, it can be demonstrated even more precisely whether a variation or mutation is present, since only the complexes are examined in which the probe oligonucleotides have actually been immobilized.

In the described embodiments, the sequence of the work steps is variable. However, it should be ensured that the unlabeled components are immobilized first and the marked ones last.

As already explained above, the method according to the invention has various advantages over the methods of the prior art. It has the advantage over the known enzymatic methods that no enzymes have to be used, which saves costs. In addition, when carrying out the process and selecting the reactants, e.g. the enzymes are not taken into account in the composition of the buffer.

In contrast to enzymatic methods, heteroduplexes for which no enzymes are available can also be used, e.g. for DNA-RNA or DNA-PNA duplexes. In contrast to SSP, many samples can be tested in parallel without a cross reaction, as is the case with many primer pairs in the same approach.

In contrast to stringent hybridization, many different sample complexes can be discriminated in one approach. The stringency is specified for each sample complex via the separating force of the reference complex. In addition, unlike stringent hybridization, no washes with temperature or salt gradients are necessary. The invention is illustrated by the figures and the following examples.

1 shows the principle of the method according to the invention, a force being exerted on a chain of receptor oligonucleotide analyte nucleic acid and probe oligonucleotide which is immobilized on both sides. The two options for separating the complex are shown.

Figure 2 shows two examples of the concatenation of a wild type receptor with analyte nucleic acid and probe oligonucleotide and a mutant receptor with analyte nucleic acid and probe oligonucleotide.

3 shows the different possibilities for coupling the components receptor oligonucleotide, analyte nucleic acid and probe oligonucleotide.

4 shows various possibilities for coupling and immobilizing the components

FIG. 5 shows the fluorescence scan of a spot which was transferred to a stamp (Example 2)

FIG. 6 shows the ratio of the fluorescence intensities for two spots transferred to the stamp. Mismatch (left) and Perfect Match (right). (Ex. 3)

FIG. 7 shows a histogram of the ratios of the fluorescence intensities for mismatch spots and perfect match spots (Ex. 3).

FIG. 8 shows the hybridization scheme of the oligonucleotides used in Example 4.

FIG. 9 shows the transfer of a PM spot and an MM spot to the stamp in the embodiment of example 4 above; below, PM and MM spots are shown on the slide after stamping; the respective histograms can also be seen. The following five variants are shown in FIG.

In variant 1, the receptor oligonucleotide is immobilized on a support. It hybridizes with the analyte nucleic acid. At its other end, the analyte nucleic acid has a specifically bindable grouping which is reacted with the corresponding binding partner. The binding partner has a binding site that can bind to a stamp. Through the coupling step, the binding partner is immobilized on the stamp, so that a chain is formed on which a tensile force can be exerted.

In variant 2, a binding partner is immobilized on a surface and reacted with the specifically binding partner that is present on an analyte nucleic acid. The analyte nucleic acid immobilized on the surface via the specific binding pair is brought into contact with a receptor oligonucleotide which has at its other end a group which can bind to a surface. Binding of the receptor oligonucleotide to the surface in turn creates a chain on which a tensile force can be exerted.

In variant 3, an analyte nucleic acid is bound via a specifically binding pair. A receptor oligonucleotide is immobilized on a second surface. Both surfaces are then brought into contact so that the complementary nucleotides of receptor oligonucleotide and analyte nucleic acid can hybridize. A force can then be exerted on the chain formed.

In variant 4, a receptor oligonucleotide is immobilized on a surface and brought into contact with an analyte nucleic acid in such a way that hybridization can take place. The analyte nucleic acid has a partner of a specific binding pair at its other end. The other partner of this specific binding pair is immobilized on a second surface. The two surfaces are then brought into contact with one another in such a way that the specifically binding pair is bound. This creates a chain again, on which a force can then be exerted. In the last variant no. 5, a receptor oligonucleotide is immobilized on a surface and is brought into contact with the analyte nucleic acid so that complementary nucleotides can hybridize with one another. A specific binding pair is immobilized on a second surface. Both the non-immobilized second partner of the specific binding pair and the analyte nucleic acid each have a binding site that can react with one another. When these two groups have reacted with each other, a chaining occurs again, on which tension can be exerted.

In a preferred embodiment of the method according to the invention, the two partners of the specifically binding pair are two complementary nucleic acid strands which form a reference complex. The nucleic acid strands are provided in such a way that they differ in a predetermined manner from the sample complex in terms of the free base pairing energy and the number of mismatches or, if appropriate, are identical in terms of the free base pairing energy and number of mismatches.

It is also possible to use another specific binding pair as a reference complex instead of the nucleic acid strands, this specific binding pair being selected in such a way that the binding force is predetermined and comes as close as possible to the binding force of the sample complex. Methods for determining the free base pairing energy or the binding force of a specific binding pair are known and are described in the literature.

The sample and reference complex are particularly preferably selected such that the free base pairing energy ΔG differs as little as possible. A reliable determination is no longer possible if the free base pairing energy of the sample complex and reference complex differs by more than 40 kcal / mol. The free base pairing energy of the sample and reference complex preferably differs by no more than 20 kcal / mol. Particularly good results are obtained if the difference between the sample and reference complex is <4 kcal / mol. Samples to determine the free base pairing energy of nucleic acid duplexes are known. One method is described, for example, in KJ Breslauer, R. Frank, H. Blöcker and LA Markie "Predicting DNA Duplexes Stability From The Base Sequence", Proc. Natl. Acad. Be. USA, vol. 83, pages 3746-3750, 1986.

