DE10207918A1 - Method for detecting nucleic acid mutations, useful e.g. for diagnosis and HLA typing, based on the force required to separate matched and mismatched hybrids - Google Patents

Abstract

A method for detecting mutations in a nucleic acid based on the application of a tensile force to separate hybridized oligonucleotides, is new. Detecting mutations in a nucleic acid (NA) comprises: (a) treating many NA strands with at least two type of oligonucleotides, receptor oligos and probe oligos, so as to form, by hybridization, sample complexes (SC) and reference complexes (RC) such that an immobilized, or immobilizable, linkage (L) between SC and RC is formed; (b) optionally immobilizing (L); (c) applying a tractive force to (L); and (d) determining which of the complexes has separated.

Description

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

The study of variations or mutations of a genomic Nucleic acid sequence becomes increasingly important and interesting as the changes draw conclusions allow that can be used for different areas.

There are different types of variations and mutations, all under the The generic term polymorphism can be summarized. Under polymorphism one variation, a genomic nucleic acid sequence that is often on the same Body can be determined. It can be both an exchange of individuals Bases, a so-called point mutation, as well as small deletions, insertions and Inversions also involve the reorganization of large sequence sections. In in any case, the sequence is in one or more places compared to the wild type changed.

Findings of polymorphisms in a genome are in different areas helpful. In genetics, polymorphisms are used as markers for the To determine the degree of kinship of two organisms by looking at inheritance which tracks 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 are used to detect certain ones Diseases in which genes are changed by single-base mutations and thereby Diseases are triggered. This is another important field of application called HLA typing, which is used to compare the similarity of two immune systems Compare individuals based on the state of many loci on the HLA genes. The HLA typing is primarily used to determine whether transplantation a graft will be tolerated.

Another area where detection of polymorphisms becomes important is Pharmacogenomics in which the detection of certain polymorphisms Drug tolerance should be predictable when treating a particular patient.

For the detection of polymorphisms, in particular single-base polymorphisms, developed various methods. A good overview is provided by Nollau et al. in Clinical Chemistry 43, No. 7 (1997), pages 1115-1128. Both methods for Identification of new SNPs, as well as methods for the detection of SNPs at known loci presented. The detection of known polymorphisms can be done by hybridization in which the sample is usually amplified by PCR, by sequencing, by primer extension, or by an oligonucleotide ligation assay. With all of these Procedures are usually complex steps necessary, for. B. electrophoretic Separations that take a lot of time.

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

One can use screening methods that serve to identify unknown SNPs from distinguish other methods used to detect polymorphisms on known Loci are suitable. DNA sequencing, on the other hand, can be used universally however, routine diagnostic testing is very costly.

The pure screening methods can either be based on the principle of different migration behavior of wild-type or mutant heteroduplexes in Electrophoresis gels, or due to the enzymatic digestion of heteroduplexes.

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

Discrimination through stringent hybridization uses oligonucleotides used under specific buffer and temperature conditions for this sequence hybridize the wild type (perfect match) but not to the mutant (mismatch). The The problem with this approach is that with a parallel execution Such tests in one approach do not require conditions to be set for all analytes are equally stringent.

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

Mini-sequencing is a modification of SSP-PCR. In this In this case, a heteroduplex is formed that has exactly one nucleotide in front of the SNP at the 3 'end. Locus ends. This primer can by the DNA polymerase by incorporation of Dideoxynucleotide triphosphates (ddATP, ddTTP, ddGTP and ddCTP) are extended, whereby there is a strand termination immediately after the installation of one of these bases. You can now color-code the four dideoxynucleotides individually determine which base has been installed or which one complementary bases on the sample molecule is present.

One is used in the so-called oligonucleotide ligation assay (OLA) similar strategy. Two oligos are tailored to be direct hybridize side by side at the SNP site with the sample molecule. The 5 'end of the first Oligos is provided with a phosphate and the 3 'end of the other oligos facing. If there is a perfect match, the oligos are now replaced by the DNA ligase connected. This reaction does not occur in the event of a mismatch. By proving the Ligation product can ultimately be based on the identity of the base at the SNP getting closed.

The object of the present invention was to provide a method with which Mutations, in particular polymorphisms, can be detected without elaborate separation steps, such as electrophoresis, are necessary. Furthermore it was Object of the invention to provide a method that the possibility of Offers parallelization of tests.

• a) eine Mehrzahl zu analysierender Nucleinsäure-Stränge mit mindestens zwei Arten von Oligonucleotiden, Rezeptoroligos und Sondenoligos, in Kontakt gebracht wird unter solchen Bedingungen, dass die Oligos mit der zu analysierenden DNA hybridisieren unter Bildung eines Probenkomplexes und eines Referenzkomplexes, so dass eine beidseitig immobilisierte oder immobilisierbare Verkettung von Probekomplex und Referenzkomplex vorliegt,
• b) anschließend gegebenenfalls die Verkettung immobilisiert,
• c) an die Verkettung eine Zugkraft angelegt wird, bis sich einer der Komplexe trennt und
• d) bestimmt wird, welcher der Komplexe getrennt wurde.

This object is achieved with a method for the detection of mutations in a nucleic acid which comprises that

• a) a plurality of nucleic acid strands to be analyzed are brought into contact with at least two types of oligonucleotides, receptor oligos and probe oligos, under conditions such that the oligos hybridize with the DNA to be analyzed to form a sample complex and a reference complex, so that one is immobilized on both sides or immobilizable chaining of sample complex and reference complex is present,
• b) subsequently, if necessary, immobilizing the chain,
• c) a tensile force is applied to the chain until one of the complexes separates and
• d) determining which of the complexes has been separated.

