WO1996027680A1

WO1996027680A1 - Sequence-specific detection of nucleic acids

Info

Publication number: WO1996027680A1
Application number: PCT/EP1996/000893
Authority: WO
Inventors: Jörg KLEIBER; Henrik ØRUM; Ane-Ullerup Lester; Albert Geiger
Original assignee: Boehringer Mannheim Gmbh
Priority date: 1995-03-04
Filing date: 1996-03-04
Publication date: 1996-09-12
Also published as: CA2214430A1; JP2004000154A; JP2000513921A; AU5002996A; EP0813610A1

Abstract

The invention concerns a method of detecting point mutations and polymorphisms in nucleic acids and of sequencing unknown nucleic acids by means of a simple method using arrays. According to the method, nucleic acid analogues are used as sequence discriminators. This method facilitates the operating method, even in complex cases.

Description

Sequence-specific detection of nucleic acids

The invention relates to a solid support, to the surface of which two or more nucleic acid analogs of different base sequences are bound at predetermined locations, and to methods for the detection of nucleic acids using such a support.

The analysis of samples has undergone rapid development in recent decades. While analytes were detected in the early years, in particular through their reaction with conventional chemical reagents and later with enzymes, tests using the immunological properties of the analyte have recently become the standard, particularly in medical diagnostics. This is especially true in the area of infectious diseases. However, only those analytes in which immunologically active compounds, such as antigens or antibodies, play a role can essentially be detected by immunological methods. With many infections by viruses and bacteria, there have been promising applications. Genetic diseases or predispositions that do not or only insufficiently change protein patterns are, however, difficult or impossible to detect by immunological methods. In many cases, nucleic acids have therefore recently been made the subject of detection. The presence of certain nucleic acids can then be used to infer the presence of an infectious agent or the genetic condition of an organism. Detections based on the presence of special nucleotide sequences have been made easier in particular by the fact that methods for amplifying nucleic acids, which are only present in small numbers, are now available. However, because of the large amount of sequence information and the sequence of two nucleotide sequences that are completely different in one base unit, the specific detection of nucleotide sequences still poses a great challenge to reagents and methods of carrying out analyzes based on the detection from Nuklein- acid sequences are based. Often, the nucleotide sequences to be detected are not known as such, but should only be determined by the nucleic acid detection.

EP-B-0-237 362 describes a method for the detection of nucleotide sequences of the HLA gene, with which a clinically relevant point mutation can be found. In this method, an oligonucleotide which is bound to a membrane and which has a nucleotide sequence which is exactly complementary to one of the two nucleic acids to be distinguished is brought into contact with the sample. If certain conditions are met, only the exactly complementary nucleic acid binds to the solid-phase bound oligonucleotide and can be detected.

In Proc. Natl. Acad. Be. USA 86, 6230-6234 (1989) describes a method in which a large number of oligonucleotides which are bound via poly-dT at predetermined different locations on a nylon membrane are used for the simultaneous detection of all known allelic variants of an amplified region of a nucleic acid.

US Pat. No. 5,202,231 describes a method in which the sequence of a nucleic acid can theoretically be determined by bringing oligonucleotides with a predetermined and known sequence into contact with the sample of the unknown nucleic acid under hybridization conditions. All possible permutations of the nucleotide sequence must be immobilized at known locations on a solid phase. By determining at which locations the nucleic acid hybridizes with the sequence to be determined, it can theoretically be determined which sequences are present in the nucleic acid.

In Nucleic Acids Research 22, 5456-5465 (1994) and Clin. Chem. 41/5, 700-6 (1995) describes the state of the art of development in the field of analysis of genetic polymorphisms using so-called oligonucleotide arrays.

The main problem of the prior art is that the melting temperatures of the selected sequence-specific oligonucleotides differ with the nucleic acid to be sequenced or detected. This has to be optimized by elaborate selection of the length of the oligonucleotides, their base composition and optimization of the position of the mismatches within the oligonucleotide and the salt concentration of the hybridization mixture. In many cases, it is still practically impossible to be closely related Differentiate sequences from each other simultaneously. The hybridization temperature is also critical. Even variations of 1 to 2 ° C can lead to modifications in the intensity or to false negative results. Faulty analysis results are very serious, especially in the diagnosis based on the presence of point mutations.