The following definitions are used in describing the examples.

Receptor oligo: Oligo, which forms a sample complex with the analyte. Probe oligo: Oligo, which forms a reference complex with the analyte or the reference strand

Analyte: oligo, PCR product or other nucleic acids on which a polymorphism or a mutation is to be detected. Sample mixture: substance mixture that contains the analyte

Mismatch: Base pair in a duplex that does not form a specific base pair. Separation force: Force that occurs on a chain immediately before it is separated by an applied tensile force

Binding partner: partner of a specific binding pair wild type: original, or mutant form of the sample sequence mutation: changed or wild type of the sample sequence, which leads to a change in the separating force of the sample complex

example 1

The following embodiment is the detection of a possible single base variation (G → C) at a known base position on genomic DNA.

An amplicon of genomic DNA is produced by PCR (sample molecule corresponds to analyte nucleic acid). The pair of primers (primer each with 18 nucleotides) flanked a sequence of 30 base pairs, which identified the site with the possible base exchange contains. Primer 1, which hybridizes to the non-codogenic strand of the genomic DNA and is extended to copy the codogenic strand, is labeled with a Cy3 molecule.

Approx. 100 femtoMol of one of two different receptor oligos are bound on two separate spots of 1 mm ² each on a slide coated with covalently bound streptavidin.

The free streptavidin surface of the coating surrounding the spots is saturated with a biotinylated poly-A-oligo of 15 bp length.

The wild-type receptor (Rez _W τ) is a 28 nucleotide-long DNA oligo that is modified at the 5 'end with biotin, whereby it is bound to the streptavidin surface. Bases 1-10 serve as poly-A spacers. Bases 11-28 (all bases counted in the 5'-3 'direction) represent the receptor sequence of Rez _W τ. This section of the sequence is complementary to bases 30 to 48 of the amplified codogenic strand of the genomic DNA in the non-mutated form ,

The mutant receptor (Rez _M ) is a 28 nucleotide long oligo which is modified at the 5 'end with biotin, with which it is bound to the streptavidin surface. Bases 1-10 serve as poly-A spacers. Bases 11-28 represent the receptor sequence of Rez _M. This sequence section is complementary to bases 30 to 48 of the codogenic strand of the mutant form of genomic DNA, which differs from the non-mutated form at base 19, in which a C against a G is exchanged. Rez _M is therefore completely complementary to a mutant DNA in which a G is replaced by a C at base 39.

The sample with the Cy3-labeled sample molecules, which are a copy of the codogenic strand, is denatured by boiling and prevented from re-hybridizing by cooling on ice. The probe oligo (probe) is added to the sample mixture. This is a 28-bp DNA oligo. The probe is at its 5 'end with a Cy5 molecule and labeled at its 3 'end with a biotin. Bases 1-18 represent the probe sequence and are complementary to bases 18-1 of the sample molecule, which represent the reference sequence.

The probe-added sample is then placed in 5 x SSC buffer for hybridization on the receptor spots of the blocked slide. After an incubation of 30 min at RT, the mixture is washed with 1 x SSC and covered with 1 x SSC.

The hybridization leads to the formation of the sample complex and the reference complex, as shown in FIG. 2, the concatenation being linked to the slide via the receptor.

A microstructured PDMS stamp coated with streptavidin is now pressed onto the area of the receptor spots with the immobilized chains under 10OmM NaCl buffer. This involves connecting the biotinylated probe oligos to the stamp or coupling the linkages between the stamp and the slide. The stamp is separated from the specimen slide after 30 minutes, the linkages being separated by separating one complex at a time. It should be noted that this process is carried out simultaneously on very many (-100 fMol) chains on each spot.

The stamp is now evaluated in a fluorescence scanner with regard to the markers Cy3 and Cy5. The probe oligos and sample molecules transferred to the stamp can thus be quantified. The amount of Cy5 transferred from the spots to a specific area of the stamp is related to the amount Cy3 transferred to the same area. The quotients obtained for the two spots are compared with one another.

According to the formula Q = (n _θ p - n _Pro ) / n _Pr0- (see below) one obtains: For the spot of Rez _τ : Qwτ = (n (Cy5 τ) - n (Cy3 _W τ)) / n (Cy3 _W τ) For the spot of Rez _M : Q _M = (n (Cy5 _M ) - n (Cy3rvi)) / n (Cy3 _M ) On the basis of the quotients determined by the stamp, two cases can now be distinguished:

Case 1)

The sample molecule is in wild-type form (no exchange from G to C at base 39).

The sample complex from Rez _W τ and the sample molecule is completely paired (Perfect

Match = PM).

The sample complex consisting of Rez _M and the sample molecule has a base mismatch between G and G at base 39 (mismatch = MM).

The separation force for the sample complex from Rez _W τ is therefore greater than the separation force for the sample complex from Rez _M :

F ^* _Pra (Rezwτ)> F ^* _Pra (Rez _M )

One obtains Q _wτ > QM

With Q _W T> Q _M the wild type can be concluded

Case 2)

The sample molecule is in the mutant form (G was exchanged for C at base 39).

The sample complex consisting of Rez _T and the sample molecule has a base mismatch between C and C at base 39 (misnatch = MM).

The sample complex from Rez and the sample molecule is completely paired (Perfect Match = PM).