According to one embodiment of the method according to the invention is used for detection of mutations in analyte nucleic acids the analyte nucleic acid with two types of Oligonucleotides, receptor oligos and probe oligos, contacted among them Conditions that the oligos hybridize to the analyte nucleic acid, the Oligonucleotides are immobilized or immobilizable, and where appropriate the Oligonucleotides are immobilized after hybridization, so that one is immobilized on both sides or immobilizable chaining of receptor oligo analyte nucleic acid probe oligo is present; a tensile force is applied to the chain, so that one of the bonds loosened and determined which of the bonds was released, the Receptor oligonucleotides have a sequence that corresponds to a sequence section of the analyte nucleic acid is essentially complementary, in which a mutation or variation is suspected. The principle of differential is used for the method of the present invention Force tests exploited. The general principle of the differential force test is described in PCT / EP01 / 09,206th It was found that the application of the differential force test reliable and highly parallelizable and miniaturizable Allows polymorphisms, with point mutations (SNP) as well as others Changes in the sample sequence can be detected, provided that they change the separating power of the hybridization with that to the wild type lead complementary strand resulting duplexes.

To summarize briefly a nucleic acid molecule in a sample, the following is also referred to as analyte nucleic acid for the presence of mutations or variations are examined. A first and a second A sample complex is formed in the binding partner. One of the two contains Binding partner - the analyte nucleic acid - a defined sequence section (sample sequence) of an analyte molecule. The other of the two binding partners contains a defined one Sequence section (receptor sequence) of a receptor oligo that essentially is complementary to the portion of the analyte sequence of interest. Through the Hybridization of the first binding partner with the second binding partner occurs 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 after the In response to the chain formed, a tensile force can be exerted.

A pulling force is applied to the ends of the chain until one of the two complexes is separated, either the first from the second binding partner, d. H. the Sample complex, or the third of the fourth binding partner, d. H. the reference complex. So the sample complex is either dissolved if its separating force is below the Test conditions is smaller than that of the reference complex or to solve the Reference complex, if its separating force is smaller under the test conditions than that of the sample complex.

If this experiment is carried out very often, preferably in the form of many concatenations on a 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 ^* _Pro / F ^* _Ref

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

F ^* _Ref / F ^* _Pro (WT) ≠ F ^* _Ref / F ^* _Pro (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 ^* _Pro (wild type) <F ^* _Ref / F ^* _Pro (mutant)

or Q ^* (WT)> Q ^* (M)

The discrimination of mutations compared to the wild type by a force comparison is therefore based on ^* _Pro that due to the change of F, the ratio F ^* _Ref / F ^* _Pro changes.

The reference complex is usually also a nucleic acid duplex, the fourth Binding partner corresponds to the probe sequence and is called probe oligo. Binding partner 3 is essentially complementary to or corresponds to 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 however, is equally applicable to the determination of variations in a nucleic acid. Therefore, when we talk about "mutations" in the following, there should also be variations be included.

According to the invention, an analyte nucleic acid on which mutations are detected are to be brought into contact with various oligonucleotides in such a way that form at least two nucleic acid duplexes which are connected to one another in such a way that forms a chain on either end of which force can be applied. Furthermore is this chaining is designed so that when a force is exerted on this chaining is exercised, one of the two hybridized duplexes or complexes separates depending on which duplex or complex under the given conditions has less binding power. This depends on those not hybridized in Unzip mode Nucleic acid strands mainly from the number of paired bases, the type of bases, especially 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. Key point of the The invention is that the binding force of two duplexes is compared with one another, wherein the analyte nucleic acid is involved in at least one of the two duplexes. By Determining which of the two duplexes has been released allows conclusions to be drawn which of the two duplexes had the stronger binding force. From this you can again conclude whether the duplex mismatch formed by the analyte nucleic acid had trained.

If one speaks of "an analyte nucleic acid", this means, of course only the description of one representative from a variety, so that at Carrying out the method according to the invention normally always involves a large number of Nucleic acids react. The description is for the sake of simplicity only to a selected representative.

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

In the following, for the sake of simplicity, we will speak of stamps and documents without that it should be determined about spatial location, nature or function, because the stamp can also serve as a base and vice versa. The same designs also apply if, instead of macroscopic surfaces, the immobilization on beads or Particle takes place.

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. It the three components are brought into contact with each other in such a way that they are in contact with each other can hybridize. For this, e.g. B. the receptor oligo on one for simplicity Base surface mentioned are immobilized in a conventional manner, for. B. through covalent bonding via functional groups located on the base. The Immobilization should take place in such a way that a duplex is formed during hybridization, which is not zipped open when using a tensile force. The The pad with the bound receptor oligos is then in contact with the analyte nucleic acid brought so that hybridization can take place, with a sample complex is formed. The probe oligonucleotide can then either simultaneously with the Analyte nucleic acid or subsequently added or on a stamp designated second surface and then immobilized on the surface bound nucleic acids are brought into contact so that there is a hybridization comes between probe oligo and analyte nucleic acid, with a reference complex is formed. In this way, a chain can be formed that consists of immobilized reference oligo, analyte nucleic acid and immobilized probe oligo. On this chaining can be carried out over the surfaces or supports on which the oligonucleotides are immobilized, a traction can be exerted. When a pull is applied, the weaker of the two bonds rises, d. H. 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 chaining orders are also possible. It is then proven which of the two bonds has risen.