The object of the present invention was therefore to provide an alternative method for the sequence-specific detection of nucleic acids and materials suitable therefor.

The invention is achieved by a solid support, on the surface of which two or more nucleic acid analogs of different base sequences are bound at predetermined, different locations. The invention also relates to a method for the sequence-specific detection of a nucleic acid using this solid support.

For the purposes of the invention, a solid support is understood to mean an object which has a surface which is so extensive that individual areas can be distinguished on it. This surface is preferably flat and larger than 5 mm ² , preferably between 10 mm ² and approximately 100 cm ² . The material of the carrier is not liquid and not gaseous and preferably does not dissolve or does not completely dissolve in sample liquids or reaction mixtures which are used for the immobilization of nucleic acids on the surface. Examples of such materials include glass, plastics (e.g. polystyrene, polyamide, polyethylene, polypropylene), gold or the like. However, the material does not necessarily have to be completely solid itself, but can be made firm, for example, by attachment to support materials.

The external shape of the solid support depends essentially on the manner in which the presence of nucleic acids on this solid support is to be detected. It has proven useful, for example, to choose an essentially planar shape, e.g. B. a tile.

Particularly suitable solid supports are, for. B. polystyrene plates with a thickness of about 1 to 5 mm, an area of about 1 to 5 cm ² . Membranes made of polyamide with a size of 4 x 2.5 cm ^{2 have} proven to be particularly suitable. Two or more nucleic acid analogs of different base sequences are bound at different points on the surface of this support. These locations or areas preferably do not overlap together. They are preferably separated from one another by surface areas to which no nucleic acid analogs are bound. The locations at which the nucleic acid analogs are bound are referred to below as binding regions. The binding regions can have different shapes, which are also essentially determined by the type of preparation of the solid support or by the method by which the nucleic acid analogs are bound in the binding regions. The minimum size of the binding regions is essentially determined by the device with which the event of the binding of a nucleic acid to nucleic acid analogues of a region is detected. Devices already exist that can detect binding in areas of approximately 1 μm. An upper limit on the size of the binding areas results from economic reasons and the desire to be able to handle the solid support in a sensible manner.

The size of the binding areas results in particular from the methods by which the nucleic acid analogs are applied to the surface. Such procedures will be described later.

The number of binding areas on the solid support depends on the intended use of the solid support. In the simplest case, only two binding areas are required for the detection of a specific point mutation. One binding region contains nucleic acid analogs which contain a base at the position at which the point mutation is to be detected, which base is complementary to the base in the position of the normal sequence, while the other binding region contains a nucleic acid analogue which corresponds to the base sequence the position contains a base that is complementary to the base of the mutated sequence. On the other hand, a solid support, to the surface of which two nucleic acid analogs that are completely different from one another in the sequence are bound, can simultaneously detect two nucleic acids or nucleic acid sequences that are not very closely related to one another.

Non-naturally occurring molecules that can recognize nucleic acids via base pairings are understood as nucleic acids analog. They therefore contain a specific base sequence that is completely complementary to a base sequence of a nucleic acid to be detected. The base sequence is therefore preferably composed essentially of the naturally occurring nucleobases. If the specificity of the base pairing is not lost, modifications to the nucleobases are also permitted. Complementary to a nucleic acid are in particular nucleic acid analogs which have a base sequence which, in the state bound to the nucleic acid, forms water bridges to a base sequence of the nucleic acid according to the principle of base pairing. This sequence is preferably at least 8 bases long and particularly preferably between 8 and 25 bases long.