The separation force for the sample complex of Rez _W τ is smaller than the separation force for the sample complex of Rez _M : F ^* _Pra (Rez _W τ) <F ^* _Pra (Rez _M )

One obtains Q _wτ <Q _M

If QWT <QM, the mutant can be concluded. Example 2

SNP type on oligonucleotides

In this example, two sample sequences with lengths of 14 and 16 nucleotides were examined for a single base exchange.

The sample molecules were oligonucleotides which were built up from 5 'to 3' from the following sequence components:

A reference sequence of 16 or 14 nucleotides, an internal spacer sequence of 16 or 20 nucleotides, a sample sequence with 16 or 14 nucleotides and a terminal spacer sequence of 4 nucleotides. Sample sequence and reference sequence were reversed to each other.

The receptors consisted of an amino-C12 linker conjugated at the 5 ^' end, a spacer of 10 nucleotides without specific binding function (here 10x adenine) and a selected receptor sequence with a length of 16 or 14 nucleotides.

To match two different wild-type receptor sequences with lengths of 14 and 16 nucleotides, receptors with a base exchange were put together. In the 14mer sample sequence, the cytosine at position 7 was replaced by adenine. In the 16mer sample sequence, cytosine at position 12 was replaced by adenine.

The probe oligos consisted of a Cy5 molecule conjugated at the 5 ^' end, a spacer of 4 nucleotides with no specific binding function (4x adenine) and a sequence that was complementary to the reference sequence (16 or 14 nucleotides), as well as a further spacer ( 10x adenine) with a 3'-terminal biotin group. The structure of the chains of the 16-mer sample molecule:

PM1S

5 'NH2 -C12-AAA AAA AAA A- gtgt gggg aggc tttg

3 'Cy3-GATC- c iamca c icmcc t icmcg a iamac GTCG GATC CTAG GCTG caaa gcct ccco acac 5'

5 'Cy5-AAAA-gitmtt cigmga gigmgg tigmtg -AAAAAAAAAA-Bio

MM16

5 'HH2 -C12-AAA AAA AAA A-gtgt gggg agga tttg imim ι ι i ^χ im

3 'Cy3-GATC- caca cccc tccg aaac GTCG GATC CTAG GCTG caaa gcct cccc acac 5'

5 'Cy5-AAAA-gitmtt cigmga gigmgg tigmtg -AAAAAAAAAA-Bio

The structure of the chains of the 14mer sample molecule:

PM14

5 'NH2 -C12-AAAAAAAAAA- eggt gccg tatg ga

3 'Cy3-GATC g icmca e igmge a itmac e nt TC TCAG GATC CTAG GACT CT tc cata egge aeeg 5'

5 'Cy5 -AAAA-a ιgι g itmat g iemcg t igmgc-AAAAAAAAAA-Bio 3'

MM14 tatg ga aitmac ent TC TCAG GATC CTAG GACT CT tc cata harrow accg 5 '

5 'Cy5-AAAA-aιgι gitmat gicmcg tigmgc-AAAAAAAAAA-Bio 3'

Connection of the receptor oligonucleotides:

0.5 μl of a 10 μM oligo solution was spotted in 0.1 M NaCl on a slide coated with epoxysilane and incubated overnight in a moist chamber. Then 2 x 5 minutes in 1 x SSC-T (T is addition of 0.05% Tween-20) and 2 x 2 minutes in dist. Washed water and dried with a stream of nitrogen.

hybridization:

The spots of the wild-type and mutant receptors were incubated for 30 minutes with a mixture of Cy3-labeled sample molecule and Cy5-labeled probe oligo in a concentration of 0.5 μM in 5xSSC, 0.05% Tween-20.

The hybridization led to the formation of the sample complex and the reference complex, as shown above, the chain being linked to the slide via the receptor. Following the hybridization, the slides were 2 x 2 minutes with 5 x SSC-T, 2 x 2 minutes with 1 x SSC-T and 2 x 2 minutes with 0.2 x SSC-T, briefly watered and dried in a stream of nitrogen.

Stamp:

A micro-structured stamp was made from PDMS (polydimethylsiloxane). The structures consisted of stamp feet of approx. 100 x 100 μm, which were separated by depressions approx. 25 μm wide and 1 μm deep. For this purpose, a mixture of a 1:10 mixture of crosslinking reagent and silicone elastomer (Sylgard 184, Dow Corning) was poured onto a correspondingly structured silicon wafer after multiple degassing and incubated for 24 hours at RT. After the polymerization, the structured surface of the stamp was exposed to H ₂ O plasma at 1 mbar in a plasma furnace for 60 s.

The oxidized surface was incubated with 3% aminosilane (3-aminopropyldimethylethoxysilane; ABCR, Karlsruhe) in 10% H ₂ O and 87% ethanol for 30 min. The silanized surface was washed with ultrapure water and blown dry with nitrogen. A bifunctional PEG was attached to the amino groups of the silane, one end of which had a carboxy group activated by NHS and the other end had a biotin group. 20 μl of a solution with 2 mg / 100 μl of NHS-PEG-Biotin (Shearwater, Huntsville) were incubated under a cover glass for 1 h on a stamp with an area of 1 cm ² . It was washed with ultrapure water and blown dry with nitrogen. 0.1 mg / ml streptavidin (Sigma) in PBS was added to the now biotinylated surface and incubated for 30 min. It was washed with ultrapure water and blown dry with nitrogen.