In another variant of the method according to the invention, a Receptor oligonucleotide immobilized and in with the sample containing the nucleic acid to be analyzed Contacted so that the analyte nucleic acid hybridize with the receptor oligo can, the order of the work steps is arbitrary. It will be a stamp prepared on which the probe oligonucleotide is immobilized. It will continue with this Probe oligonucleotide hybridizes to a reference nucleotide that has a base sequence, that result in a sequence of the analyte nucleic acid that is outside the range in which Mutations are suspected. Here too, the order of the work steps is arbitrary. The number of base pairs hybridizing between them Reference strand and the analyte nucleic acid is far higher than that of to be formed Reference complex and sample complex and only serves to reliably connect the two checking complexes. This additional hybridization strand should definitely be like this many binding base pairs show that its separating force is well above the separating power of the two complexes is so that it remains in any case. How in the previous variant, a tractive force is then applied to the Formed chaining exercised and then demonstrated which of the two complexes has solved.

In a further variant (in which the sequence of the work steps so far the analyte nucleic acid is placed on a support immobilized. The base with immobilized analyte nucleic acid is with the Receptor oligonucleotide contacted under conditions such that hybridization can be done. A probe oligonucleotide is immobilized on a stamp and then a reference strand, as stated in the previous variant, hybridizes. Both the receptor oligonucleotide and the reference strand point to one another complementary bases. The pad and stamp are then in contact with each other brought that the complementary bases of reference strand and Receptor oligonucleotide can hybridize with each other. The number of hybridizing with each other Base pairs between this reference strand and the receptor oligonucleotide are far higher than that of the reference complex and sample complex to be formed and only serves the reliable connection of the two complexes to be checked. This additional In any case, the hybridization strand should have as many base pairs that bind together have that its separating force is far above the separating force of the two complexes, so that it remains in any case. After hybridization, there will be another Tensile force on the chain formed from analyte nucleic acid, receptor oligonucleotide, Reference strand, probe oligonucleotide exerted, whereby the bond is released, which has the lowest separation force.

Further variants of the described method variants are possible can be easily found by the expert. As stated, an essential feature that two duplexes can be compared in their binding force and From which bond is released, conclusions can be drawn about the type of hybridization can be.

For the method according to the invention, one nucleic acid to be examined is used with two different oligonucleotides in contact, one of which is an oligonucleotide has defined sequence section that is complementary to the range of analyzing nucleic acid 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 one Sequence section that is complementary to another section. This Oligonucleotide is called probe oligo. The sequence of the probe oligo should not include the same section as the receptor oligo hybridize to cross reactions avoid. In addition, the single strands should not be mutually exclusive in hybridization hinder. In a preferred embodiment, however, the probe oligo can have reverse sequence to the sequence of the receptor oligo. This has several advantages. It simplifies the production of the oligonucleotides, it prevents cross reactions and it creates complexes that are comparable in terms of binding power.

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

In a preferred embodiment, the nucleic acid to be analyzed is labeled with a Mark it. There are various ways of doing this. The type of marking depends, among other things, on how the nucleic acid to be investigated won. The label can be directly attached to the nucleic acid to be examined be bound. In a preferred embodiment, the marking is introduced by generating the nucleic acid to be examined using PCR, the primer to be used for this has a label, the pair of primers, that is used for the PCR has a sequence that flanks the site that the contains 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 verifiable 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 which bring about a change in optical or electrical properties are suitable for the marking. 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. Preferably acts the marking is 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 a very small amount.

In a preferred embodiment, two marks are per chain used, which are preferably different from each other. When using Different fluorescent labels can be used, each one by light of the same wavelength or by radiation of different wavelengths be stimulated.

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 exhibits 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 can be, it can also be c-DNA. For the investigation, the Nucleic acid can be obtained directly from cells and, if enough copies of the Interesting sequence are present in the cell, this sequence directly for the test be used.

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

The length of the nucleic acid used depends, among other things, on whether one Mutation or variation or multiple variations or mutations can be detected and in which area they are located. The method according to the invention is particularly well suited for nucleic acid sequences with up to 100 nucleotides, especially for those sequences which have 30 to 50 nucleotides. With proof Known single base mutations can also have shorter nucleic acid sequences be used.

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 which 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 called the receptor _WT- oligos, whereas those receptor oligos that contain variations and mutations are called the 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 act that leads to a change in attachment behavior. It is called a mutation or variation any conceivable change in the chemical structure of the nucleotide that have a separation force difference compared to an original condition a force comparison.

The probe oligo is used with another strand that contains the analyte nucleic acid or but can be a reference strand to form a reference complex. The Reference complex should be as similar as possible to the sample complex in order to reduce the differentiation To enable separation force differences between two sample complexes.

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

The length of the hybridizing sequence of the probe oligo is preferably so selected that the force that is necessary to separate the reference complex, the force that is necessary to separate the sample complex comes very close. If you for the Reference complex selects a sequence that has the same GC ratio as the Has sample complex, the number of base pairs is preferably in the same range and preferably only differs by up to 2 base pairs. If the GC ratio is between distinguishes between two complexes, the length of the sequence to be hybridized then be selected so that the separation force matches that of the sample complex approaches. The insertion of one or more mismatches can also be used to set the serve optimal reference complex.

In one embodiment, the nucleic acid to be analyzed with receptor oligos and Probe oligos contacted under conditions such that hybridization can take place. The probe oligo hybridizes with one end of the analyzing DNA to form a perfect match while the other end of the to investigating DNA hybridized with receptor oligos, making it a perfect match or a mismatch occurs depending on whether the sequence of the receptor is too corresponds to investigating DNA or not.

By bringing the two types of oligos into contact, a chain of receptor oligo analyte nucleic acid probe oligo is formed in the method according to the invention in the embodiment described, 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.