The nucleic acid analogs are further defined by the fact that they differ structurally from nucleic acids, at least in the backbone. A basic structure in nucleic acids or in a nucleic acid analog is understood to be a structure of essentially identical units, each of which contains a base bound. In the naturally occurring nucleic acids, the basic structure is a sugar phosphate basic structure. In nucleic acid analogs, this backbone is structurally modified, for example by completely or partially replacing the sugar or phosphate portion with other chemical units, e.g. B. non-cyclic components. In the basic structure, essentially identical units can also alternate.

Some properties of nucleic acid analogs are given below, which are intended to facilitate the selection of nucleic acid analogs suitable for the present invention. It is an advantageous property of nucleic acid analogs if they have a higher affinity for sequence-complementary nucleic acids than an oligonucleotide with an identical base sequence. Furthermore, nucleic acid analogs are preferred which carry fewer charges than a corresponding oligonucleotide of the same length or which are able to compensate charges with opposite charges. Essentially uncharged nucleic acid analogs are particularly preferred. Particularly preferred nucleic acid analogs are those whose affinity for complementary nucleic acids essentially does not depend on the salt content of the hybridization mixture.

Particularly suitable nucleic acid analogs are the compounds described in WO 92/20702 and WO 92/20703 (peptides nucleic acid, PNA, e.g. Nature 365, 566-568 (1993) and Nucl. Acids Res. 21, 5332-5 (1993)). For a description of the structure of the nucleic acid analogs, reference is hereby made to the patent applications mentioned. Preferred nucleic acid analogs are compounds which have a polyamide backbone which contains a multiplicity of bases bonded along the backbone, each base being bound to a nitrogen atom of the backbone. Under However, nucleic acid analogs are also said to fall into compounds as described in EP-A-0 672 677. Further nucleic acid analogs are described in Recueil 91, 1069-1080 (1971), Methods in Molecular Biology 29, 355-389 (1993), Tetrahedron 31, 73-75 (1975), J. Org. Chem. 52, 4202-4206 ( 1987), Nucl. Acids Res. 17, 6129-6141 (1989), Unusual Properties of New Polymers (Springer Verlag 1983), 1-16, Specialty polymers (Springer Verlag 1981), 1-51, WO 92/20823, WO 94/06815, WO 86/05518 and WO 86/05519. Further nucleic acid analogs are described in Proc. Natl. Acad. Be. USA 91: 7864-7868 (1994), Proc. Natl. Acad. Be. USA 92: 6097-6101 (1995) and J. Am. Chem. Soc. 117, 6140-6141 (1995). The nucleic acid analogs mentioned have a length of 8 to 30 bases, a length of 10 to 25 bases is particularly advantageous. The nucleic acid analogs mentioned are bound directly or indirectly to the surface of the solid support. The type of binding essentially depends on which reactive groups on the solid support are available for binding and which reactive groups on the nucleic acid analog are available for binding without inhibiting the ability of the nucleic acid analog to bind to a complementary nucleic acid, and whether the nucleic acid analogs are to be simultaneously bound to different sites or to be built up at these sites. In addition, it may be expedient to coat the surface of the solid support with a layer of a more bindable material or to activate the surface by a chemical reaction. Reactive groups on the surface of a solid support are usually selected from the group -OH, -NH ₂ and SH. Reactive groups of nucleic acid analogs are preferably selected from the group -OH, -NH ₂ , -SH, -COOH, -SO ₃ H and -PO ₃ H ₂ .

The reactive groups of the surface and the nucleic acid analog are particularly preferably covalently bonded to one another, in particular by means of a linker of more than 15 atoms and less than 200 atoms in length. A linker is understood to mean a part of a molecule which essentially has the function of removing the nucleic acid analogs in a sterically accessible manner on the surface of the solid support. The linker is usually selected so that it contains, in addition to carbon atoms (eg in alkylene units), a plurality of hetero atoms (eg -O- or -NH- or -NR-) which facilitate solvation. The linker preferably contains one or more ethyleneoxy units and / or peptide groups. The linker particularly preferably contains one or more units as described in DE-A 3924705. Particular preference is given to the units described by way of example, hereinafter referred to as ado (8-amino-3,6-dioxa-octanoic acid) be designated. A slight dependence of the binding of nucleic acids on the PNA surface can be reduced by longer linkers between PNA and the solid support.