A freshly prepared stamp and a base were pressed onto the spots with a solution of 30 mM NaCl under a pressure of 100 g / cm ² . The stamp was lifted off after 30 minutes. Slides and stamps were washed with ultrapure water and blown dry with nitrogen. Measurement:

The stamp was measured in a fluorescence scanner (Axon, Genepix 4000 B) with a spatial resolution of 5 μm with regard to the intensities of Cy3 (excitation at 532 nm) and Cy5 (excitation at 635 nm).

Evaluation:

The fluorescence scan of a spot that was transferred to the stamp is shown in FIG. 5.

The intensity distributions for Cy3 and Cy5 of the entire spot area were determined

I and shown in a histogram. 5 shows a typical histogram of a spot for one of the two markings. The histograms have two distinct maxima. The maximum at low intensity corresponds to the fluorescence background (dark grid), that at higher intensity corresponds to the actual signal (light areas).

The difference between signal and background was calculated for each spot and for each marker.

In contrast to Experiment Example 1 was not here

Q = (n (Cy5) - n (Cy3)) / n (Cy3) calculated, but the ratio V = n (Cy5) / n (Cy3). VM (mismatch) <V _PM (perfect match) or V _MM / V _PM <1 applies. The ratio V for each of the 15 measured spots is listed in Table 1. The mean values of V for 14- and 16-mer with and without mismatch were then calculated in Table 2. Finally, the mean values for Perfect Match and Mismatch (each for 14 and 16 mer) were compared.

V _M M / V _PM <1 is found for both examined sample molecules (14 and 16 mer), which clearly shows that PM and MM can be clearly distinguished here. Table 1

Table 2

Example 3

SNP type on oligonucleotides

In this example, a sample sequence of 16 nucleotides in length was examined for a single base exchange.

The sample molecule was an oligonucleotide that was built up from 5 'to 3' from the following sequence components:

A reference sequence of 16 nucleotides, an internal spacer sequence of 16 nucleotides, a sample sequence with 16 nucleotides and a terminal spacer sequence of 4 nucleotides. Sample sequence and reference sequence were reversed to each other. The receptors consisted of a biotin group conjugated at the 5 ^' end, a spacer of 10 adenines and a selected receptor sequence with a length of 16 nucleotides. Matching the wild-type receptor sequence with a length of 16 nucleotides, a mutated receptor with a base exchange was derived, in which cytosine at position 12 was replaced by adenine.

The probe oligo consisted of a Cy5 molecule conjugated at the 5 ^' end, a spacer consisting of 4 adenines and a sequence with 16 nucleotides, which is complementary to the reference sequence, and a further spacer composed of ten adenines with a 3'-terminal biotin group.

The structure of the chains of the 16-mer sample molecule:

PM16

5 'Bio-AAAAAAAAAA- gtgt gggg aggc tttg

3 'Cy3-GATC- c iamca c iemec t iemeg a iamac GTCG GATC CTAG GCTG caaa gect ecec acac 5'

5 'Cy5-AAAA-gitmtt cigmga gigmgg tigmtg -AAAAAAAAAA-Bio

MM16

5 'Bio-AAAAAAAAAA- gtgt gggg agga tttg

3 'Cy3-GATC- ciamca cicmcc ticnc * g aiamac GTCG GATC CTAG GCTG caaa gcct ccce aeac 5'

5 'Cy5-AAAA-gitmtt cigmga gigmgg tigmtg -AAAAAAAAAA-Bio

Connection of the receptor oligonucleotides:

0.5 μl portions of a 1 μM SSC solution of the receptor oligos were spotted on a slide coated with streptavidin (Greiner Bio-One) and incubated for 30 minutes in a moist chamber. The slide was washed 2 x 5 minutes in 1 x SSC-T (T is addition of 0.05% Tween-20) and the remaining drops were removed with a stream of nitrogen (not dried).

Block:

In order to saturate the free streptavidin binding sites remaining after the binding of the receptor oligos with biotin, the slide was covered with a 2μM solution of a biotinylated DNA oligo (in 1x SSC, 1% Tween) and incubated for 15 min. It was Washed 2 x 10 min with 1 x SSC-T (T is addition of 0.05% Tween-20) and the remaining drops were removed with a stream of nitrogen (not dried).

hybridization:

The spots of the wild-type and mutant receptors were incubated for 30 minutes with a mixture of Cy3-labeled sample molecule (0.25μM) and Cy5-labeled probe oligo (1 μM) in 5xSSC.

The hybridization led to the formation of the sample complex and the reference complex, as shown above, the chain being linked to the slide via the receptor oligo. It was washed 2 × 10 min with 1 × SSC-T (T is addition of 0.05% Tween-20) and the remaining drops were removed with a stream of nitrogen (not dried).

Stamp:

A micro-structured stamp was made from PDMS (polydimethylsiloxane). The structures consisted of stamp feet of approx. 100 x 100 μm, which were separated by depressions approx. 25 μm wide and 1 μm deep. For this purpose, a mixture of a 10: 1 mixture of silicone elastomer (Sylgard 184, Dow Corning) and crosslinking reagent was poured onto a correspondingly structured silicon wafer after repeated degassing and incubated for 24 hours at RT. After the polymerization, the structured surface of the stamp was exposed to H2O plasma at 1 mbar in a plasma oven for 60s.