The oligonucleotides can be immobilized in a manner known per se, by the nucleotides via spacers or bridging molecules to a support or a Surface bound. In any case, the support or the surface must be like this be designed so that a tensile force can be applied. It can be about macroscopic surfaces, but also around other solids, such as beads, or Act molecules. Immobilization is particularly preferably carried out by the Receptor oligonucleotides are or are immobilized on a first surface while the Probe oligos are immobilized on a second surface or are immobilized.

If surfaces are used for the immobilization, the material can be made of whose surfaces are the same or different and one of the two or both surfaces can be coated in a suitable manner. Either is preferred one surface of a rigid material and the other of an elastic Material formed or both surfaces are made of elastic material, so that the respective surface sections of the first and second surfaces when brought into contact exactly can adapt to each other and thus an optimal contact of the individual Nucleic acid strands and optionally binding partners is possible. The surfaces can for example made of glass, silicone, in particular polydimethylsiloxane (PDMS), nylon, Polystyrene or other plastics are formed. At least one of the is preferred both surfaces made of an elastic material, preferably an elastic Plastic. At least one surface made of a siloxane is particularly preferred, especially polydimethylsiloxane. There are various other flexible ones Materials or mixtures thereof possible. Another possible material is Polyacrylamide gel, the elastic properties of which by molecular weight and Degree of crosslinking can be adapted to experimental needs. The surface can from a single material, a mixture of materials or even a system be constructed from elements of one or different materials.

The immobilization of the oligonucleotides or nucleic acids can be done in a manner known per se Way, there are both covalent and non-covalent bonds and Bonds directly to the surface or via bridge molecules. Is possible also immobilization via the partners of a specifically binding couple. The Immobilization should take place in such a way that a duplex is formed during hybridization, which is not zipped open when using a tensile force. In the latter Fall is one of the two partners of the specific binding couple permanent immobilized and the other bound to a nucleotide or the analyte nucleic acid, under permanent bond here is understood to be a bond that comes before contact of the surfaces remains essentially stable and also by binding the Does not solve binding partner to its specific counterpart. A permanent bond can z. B. via functional groups that the binding partner has or in the molecule were introduced to functional groups that already provide the surface.

A preferred method is to biotinylate a partner and use one Streptavidin molecule to connect to the also biotinylated surface. monoclonal Antibodies can be activated chemically by targeting certain groups of their Glycosylations oxidized to aldehyde groups. These aldehyde groups can in turn Bind amino groups or hydrazide groups on a modified surface (see Solomon et al., Journal of Chromatography, 1990, Vol. 510, 321-329). Another method the skilled worker is familiar with the conjugation of amino groups of the antibody with carboxy groups on a surface through the use of 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 specialist knows ways in which Nucleotides can be bound to surfaces. The immobilization should be like this take place that the sequence complementary to the nucleic acid to be analyzed for the Hybridization is accessible.

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

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

As immobilizable nucleotides, those are used which have one end Have partners of a specifically binding couple, the other partner of the specifically binding pair is covalently bound to a surface. When in- Bringing the nucleotides into contact with the surface then results in binding specifically binding partner, whereby immobilization takes place. As specific bindable couples come into consideration the most diverse bindable partners, where the specificity of the bond can relate to a specific partner, such as B. antigen-associated antibody or biotin-avidin / streptavidin, as well as on a Group or class of connections, e.g. B. 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 means antibody fragments or antibody derivatives, as well as functional fragments of antibodies or derivatives thereof, which can recognize and bind the epitope. 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) included that recognize an epitope of the sample.

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

Various variants are available for carrying out the method according to the invention possible, depending on the application and the circumstances of the respective test can be selected. Below are some variants of the order and Type of chaining specified. In a first variant, the DNA to be analyzed with receptor oligonucleotide and probe oligonucleotide under conditions that a Allow hybridization, implemented and then by bringing the hybridized molecules with two surfaces, each specifically binding partner for the provide both oligonucleotides, immobilized.

In a second embodiment, one of the two oligonucleotides, either Receptor oligonucleotide or probe oligonucleotide immobilized on a surface, then with the other type of oligonucleotide and the one to be analyzed Contacted nucleic acid so that there is binding of receptor oligonucleotide Analyte nucleic acid probe oligonucleotide immobilized on a surface comes and goes then the second surface is brought into contact with this concatenation, so that a binding partner bound to the second surface of a specific binding Couple with the one not yet immobilized with a binding partner Can bind nucleotide.

In a further variant of the method according to the invention Receptor oligonucleotides immobilized on a first surface, while probe oligonucleotides on one second surface can be immobilized. It will then either be the first surface or the second surface with the analyte nucleic acid under such conditions in Contacted that one hybridization takes place and then the other Surface with the analyte nucleic acid now bound to an oligonucleotide Contacted that a further hybridization can take place, so that in turn forms a chain.

In all variants, when the chain has been formed and the Immobilization of the two types of oligonucleotides has occurred, separation of the two Surfaces carried out so that one of the bonds comes loose.

Then it is determined which of the bonds has mostly broken, from which conclusions can then be drawn as to whether the one to be analyzed Nucleic acid corresponds to the wild type or has a mutation or variation. To it is checked where the marker is located. The marking is on the The surface on which the probe oligonucleotide is bound must bind to the Embodiment between probe oligo and analyte nucleic acid described above be stronger than the bond between receptor oligo and analyte nucleic acid. Since it is hybridization of probe oligonucleotide with analyte nucleic acid usually around is a perfect match, this is the case if the hybridization between Receptor oligonucleotide and DNA to be analyzed at least one mismatch is present or a nucleotide is modified so that the binding is weaker than in one "normal" nucleotide.

Significant results are obtained when the chains are separated was carried out simultaneously on many complexes under the same conditions. If then the number of separated sample complexes and the number of separated If reference complexes are put in relation, one obtains a relationship that draws conclusions on the type of hybridization, as already explained above.