The nucleic acid analog that is bound at one site can also be a mixture of two or more analogs of different but known sequences. This can reduce the number of digits required for a multiple determination.

The surface of the carrier is preferably not charged and is preferably hydrophilic. It has been shown as a result of the invention that the use of essentially uncharged surfaces is advantageous for the detection of nucleic acids.

A solid support loaded with nucleic acid analogs at different locations in the sense of the invention can be produced in different ways. In a first embodiment, suitable amounts of solutions, each containing different nucleic acid analogs, are applied to the surface of the solid support at different points, e.g. B. pipetted. The amounts of liquid added should not mix here. This can be achieved, for example, by the fact that the feed points are far apart or the spreading of the liquid is stopped by a hydrophobic barrier between the different points. Either the nucleic acid analogs or the surface of the solid support are preferably activated for the reaction. Such activation can be carried out, for example, in that one of the abovementioned groups, by generating a reactive species, in the case of a carboxyl group, for example an activated ester (for example an N-hydroxysuccinimide ester), which rapidly binds an ester to a hydroxyl group without further activation is activated. Suitable activated polyamide membranes carry, for example, triazm groups which can react with amino groups of nucleic acid analogs to form a covalent bond. The activation can also take place via bifunctional reagents, squaric acid derivatives according to WO 95/15983 or by means of glutaraldehyde (GB 2197720).

However, the binding of the analogs can also be achieved by coating the support surface with nucleotide sequences that are complementary to part of the sequence the nucleotide analogs. The different nucleic acid analogs can be bound to the binding regions simultaneously or in succession.

After sufficient time for the binding, it has proven to be advantageous to remove any nucleic acid analogs which have not been bound, or have been bound insufficiently, and any binding reagents which have been used, by washing. This is preferably done under conditions in which unbound nucleic acid analogs cannot form bonds with nucleic acid analogs bound to other regions.

In principle, however, it is also possible to build up the nucleic acid analogs successively from monomer units at the different locations on the surface. The technology described in WO 92/10092 or WO 90/15070 can be used for this purpose. Corresponding monomers are z. B. described in WO 92/20702.

The invention also relates to a method for the sequence-specific detection of a nucleic acid with the aid of the solid support according to the invention.

Detectable nucleic acids are natural or artificial nucleic acids. Nucleic acids are accordingly also understood to mean nucleic acid analogs. In particular, however, the nucleic acid to be detected is RNA or DNA, which is suitable for a nucleic acid-containing organism, e.g. B. a virus, a bacterium, a multi-cell, a plasmid or a genetic state, e.g. B. a predisposition or predisposition to a certain disease or spontaneous mutation in a gene are characteristic. These are essentially RNA and DNA, both genomic and derived from them. An important class of nucleic acids in the sense of the invention are the results of a nucleic acid amplification. These are also referred to below as amplicons or amplicons. The nucleic acids can either be in their raw form or in a purified or processed form. A purification can be present, for example, in that the nucleic acids are separated from cell components in a prepared step, for example by an affinity separation step. In addition, the nucleic acids can be enzymatically extended, specifically amplified or transcribed.

For the sequence-specific detection of the nucleic acid, the carrier according to the invention has a nucleic acid analog bound to one site which has a base sequence which is complementary to a base sequence of the nucleic acid to be detected. This base sequence is chosen so that it allows a statement to be made about the specific presence of the nucleic acid. As a rule, this means that as little as possible, but preferably no further nucleic acid of the same total sequence is present in the mixture. However, it must be established that it is also possible with the aid of the carrier according to the invention to specifically detect groups of nucleic acids. For example, it may be the task of the method to include any members of a particular taxonomic group, e.g. B. a bacterial family to detect their nucleic acids. In this case, the base sequence of the nucleic acid analog can be chosen so that it lies in a conserved area, but this sequence only occurs in members of this taxonomic group.