The oxidized surface was incubated with 3% aminosilane (3-aminopropyldimethylethoxysilane; ABCR, Karlsruhe) in 10% H2O and 87% ethanol for 30 minutes. The silanized surface was washed with ultrapure water and blown dry with nitrogen. A bifunctional PEG was attached to the amino groups of the silane, one end of which had a carboxy group activated by NHS and the other end had a biotin group. 20 μl of a solution with 2 mg / 100 μl of NHS-PEG-Biotin (Shearwater Polymers, AL) were incubated under a cover glass for 1 h on a stamp with an area of 1 cm ² biert. It was washed with ultrapure water and blown dry with nitrogen. 0.1 mg / ml streptavidin (Sigma) in PBS was added to the now biotinylated surface and incubated for 30 min. It was washed with ultrapure water and blown dry with nitrogen.

A freshly prepared stamp and a base were pressed onto the spots with a solution of 30 mM NaCl under a pressure of 100 g / cm ² . The stamp was lifted off after 30 minutes. Slides and stamps were washed with ultrapure water and blown dry with nitrogen.

Measurement:

The stamp was measured in a fluorescence scanner (Axon, Genepix 4000 B) with a spatial resolution of 5 μm with regard to the intensities of Cy3 (excitation at 532 nm) and Cy5 (excitation at 635 nm).

Evaluation:

A fluorescence background was determined and subtracted for both measurement images. The measurement images adjusted for the background were processed further by determining the quotient of the fluorescence intensities (635nm / 532nm) for each pixel. As a result, a quotient image of the fluorescence intensities was obtained, as shown in Figure 6 for two spots.

A histogram of the intensity quotients V (V = intensity (Cy5) / intensity (Cy3)) was created from the quotient image (FIG. 7), the scaling of the intensity quotients being arbitrary.

The low peaks in the histogram corresponded to the stamp areas (dark rectangles in FIG. 6), the high peaks to the depressions in the stamp, to which no fluorophores were transferred (light grid in FIG. 6). The mean intensity quotients V (maxima of the low peaks) were determined for all spots. Then VM (mutant) and VPM (wild type) were compared.

It was to be expected that: VMM (mutant) <VPM (wild type) or VMM VPM <1 • For the present example, the histogram (FIG. 7) determined: VMM PM = 124/154 = 0.81.

The difference from a single base mismatch in a duplex of 16 nucleotides could be reliably detected.

Example 4

SNP typing on oligonucleotides

In this example, a sample sequence of 20 nucleotides in length should be checked for a possible exchange of a single guanine for a cytosine. The Pfob molecule (analyte) was an oligonucleotide with a Cy5 molecule conjugated at the 5 ^' end, which was constructed from 5' to 3 'from the following sequence components:

A reference sequence of 20 nucleotides, an internal spacer sequence of 11 nucleotides, a sample sequence of 20 nucleotides and a terminal spacer sequence of 5 nucleotides (here 5 x adenine). Sample sequence and reference sequence were reversed to each other.

The receptors consisted of an amino-C6 linker conjugated at the 5 ^' end, a spacer of 10 nucleotides without specific binding function (here 10x adenine) and a selected receptor sequence with a length of 20 nucleotides. Matched to the wild-type receptor sequence (receptor PM) with a length of 20 nucleotides derived a mutated receptor (receptor MM) with a base exchange in which guanine at position 13 was replaced by cytosine.

The probe oligo consisted (in 5 ^' -> 3 ^' direction) of a spacer of 5 nucleotides (here 5x adenine) without a specific binding function and of a sequence that was complementary to the reference sequence (20 nucleotides), as well as a further spacer (10xAdenine) a 3'-terminal biotin group.

Figure 8 shows the hybridization scheme of the oligonucleotides used.

Connection of the receptor oligonucleotides:

First, 18 mM bis-NHS-PEG (MW 3000 Da, Rapp Polymer Tübingen; 54 mg / ml) was dissolved in anhydrous DMF. 150 μl of the solution were pipetted onto a slide functionalized with amino groups (Quantifoil, Jena) and covered by a second slide. After an hour of incubation in a chamber saturated with DMF, the slides were cleaned with acetone.

For the PM receptor 5 μl of a 0.5 μM, for the MM receptor 5 μl of a μM oligo solution 1: 1 with a 100 mM EDC solution (=> final concentration 50 mM). 1 μl of both batches were spotted alternately with a hand pipette onto the slide (water vapor atmosphere) coated with Bis-PEG-NHS ₃ 00 ₀ . Incubation was carried out at room temperature for 1 h in a moist chamber. The mixture was then washed 2 × 20 minutes in 1 × SSC / 0.5% SDS and briefly with dist. Rinsed off water.

Block:

In order to avoid non-specific binding, the slide was swirled in a 2% BSA solution (w / v in distilled water) for 1 h at room temperature. Thereafter, with dist. Washed water for 5 minutes and rinsed briefly. hybridization:

The spots of the wild-type and mutant receptors were incubated for 1 hour with a mixture of Cy5-labeled sample molecule and unlabeled probe oligo in a concentration of 0.1 μM in 5xSSC.

The hybridization led to the formation of the sample complex and the reference complex, as shown above, the chain being linked to the slide via the receptor. Following the hybridization, the slides were washed for 10 minutes with SSC / 0.1% SDS or 1xSSC minutes and briefly blown dry in a stream of nitrogen.

Manufacture of stamps:

A micro-structured stamp was made from PDMS (polydimethylsiloxane). The structures consisted of stamp feet of approx. 100 x 100 μm, which were separated by depressions 41 μm wide and five μm deep.

A mixture of a 1:10 mixture of crosslinking reagent and silicone elastomer (Sylgard 184, Dow Corning) was poured onto a correspondingly structured silicone wafer after multiple degassing and incubated for 24 hours at RT. After the polymerization, the PDMS was _de st. In an EtOH / water. Mixture (1: 1) washed for 3 minutes in an ultrasonic bath. Before and after activation overnight in a 12.5% HCI solution, briefly with dist. Rinsed off water.