This can be done by selecting the appropriate probe oligonucleotide for each desired analyte nucleic acid a sensitive detection method for variations and Perform mutations by matching the analyte DNA with receptor oligonucleotides Brings in contact, some of which are of the wild type and others at least one Show change in the sequence so that a comparison between wild type and Change is possible.

In a preferred embodiment, for example, for the detection of a SNPs use four different receptor oligos that are dedicated to the SNP differentiate suspected location in the base, so that each base at this location once occurs. The method according to the invention is then carried out with the four receptor oligos at the same time, and then compares the frequency with which complexes with Receptor oligos separated, it can be expected that in the three oligos in which one Mismatch is expected to result different from the one oligo in which a perfect match has arisen. This embodiment therefore leads to special 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 can. The essence of the invention is that many hybridizations occur simultaneously can be performed and the separation force of the hybridized strands on many Molecules can be detected at the same time.

In a further embodiment, the method becomes even more meaningful Use a second marker. In this embodiment, both the Analyzing nucleic acid with a label, as well as the Probe oligonucleotides. This is an embodiment in which the probe oligonucleotides are immobilizable are, but not yet immobilized during the implementation with the DNA to be detected are. The probe oligonucleotides are provided with a label which differs from the Marking with which the DNA to be analyzed is distinguished. To Implementation of the method according to the invention, in which in turn the analyte nucleic acid is brought into contact with receptor oligonucleotides and probe oligonucleotides, a Hybridization and then immobilization is brought about and then the A linkage is applied to either side of the bindings of the chain Solve chaining. Then a surface is analyzed to see how much of it the probe oligonucleotide has bound to the surface and how much of that too has bound analyzing DNA. By determining the quotient of bound Probe oligonucleotides to bound analyte nucleic acids can be more precisely prove whether there is a variation or mutation, since only the complexes are examined which actually immobilize the probe oligonucleotides has taken place.

In the described embodiments, the order of the steps is variable. However, it should be ensured that the unmarked ones first Components are immobilized and finally the marked ones.

As already explained above, the method according to the invention points towards the Prior art methods have various advantages. So it has towards the known enzymatic methods the advantage that no enzymes are used need, which saves costs. In addition, when performing the procedure and the choice of reactants, e.g. B. the composition of the buffer not on the Enzymes are taken into account.

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

In contrast to stringent hybridization, many different ones can be used Sample complexes are discriminated against in one approach. The stringency is for each sample complex quasi specified via the separating force of the reference complex. Besides, are different from in stringent hybridization, no washes with temperature or Salt gradients necessary.

The invention is illustrated by the figures and the following examples.

Fig. 1 shows the principle of the inventive method, wherein a force is applied to both sides of a concatenation of immobilized analyte nucleic Rezeptoroligonucleotid-and probe oligonucleotide. 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.

Fig. 3 shows the various options for coupling of the components Rezeptoroligonucleotid, and analyte nucleic probe oligonucleotide.

Fig. 4 shows various possibilities of coupling and immobilization of the components

Fig. 5 shows the fluorescence scan of a spot, which was transferred to a stamp

Fig. 6 shows the ratio of fluorescence intensities at two spots on the stamp transmitted. Mismatch (left) and Perfect Match (right).

Fig. 7 shows a histogram of the ratios of fluorescence intensities for mismatch and perfect match spots spots

The following five variants are shown in FIG. 3.

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

In variant 2, a binding partner is immobilized on a surface and with the specifically binding partner that is present on an analyte nucleic acid. The one immobilized on the surface via the specific binding pair Analyte nucleic acid is contacted with one receptor oligonucleotide, the other End has a group that can bind to a surface. By binding the Receptor oligonucleotides on the surface in turn form a chain to which one Tractive 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 ones Can hybridize nucleotides of receptor oligonucleotide and analyte nucleic acid. On the a chain can then be exerted again.

In variant 4, a receptor oligonucleotide is immobilized on a surface and with an analyte nucleic acid so that hybridization takes place can. The analyte nucleic acid has a partner at its other end specific binding pair. The other partner of this specific binding couple is immobilized on a second surface. The two surfaces will be like this brought into contact with each other that it is for binding the specific binding pair comes. In this way, a chain is created again, and then a force can be exercised.

In the last variant No. 5 there is a receptor oligonucleotide on one surface immobilized and brought into contact with the analyte nucleic acid so that complementary Can hybridize nucleotides with each other. On a second surface is a immobilized specifically binding pair. Both the non-immobilized second partner of the specifically binding pair as well as the analyte nucleic acid each have one Binding site that can react with each other. If these two groups have reacted with each other, a chaining takes place again, to which in turn train can be exercised.

The two partners of the specific binding pair are in a preferred one Embodiment of the method according to the invention two complementary Nucleic acid strands that form a reference complex. The nucleic acid strands are like this provided that they differ in terms of the free base pairing energy and the number of Distinguish mismatches from the sample complex in a predetermined manner or possibly with regard to the free base pairing energy and number of mismatches are identical.

It is also possible to use another specific binding instead of the nucleic acid strands Pair to use as a reference complex, this specific binding pair so is selected that the binding force is predetermined and the binding force of the Trial complex comes as close as possible. Procedure for determining the free Base pairing energy or the binding power 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 differs free base pairing energy ΔG as little as possible. A reliable one Determination is no longer possible if the free base pairing energy of Sample complex and reference complex differ by more than 40 kcal / mol. Prefers the free base pairing energy of the sample and reference complex does not differ more than 20 kcal / mol. Particularly good results are obtained if the 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. A procedure will for example in K. J. Breslauer, R. Frank, H. Blöcker and L. A. Markie "Predicting DNA Duplexes Stability From The Base Sequence ", Proc. Natl. Acad. Sci. USA, Vol. 83, pages 3746-3750, 1986.