Another nucleic acid analog is preferably bound at a different location on the surface, which has a base sequence that is not complementary to the same base sequence. It can be a nucleic acid analogue whose base sequence can be shorter or longer or which differs in its base sequence from the first nucleic acid analogue in one or more bases. The difference in the base sequence depends on the task to be solved. The differences may include e.g. B. point mutations, minor deletions and insertions. With some inherited diseases, e.g. B. cystic fibrosis, the sequences of the nucleic acid analogs differ in individual positions (point mutations) and several positions (deletions, see at Δ 508).

The mutation to be detected is preferably positioned near the center of the base sequence of the analog.

The sequences of the analogs can also be chosen such that their hybridization positions differ by one base each (overlapping) while the length remains the same. The sequences can also be chosen so that the hybridization areas are adjacent to the nucleic acid to be detected.

Several mutations in the same or different nucleic acids can also be detected by selecting nucleic acids analogous, the sequence of which is complementary to sequences on these nucleic acids. In a particularly simple case, in which it is to be determined which of two alleles is present in a mixture, two nucleic acid analogs are bound at different locations on the surface of the solid support, the base sequence of which differ exactly in position which also differentiate the alleles. Thus, a nucleic acid analogue complementary to a certain sequence of the one alley and the other nucleic acid analogue complementary to the sequence of the other alien are chosen. The length and hybridization site of the nucleic acid analogs are the same.

In the case of cystic fibrosis, for example, detection of all alleles is usually carried out. The wild-type contains two healthy alleles, heterozygotes contain a mutated and a wild-type sequence and homozygous mutants contain two mutant nucleic acids. In this case, it should not only be determined whether there are mutants, but whether it is a heterozygous or homozygous case. According to the present invention, it is possible to quantitatively detect both alleles simultaneously and thus differentiate between the three cases mentioned.

In some cases, especially in oncology and when determining infection parameters, mutated cells / particles are often present in the background of non-mutated / normal cells. In such cases, the selective detection is not certain or not possible with methods of the prior art. For example, the analysis of ras mutations from DNA from stool requires the reliable detection of a mutated sequence in the presence of approximately 100 normal sequences (Science 256, 102-105, (1992)). In the area of infection parameters, it would be desirable to determine different HIV populations in an infected patient. However, some of these mutants are less than 2% of all HIV sequences. It is therefore particularly possible with the method according to the invention to investigate mixtures of nucleic acids which are very similar to one another, even if one of the nucleic acids is in a very large excess compared to the nucleic acid to be determined.

The lengths of the bound nucleic acid analogs are preferably the same. The selection of a length of between 10 and 100, preferably 10 and 50, bases has proven to be expedient. Particularly good results can be achieved if nucleic acid analogs with a length of between 10 and 25 bases are used. To carry out the method according to the invention, the nucleic acid-containing sample is brought into contact with the sites on the surface of the support which have bound the nucleic acid analogs. This can be done, for example, by introducing the solid support into the sample liquid or pouring the sample liquid onto the solid support in one or more portions. The nucleic acids of the sample liquid can be in a denatured (single-stranded) state before being brought into contact with the support. A great advantage of the invention is, however, that, for. B. by using PNAs, denaturation before contacting can be avoided. The PNAs displace a strand of possibly double-stranded nucleic acid to be determined. It is only essential that the contacting takes place under conditions in which the nucleic acid to be detected specifically binds to the relevant location on the surface via the nucleic acid analogs which are complementary to a sequence of the nucleic acid to be detected. Although these conditions can be different for different types of nucleic acid analogs, they can be obtained in a simple manner by testing for given nucleic acid analogs. As a rule, these conditions will be based on the conditions known for carriers loaded with oligonucleotides. If nucleic acid analogs according to WO 92/20702 are used, however, conditions can be selected which are significantly different from the hybridization conditions of corresponding oligonucleotides. In particular, it has proven to be advantageous to use significantly fewer salts than when using corresponding oligonucleotides. For example, the presence of less than 100 mM, particularly preferably less than 50 mM and very particularly preferably less than 10 mM salts should be emphasized. Under these conditions, sufficient differentiation between sequence-like nucleic acids could not be achieved with oligonucleotides of the same sequence.