The activated surface was incubated with 3% aminosilane (3-aminopropyldimethylethoxysilane; ABCR, Karlsruhe) in 10% H ₂ O and 87% ethanol for 30 min. The silanized surface was washed first with ethanol and then with ultrapure water and blown dry with nitrogen. A bifunctional PEG was attached to the amino groups of the silane, one end of which had a carboxy group activated by NHS and the other end had a biotin group. In order to reduce the density of the biotin groups, the NHS-PEG-biotin (6 mM; Shearwater, Huntsville) was 1:20 with 6 mM NHS- methoxy-PEG ₂ ooo thinned. 20 μl of this solution were incubated under a cover glass for 1 h on a stamp with an area of 1 cm ² . It was washed with ultrapure water and blown dry with nitrogen and blocked with 6 mM NHS-methoxy-PEG for ₂ hours. 0.2 mg / ml streptavidin (Sigma) in PBS / 1% BSA was added to the now biotinylated surface and incubated for 1 h. Each was washed for 10 minutes with SSC / 0.1% SDS or 1xSSC minutes and briefly blown dry in a stream of nitrogen.

Stamp:

A freshly prepared stamp was pressed in 1xSSC under a pressure of 125 g / cm ² onto the spots of the support (slide). The stamp was lifted after 10 minutes. Slides and stamps were blown dry with nitrogen.

Measurement:

The stamp and the slide were measured in a fluorescence scanner (Virtek, ChipReader ™; 3 μm) with a spatial resolution of 6 μm with regard to the intensity of Cy5 (excitation at 635 nm). Since a higher photomultiplier voltage was used for the measurement of the stamp than for the base, the measured intensities of the transfer to the stamp (“ABOUT”) are greater than that of the hybridization on the slide (“VOR”). Since the evaluation in the present example is based on the "OVER / BEFORE" conditions and not on a comparison of absolute measured values, the intensities could not be normalized.

Evaluation:

The scans are shown in Fig. 9. In the upper half you can see the transfer of a mismatch spot (left) and that of a perfect match spot (right). A pattern of 5x5 boxes was selected for both spots and their intensities displayed in a histogram.

Below you can see the image of the two spots on the slide, which is complementary to the stamp. Here, the remaining grid was selected in the area of the 5x5 boxes and displayed in the histogram for both spots. It can clearly be seen that the intensity values of the hybridization (PMV _OR = 14000; MM _VOR = 15500) are only slightly that of the carry (PMQ _B ER = 23100; MM O _B ER = 39400) differ significantly from each other.

Since here, in contrast to the other examples, only the sample molecule, but not the probe molecule, was marked by a fluorophore, the evaluation is carried out only on the basis of the ratio of transferred (Uber) to offered (VOR) sample molecule.

Since a higher transfer of sample molecule to the stamp is expected with a mismatch in the sample duplex than is the case with a perfect match, the following applies:

QÜBER = PMQBER / MMQBER <0

However, this only applies on the condition that the intensities of the hybridization for

PM and MM are the same size. If this is not the case, use:

QÜBERΛ / OR = (PMQBER / PM _V0R ) / (MMQ _BE R / MM _VO R) <0

In this example you get:

Q _ÜBE R _{Λ / O} R = (23100/14000) / (39400/15400) = 0.65

The resulting Q _{OVERΛ / OR} <1. clearly shows that the analyte forms a perfect match with receptor PM and a mismatch with receptor MM. From this it can be concluded that the sample corresponds to the wild type and consequently G is present at position 13.

Variants of execution

Many variations are possible for the described method, which can be selected depending on the situation.

Apply the traction

As described in the preferred embodiment, the easiest way to compare the forces is to couple a chain between two approximated surfaces and then separate the surfaces. Instead of immobilizing on surfaces, the binding partners can also be immobilized on other solids, e.g. beads, or via molecules to which a tensile force can be applied.

The coupling step

Coupling is the step by which a chain is linked with the two

Force application points is connected.

A distinction is made between several cases in the coupling step, which are illustrated in FIG. 3: Case 1 corresponds to the example given above, the coupling step corresponding to the formation of the biotin-streptavidin bond between probe oligo and the stamp.

As in case 1, in case 2 the coupling step only takes place after the chaining has been formed, i.e. only after the sample complex and reference complex have already been linked. The only difference is that in case 1 there is coupling on the side of the reference complex and in case 2 on the side of the sample complex.

In cases 3, 4 and 5, the coupling and the formation of the chain are carried out by the same step. In case 3 the coupling takes place through the formation of the sample complex and in case 4 through the formation of the reference complex.

In case 5, both complexes are already formed and connected to the force application points (surfaces) when there is a coupling between the complexes. Position of the sample molecule in the chain

The above exemplary embodiment describes a case in which the analyte nucleic acid or sample sequence represents the second binding partner of the sample complex and is consequently directly connected to binding partner 3, the reference sequence. The sample sequence takes on a "central position" within the chain.

Deviating from this, the sample sequence can also correspond to binding partner 1. In this case it occupies a "peripheral position" in the chain. For this embodiment, the sample molecule is therefore immobilized on a surface. This is illustrated in FIG. 4. Cases A and B show a structure in which the sample is placed centrally ( Case A corresponds to the above exemplary embodiment.) With regard to the coupling, A can be compared with Case 1 from Figure 3 and B with Case 5 in Figure 3. At C, on the other hand, the sample molecule is placed peripherally and immobilized on the support Case 5 of Figure 3 comparable.