The following definitions are used in describing the examples.
Receptor oligo: oligo that forms a sample complex with the analyte
Probe oligo: Oligo that 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 containing the analyte
Mismatch: base pair in a duplex that does not form a specific base pair
Release force: force that occurs on a chain immediately before it is separated by an applied tensile force
Binding partner: partner of a specific binding couple
Wild type: original or different form of the sample sequence from the mutant
Mutation: modified or different form of the sample sequence, which leads to a change in the separating power of the sample complex

example 1

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

An amplicon (sample molecule corresponds to genomic DNA by PCR Analyte nucleic acid). The pair of primers is flanked (primer with 18 Nucleotides) a sequence of 30 base pairs, which the site with the possible Contains base exchange. Primer 1 attached to the non-codogenic strand of genomic DNA hybridized and extended to copy the codogenic strand is with a Cy3 Marked 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 also covered saturated a biotinylated poly-A-oligo of 15 bp length.

The wild-type receptor (Rez _WT ) is a 28 nucleotide long DNA oligo that is modified at the 5 'end with biotin, whereby it is bound to the strepavidin 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 _WT . This sequence section 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 strepavidin 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 thus 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 is a copy of the codogenic Making a strand is denatured by cooking and by cooling on ice Rehybridization prevented. The probe oligo (probe) is added to the sample mixture. there it is a 28-bp DNA oligo. The probe is at its 5 'end 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 that represents the reference sequence.

The sample added with the probe is now in 5 × SSC buffer for hybridization on the Given receptor spots of the blocked slide. After an incubation of 30 min at RT is washed with 1 × SSC and covered with 1 × SSC.

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

A microstructured PDMS stamp coated with streptavidin is now under 100 mM NaCl buffer on the area of the receptor spots with the immobilized Chains pressed on. This involves the connection of the biotinylated probe oligos the stamp, or for coupling the links between the stamp and the slide. The stamp is separated from the slide after 30 minutes, whereby it is used to loosen the Concatenations by separating one complex each comes. It should be noted that this process simultaneously on a large number (~ 100 fMol) of chains on each spot is performed.

The stamp is now in a fluorescence scanner for the markers Cy3 and Cy5 evaluated. The probe oligos and sample molecules transferred to the stamp can be quantified in this way. That of the spots on a certain area of the stamp transferred amount of Cy5 is with the transferred amount Cy3 on the same area related. The quotients obtained for the two spots will be compared with each other.

According to the formula Q = n _Kop - n _Pro / n _Pro (see below) you get:
For the Rez _WT spot: Q _WT = n (Cy5 _WT ) - n (Cy3 _WT ) / n (Cy3 _WT )
For the Rez _M spot: Q _M = n (Cy5 _M ) - n (Cy3 _M ) / n (Cy3 _M )

On the basis of the quotients determined by the stamp, two cases can now be found distinguished:

Case 1)

The sample molecule is in wild-type form (no exchange from G to C at base 39). The sample complex of Rez _WT 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 _WT is therefore greater than the separation force for the sample complex from Rez _M :

F ^* _Pro (Rez _WT )> F ^* _Pro (Rez _M )

One obtains Q _WT > Q _M

With Q _WT > Q _M the wild type can be concluded

Case 2)

The sample molecule is in the mutant form (at base 39, G was converted to C ) Replaced.

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

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

The separation force for the sample complex from Rez _WT is smaller than the separation force for the sample complex from Rez _M :

F ^* _Pro (Rez _WT ) <F ^* _Pro (Rez _M )

One obtains Q _WT <Q _M

If Q _WT <Q _M , the mutant can be concluded.

Example 2 SNP typing on oligionucleotides

In this example, two sample sequences with lengths of 14 and 16 were used Nucleotides 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, one Spacer of 10 nucleotides without specific binding function (here 10 × adenine) as well from a selected receptor sequence with a length of 16 or 14 nucleotides.

Suitable for two different wild-type receptor sequences with lengths of 14 and 16 Nucleotides were put together with receptors each with a base exchange. 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 without a specific binding function (4 × adenine) and a sequence that was complementary to the reference sequence (16 or 14 nucleotides), as well as a further spacer (10 × adenine) with a 3'-terminal biotin group. The structure of the chains of the 16mer sample molecule

Building chains of the 14mer sample molecule

Connection of the receptor oligonucleotides

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

hybridization

The wild-type and mutant receptor spots were mixed for 30 minutes from Cy3-labeled sample molecule and Cy5-labeled probe oligo in one Concentration of 0.5 µM in 5 × SSC, 0.05% Tween-20 incubated.

The hybridization led to the formation of the sample complex and the Reference complex, as shown above, the chaining via the receptor to the Slide is attached. Following the hybridization, the Slides 2 × 2 minutes with 5 × SSC-T, 2 × 2 minutes with 1 × SSC-T and 2 × 2 minutes with 0.2 × SSC-T washed, briefly watered and dried in a stream of nitrogen.

Stamp

A micro-structured stamp made of PDMS (polydimethylsiloxane) was manufactured. The structures consisted of stamp feet of approx. 100 × 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 h 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 ul of a solution with 2 mg / 100 ul 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 in a fluorescence scanner (Axon, Genepix 4000 B) with a Local resolution of 5 µm with regard to the intensities of Cy3 (excitation at 532 nm) and Cy5 (Excitation at 635 nm) measured.

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 and displayed in a histogram. In Fig. 5 is a typical histogram is shown a spot for one of the two markers. 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).