The sample molecule can be immobilized by introducing a biotin into one of the PCR primers used to amplify the sample molecule. The binding then takes place via the binding of this biotin to the streptavidin coating of the support.

Alternatively, the immobilization can also be carried out by hybridization with a capture oligo. For this purpose, the capture oligo is bound to the support and forms a complex with the sample molecule that is more stable than the reference or sample complex. In case C, the sample molecule is not labeled.

Cases B and C are evaluated by determining the amount of marker 1 that is transferred to the stamp and the amount of marker 2 that is transferred to the base. The following applies: Q ^* = n _Rβ . / n _Pra- ≡ Q = marker 2 (stamp) / marker 1 (pad) The reference complex

The reference complex can consist of a duplex of nucleic acids or also a complex of any other binding partner, e.g. Partners of a specifically binding couple.

It is advantageous to choose a reference complex that has a separation force similar to the sample complex under the given test conditions. For this reason, reference complexes are preferably chosen that are similar to the sample complex in chemistry, base composition and GC content. However, reference complexes can also be used which differ in length and base composition compared to the sample complex, the conditions given above with regard to the free base pairing energy then having to be taken into account.

The reference sequence

In the above embodiment, the reference sequence is part of the sample molecule. However, this would not be the case with a peripheral position of the sample molecule.

In the case of the exemplary embodiment, the reference sequence primer 2 corresponds to the amplification or the original sequence of the genomic DNA. However, the reference sequence can also consist of a sequence that was not contained on the genomic DNA and was only inserted by a degenerate primer during the amplification.

detection

The determination of Q ^* = n _Ref- / n _Pr0- (see _below ) can be carried out either by the detection of separate binding partners of a separate complex or by the detection of still intact complexes.

The detection of the binding partner is done via the marking. The property of the detector molecule to be determined is therefore dependent on the marking used certainly. This is usually done by evaluating optical or electrical parameters. The use of mass spectroscopy is also possible.

Determination of Q

Since it is very difficult in practice to determine the ratio of the average separation forces exactly, instead of the ideal Q ^* = n _Ref . / n _Pro ^Λ = F ^* _Rβf / F ^* _Pra approximately determines the quotient Q, ie Q «Q ^* .

Under ideal conditions, the ratio of separate sample complexes to the separate reference complexes can easily be determined from the distribution of the sample molecule between the two surfaces. In the exemplary embodiment, this would mean using only the Cy3 marker on the sample molecule. Q ^* = n _Rθf . / N _production would correspond in this case, approximately the ratio of Cy3, which is assigned to the punch to Cy3, which has remained on the slide.

However, this presupposes that the coupling, i.e. the connection of both surfaces through the chains, is reproducibly efficient in every experiment. This is not always the case and therefore two markings are preferred.

The following example is intended to illustrate why a second marking is often useful: An experiment in which the reference complex and the sample complex have the same separation forces is carried out twice. In Experiment 1, all of the 100 chains are coupled (n _Ko = 100). After separation, 50 sample molecules can be found on the stamp and 50 on the support. In Experiment 2, only 50 out of 100 chains are coupled. After separation, 25 sample molecules can be found on the stamp and 75 on the support. For experiment 1 one measures Q = n _Ref- / n _Pro = 50/50 = 1. For experiment 2 one measures Q = n _Ref- / n _Pro = 75/25 = 3. In fact, however, it is n _{Ref -} = 25 and n _Pro = 25 as well as 50 chains that were not coupled and separated. The different coupling efficiency, which can be caused, for example, by quality fluctuations in the stamp surface, leads to a different result. In the preferred embodiment, therefore, a second marker is used, which is attached to the probe and is used to determine the amount of coupled linkages (n _Kop ).

In this case, the ratio of the separation forces is obtained from n _Kop = n _Rβf - + n _Pro and Q ^* = n _βf - / n _Pro : Q ^* = (n _Kop - n _Pro ) / n _Pro .

When evaluating the stamp, the amount of the probe marker transferred to a specific area of the stamp is compared with the sample marker transferred to the same area. The amount of the probe marker corresponds to the amount of couplings (n _Kop ): n (probe marker) / n (sample marker) = n _Kθ p / _{n- Pro} for the above example:

Experiment 1: Q = (n _Kop - n _Pro ) / n _Pro = (100 - 50) / 50 = 1 Experiment 2: Q = (n _Kop - n _Pro ) / n _Pro = (50 - 25) / 25 = 1

By including the coupling number n _Kop , the correct quotient is also obtained for non-reproducible coupling efficiency.

The receptor oligo

The receptor oligonucleotide can consist of DNA, RNA, PNA, or other artificial nucleotide building blocks. As a rule, it is between 15 and 25 base pairs in length.

The discrimination

A mutation is discriminated against a wild type as a rule via a difference in the quotient Q. In the exemplary embodiment, two Q values are determined and compared with one another (reference experiment). Alternatively, one could also do without the reference experiment if, for example, the quotient for the wild type (Q _W τ) is already known. A significant change compared to Q _W τ would then indicate a mutation. However, this only makes sense if a very good reproducibility of all test parameters (eg constant quality of the markers) is guaranteed, which is not always the case.

The sample molecule

The sample molecule or the analyte nucleic acid can be both DNA and RNA as well as synthetic NA of various origins. Whether an amplification is necessary always depends on the amount of sample material available. The sample material is preferably amplified by PCR in order to have a sufficient amount available.