For each spot and for each marker, the difference between the signal and the background was determined calculated.

In contrast to Experiment Example 1, for practical reasons it was not Q = n (Cy5) - n (Cy3) / n (Cy3) that was calculated, but the ratio V = n (Cy5) / n (Cy3). V _MM (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.

In Table 2 the mean values of V for 14- and 16-mer were then with and without Mismatch calculated. Finally, the averages for Perfect Match and Mismatch (each for 14- and 16-mer) in relation to each other.

V _MM / V _PM <1 is found for both 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 typing on oligonucleotides

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

The sample molecule was an oligonucleotide which 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.

Matched to the wild-type receptor sequence with a length of 16 nucleotides mutated receptor derived with a base exchange with the cytosine at position 12 was replaced by adenine.

The probe oligo consisted of a Cy5 molecule conjugated at the 5 'end, a spacer composed 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 16mer sample molecule

Connection of the receptor oligonucleotides

0.5 µl of a 1 µM SSC solution of the receptor oligos was applied to one with streptavidin coated slides (Greiner Bio-One) spotted and in a moist for 30 min Chamber incubated. The slide was spun 2 × 5 minutes in 1 × SSC-T (T is addition of 0.05% Tween-20) and remaining drops with a stream of nitrogen removed (not dried).

Block

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

hybridization

The wild-type and mutant receptor spots were mixed for 30 minutes from Cy3-labeled sample molecule (0.25 µM) and Cy5-labeled probe oligo (1 µM) in 5 × SSC incubated.

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

Stamp

A micro-structured stamp made of PDMS (polydimethylsiloxane) was manufactured. The structures consisted of stamp feet of approx. 100 × 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 multiple degassing and incubated for 24 h at RT. After the polymerization, the structured surface of the stamp was exposed to H ₂ O plasma at 1 mbar in a plasma oven for 60 s.

The oxidized surface was incubated with 3% aminosilane (3-aminopropyldimethylethoxysilane; ABCR, Karlsruhe) in 10% H2O 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 ul of a solution with 2 mg / 100 ul 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 . 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 in a fluorescence scanner (Axon, Genepix 4000 B) with a Local resolution of 5 µm with regard to the intensities of Cy3 (excitation at 532 nm) and Cy5 (Excitation at 635 nm) measured.

evaluation

A fluorescence background was determined and subtracted for both measurement images. The measurement images adjusted for the background were further processed by determining the quotient of the fluorescence intensities (635 nm / 532 nm) for each pixel. As a result, a quotient picture of the fluorescence intensities was obtained, as shown in FIG. 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. certainly. Then VMM (mutant) and VPM (wild type) were compared set.

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

V _MM / V _PM = 124/154 = 0.81.

This was the difference between a single base mismatch in a duplex of 16 nucleotides can be reliably detected.

Variants of execution

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

Apply the traction

As described in the preferred embodiment, the comparison of forces is easiest by coupling a chain between two approximate surfaces and then separating the surfaces. Instead of immobilization Surfaces, the immobilization of the binding partners but also on other solids, e.g. B. 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 the probe oligo and the stamp.

As in case 1, in case 2 the coupling step takes place only after the formation of the Concatenation, d. H. only after the sample complex and the reference complex are already together were connected. The only difference is that in case 1 on the side of the Reference complex and in case 2 on the side of the sample complex is coupled.

In cases 3, 4 and 5, the coupling and the formation of the chain is done by the same step. In case 3, the coupling takes place through the formation of the Sample complex and in case 4 by the formation of the reference complex.

In case 5 both complexes are already formed and with the Force application points (surfaces) connected when there is coupling between the complexes.

Position of the sample molecule in the chain

The above embodiment describes a case in which the analyte nucleic acid or Sample sequence represents the second binding partner of the sample complex and consequently directly with binding partner 3, which is linked to the reference sequence. The Sample sequence occupies 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 Figure 4. Cases A and B show a structure in which the sample is placed centrally (case A corresponds to the above embodiment). Regarding the coupling, one can compare A with case 1 of FIG. 3 and B with case 5 in FIG. 3. At C, on the other hand, the sample molecule is placed peripherally and immobilized on the support. With regard to the coupling, C is comparable to case 5 of FIG. 3.

The sample molecule can be immobilized by placing it in one of the PCR primers that are used to amplify the sample molecule are a biotin introduces. The connection then takes place via the binding of this biotin to the Streptavidin coating of the underlay.

Alternatively, the immobilization can also be carried out via hybridization with a capture oligo respectively. For this purpose, the capture oligo is bound to the base and forms the sample molecule from a complex 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 _Ref- / n _Pro ∼ Q = Marker 2 (stamp) / Marker 1 (pad)

The reference complex

The reference complex can consist of a duplex of nucleic acids or also of one Complex any other binding partner, e.g. B. partners of a specific binding Couple be built.

It is advantageous to choose a reference complex that is among the given Test conditions has a separation force similar to the sample complex. Therefore reference complexes are preferably chosen that correspond to the sample complex in chemistry, Base composition and GC content are similar. However, it can also Reference complexes are used that are opposite the sample complex in length Base composition differ, subject to the conditions given above regarding the free base pairing energy should then be considered.

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 the case.

In the case of the exemplary embodiment, the reference sequence Primer 2 corresponds to Amplification or the original sequence of the genomic DNA. The reference sequence may also consist of a sequence that is not on the genomic DNA was included and only inserted by a degenerate primer during the amplification has been.

detection

The determination of Q ^* = n _Ref- / n _Pro (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. Depends on the The marking used therefore becomes the property of the detector molecule to be determined certainly. This is usually done by evaluating optical or electrical Parameter. 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 separating forces exactly, instead of the ideal Q ^* = n _Ref- / n _Pro ^ = F ^* _Ref / F ^* _{Pro, the} approximate quotient Q is determined, 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 _Ref- / n _production would correspond in this case, remaining on the slide is approximately the ratio of Cy3, which is assigned to the punch to Cy3.