Claims

P a t e n t a n s r u c h e

A method of detecting mutations in a nucleic acid comprising a) contacting a plurality of nucleic acid strands to be analyzed with at least two types of oligonucleotides, receptor oligos and probe oligos, under conditions such that the oligos hybridize to the DNA to be analyzed to form a sample complex and a reference complex, so that there is a chain of sample complex and reference complex immobilized or immobilizable on both sides, b) the chain is then optionally immobilized, c) a tensile force is applied to the chain until one of the complexes separates and d) is determined which of the complexes was separated.

A method for the detection of mutations in analyte nucleic acids, in which the analyte nucleic acid is brought into contact with at least two types of oligonucleotides, receptor oligos and probe oligos, under conditions such that the oligos hybridize with the analyte nucleic acid to form a sample complex and a reference complex, immobilizing the oligonucleotides or can be immobilized, and where appropriate the oligonucleotides are immobilized after the hybridization, so that a mutually immobilized chain of receptor oligo - analyte nucleic acid - probe oligo is formed; a tensile force is applied to the linkage so that one of the complexes formed dissolves and it is determined which of the complexes has been dissolved, the receptor oligonucleotides having a sequence which is complementary to a sequence section of the wild-type analyte nucleic acid in which a mutation or variation is suspected.

A method according to claim 1 or claim 2, characterized in that the connection of analyte nucleic acid and probe oligo takes place via a coupling sequence.

4. A method for the detection of mutations in analyte nucleic acids, in which sample complexes and reference complexes are formed from analyte nucleic acid, two types of oligonucleotides, receptor oligos and probe oligos, and optionally a coupling sequence in such a way that the two complexes are in each case linked to one another; a tensile force is exerted on the chains formed, one of the complexes separating in each case and then determining which of the complexes has separated in each case.

5. The method according to any one of the preceding claims, characterized in that nucleic acids are selected for the sample complex and reference complex that the free base pairing energy ΔG of the hybridized complexes differs by <40 kcal / mol.

6. The method according to the preceding claim, characterized in that the free base pairing energy ΔG of the sample and reference complex differs by less than 20 kcal / mol.

7. The method according to the preceding claim, characterized in that the free base pairing energy ΔG differs from the sample and reference complex by less than 4 kcal / mol.

8. The method according to any one of the preceding claims, characterized in that in the sample and reference complex, the number of hybridizing pairs differ by a maximum of two bases in length for the same GC content.

9. The method according to any one of the preceding claims, characterized in that in the sample and reference complex, the number of hybridizing pairs differ by a base length for the same GC content.

10. The method according to any one of the preceding claims, characterized in that the number of mismatches in the sample and reference complex differs only by one mismatch.

11. The method according to any one of the preceding claims, characterized in that the method is carried out simultaneously and in parallel on a plurality of analyte nucleic acids.

12. The method according to any one of the preceding claims, characterized in that the analyte nucleic acids were obtained by PCR.

13. The method according to claim 10, characterized in that the analyte nucleic acid is labeled by using for the PCR primers that carry a label.

14. The method according to any one of the preceding claims, characterized in that receptor oligo and / or probe oligo have at one end a partner of a specific binding pair, the other partner of the specific binding pair being bound to a surface.

15. The method according to any one of the preceding claims, characterized in that receptor oligo and / or probe oligo are bound to a surface.

16. The method according to any one of claims 1 to 14, characterized in that analyte and probe oligo are bound to a surface ^

17. The method according to any one of the preceding claims, characterized in that receptor oligo and probe oligo are immobilized on a surface and then hybridized with the analyte nucleic acid.

18. The method according to any one of the preceding claims, characterized in that receptor oligo and / or probe oligo are first brought into contact with the analyte nucleic acid under conditions such that hybridization takes place and then immobilized.

19. The method according to any one of the preceding claims, characterized in that at least one of the two surfaces used for immobilization consists of an elastic material or is coated with an elastic material.

20. The method according to claim 19, characterized in that at least one of the two surfaces is coated with polydimethylsiloxane or a derivative thereof.

21. The method according to any one of the preceding claims, characterized in that receptor oligo and / or probe oligo is immobilized on a surface either directly covalently, via a bridge molecule and / or via a specifically binding pair.

22. The method according to any one of the preceding claims, characterized in that the complementary nucleic acid strands are DNA, RNA and / or LNA strands.

23. The method according to any one of the preceding claims, characterized in that the partners of the specifically binding pair are biotin and streptavidin or antibiotin antibodies.

24. The method according to any one of the preceding claims, characterized in that a group is used as the marking, which is optically and / or electrically detectable.

25. The method according to any one of the preceding claims, characterized in that a radioactive, fluorescent, luminescent, chromophoric label or a dye or a conductive group is used as the label.

26. The method according to claim 25, characterized in that a fluorescent group is used as the label.

27. The method according to claim 26, characterized in that fluorescein isothiocyanate, fluorescein, rhodamine, tetramethylrhodamine-5- (and-6) - isothiocyanate, Texas Red or a cyanine dye is used as the label.

28. The method according to any one of the preceding claims, characterized in that two labels are used which are bound to analyte nucleic acid and probe oligo or reference strand.

29. The method according to any one of the preceding claims, characterized in that two labels are used which are bound to the receptor oligo and the reference strand.

30. The method according to any one of the preceding claims, characterized in that the unlabeled components are immobilized and the labeled components are added last to the reaction mixture.