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

The following example shows 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 _Kop = 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, the z. B. can be caused by quality fluctuations in the stamp surface, so 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 _Ref- + n _Pro and Q ^* = n _Ref- / 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 _Kop / n _pro

So 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 be made of DNA, RNA, PNA, or other artificial Nucleotide building blocks exist. As a rule, it is between 15 and 25 in length Base pairs.

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 e.g. B. the quotient for the wild type (Q _WT ) is already known. A significant change compared to Q _WT would then indicate a mutation. However, this only makes sense if a very good reproducibility of all test parameters (e.g. 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 also act as RNA as well as synthetic NA of various origins. If one Amplification is necessary, always depends on the amount of available Sample material. The sample material is preferably amplified by PCR in order to to have enough available.

Claims (30)

1. A method for detecting mutations in a nucleic acid comprising that a) a plurality of nucleic acid strands to be analyzed are brought into contact with at least two types of oligonucleotides, receptor oligos and probe oligos, under conditions such that the oligos hybridize with the DNA to be analyzed to form a sample complex and a reference complex, so that one is immobilized on both sides or immobilizable chaining of sample complex and reference complex is present, b) subsequently, if necessary, immobilizing the chain, c) a tensile force is applied to the chain until one of the complexes separates and d) determining which of the complexes has been separated. 2. Method for the detection of mutations in analyte nucleic acids, in which the Analyte nucleic acid with at least two types of oligonucleotides, Receptor oligos and probe oligos, is brought into contact under such conditions that the oligos hybridize with the analyte nucleic acid to form a Trial complex and a reference complex, wherein the oligonucleotides immobilized or are immobilizable, and where appropriate the oligonucleotides after Hybridization can be immobilized so that one is immobilized on both sides Concatenation of receptor oligo analyte nucleic acid probe oligo is formed; to the Concatenation a tensile force is applied so that one of the complexes formed resolves and determines which of the complexes has been dissolved, wherein the receptor oligonucleotides have a sequence which leads to a Sequence section of the wild-type analyte nucleic acid is complementary in which a mutation or variation is suspected. 3. The method according to claim 1 or claim 2, characterized in that the Connection of analyte nucleic acid and probe oligo via a Coupling sequence takes place. 4. Method for the detection of mutations in analyte nucleic acids, in which Analyte nucleic acid, two types of oligonucleotides, receptor oligos and Probe oligos, and optionally a coupling sequence by hybridization Sample complexes and reference complexes are formed in such a way that the the two complexes are linked together; on the educated Chains a tensile force is exerted, with each of the complexes separating and then determining which of the complexes separated. 5. The method according to any one of the preceding claims, characterized in that such nucleic acids were selected for the sample complex and reference complex that the free base pairing energy ΔG of the hybridized Complexes differ 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 less than 20 kcal / mol. 7. The method according to the preceding claim, characterized in that the free base pairing energy ΔG of the sample and reference complex differs less than 4 kcal / mol. 8. The method according to any one of the preceding claims, characterized in that the number of hybridizing pairs in the sample and reference complex 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 that the number of hybridizing pairs in the sample and reference complex differentiate by one 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 is only about distinguishes a mismatch. 11. The method according to any one of the preceding claims, characterized in that the process simultaneously and in parallel on a variety of Analyte nucleic acids is carried out. 12. The method according to any one of the preceding claims, characterized in that 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 labeling the PCR primers be used. 14. The method according to any one of the preceding claims, characterized in that receptor oligo and / or probe oligo have a partner at one end specific binding pair, the other partner of the specific binding pair is bound to a surface. 15. The method according to any one of the preceding claims, characterized in that 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 one surface. 17. The method according to any one of the preceding claims, characterized in that that receptor oligo and probe oligo are immobilized on one 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 in first with the analyte nucleic acid Be contacted under such conditions 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 be made of an elastic material or with an elastic Material is coated. 20. The method according to claim 19, characterized in that at least one of the coated on both surfaces with polydimethylsiloxane or a derivative thereof is. 21. The method according to any one of the preceding claims, characterized in that receptor oligo and / or probe oligo are either directly covalent, via a Bridge molecule and / or via a specific binding pair on a surface is immobilized. 22. The method according to any one of the preceding claims, characterized in that that the complementary nucleic acid strands DNA, RNA and / or LNA Strands are. 23. The method according to any one of the preceding claims, characterized in that that the partners of the specifically binding pair biotin and streptavidin or Antibiotin antibodies are. 24. The method according to any one of the preceding claims, characterized in that a group is used as the marking, the optical and / or electrical is detectable. 25. The method according to any one of the preceding claims, characterized in that a radioactive, fluorescent, luminescent, chromophore marker or a dye or a conductive group used becomes. 26. The method according to claim 25, characterized in that as a marker fluorescent group is used. 27. The method according to claim 26, characterized in that as a marker Fluorescein isothiocyanate, fluorescein, rhodamine, tetramethylrhodamine-5- (and-6) - Isothiocyanate, Texas Red or a cyanine dye is used. 28. The method according to any one of the preceding claims, characterized in that that two labels are used that are attached to analyte nucleic acid and Probe oligo or reference strand are bound. 29. The method according to any one of the preceding claims, characterized in that that two labels are used that are attached to receptor oligo and Reference strand are bound. 30. The method according to any one of the preceding claims, characterized in that that the unmarked components are immobilized and the marked Components are added last to the reaction mixture.