The sample is kept in contact with the surface for as long as is necessary for sufficient binding of the nucleic acid to the relevant point on the surface. This period is usually in the range of a few minutes.

It is then determined whether and at what point a nucleic acid has bound to the surface. This is regarded as a sign of the presence of a nucleic acid which is an analogue of the nucleic acid bound at this point contains complementary base sequence. The determination of the binding that has taken place can be done in different ways.

There are devices that can be used to directly detect changes in surfaces at very specific points. For methods using such devices, it is not even necessary to remove the nucleic acid-containing sample applied to the surface from the surface again. As a rule, however, the liquid which is on the surface after being brought into contact is preferably removed from the surface and any remaining reagent residues are removed by a washing solution. This also has the advantage that sample components that could interfere with the determination of the binding are washed off.

The nucleic acid to be detected can be bound to the nucleic acid analog in an advantageous manner by detecting a mark made in the nucleic acid to be detected in a step prior to contacting the surface. This can be, for example, a detectable group, e.g. B. act a fluorescent residue. This determination can be made optically using a microscope or in a measuring cell provided for this purpose. While the location of the binding that took place is an indication of the presence of a nucleic acid with a certain sequence, the amount of label at a predetermined location can be used as a sign of the amount of the presence of the nucleic acid to be detected.

The nucleic acids to be detected are particularly preferably results of a nucleic acid amplification reaction such as the polymerase chain reaction according to EP-B-0 202 362 or NASBA according to EP-A-0 329 822. It is essential for the amplification that the nucleic acid sequence, the is intended for binding to the nucleic acid analogs, is amplified by the amplification process. The more true to the sequence the amplification takes place, ie the fewer errors are built into the sequence during the amplification, the more suitable the amplification method is. The polymerase chain reaction has proven to be particularly suitable. The primers for the amplification are chosen such that the nucleic acid sequence to be detected lies in the area between the primer hybridization sites. In addition, it has proven to be advantageous to incorporate the marking required for the detection reaction into the amplificates during the amplification. This can be done, for example, by using labeled primers or labeled mononucleoside triphosphates.

A direct detection of the binding that has taken place without incorporation of a label is possible, for example, by using an intercalating agent. These agents have the property of being selectively incorporated into double-stranded base-containing compounds, including the complex of the nucleic acid analogs and the sequence-specifically bound nucleic acid. Based on specific properties of the intercalating agents, e.g. B. a fluorescence, the occurrence of the complexes can be detected. A particularly suitable agent is ethidium bromide.

Another possibility of detecting the hybrids without installing a label is given by means of the surface plasmon resonance, e.g. B. according to EP-A-0 618 441.

In another possibility of determining the binding that has taken place, the surface is coated with a solution of a detectably labeled antibody against the complex of nucleic acid analogs. and the nucleic acid to be detected. Such antibodies are described, for example, in WO 95/17430.

The detection of the hybrid depends on the type of marking. It can be, for example, a scanner, a CCD camera or a microscope.

Many advantages can be realized with the present invention. In particular, it is possible to also detect sequence differences in nucleic acid areas that lie within secondary structures. According to the invention, it is also possible to increase the sensitivity of the detection, since with it the absolute amount of bound nucleic acid to be detected can be higher than in conventional methods. In particular, it is also possible to increase the signal-to-noise ratio compared to methods using oligonucleotides. In the first trial, a signal to noise ratio of less than 1: 1000 was available.

Applications of the invention are in at least two areas. In the first case, the solid support is used to detect known mutations and polymorphisms. For this the number of mutations and polymorphisms to be determined is one Measure of the required number of different nucleic acid analogs or sites on the surface. The sequence of the nucleic acid analogs is specially matched to the sequence of the nucleic acids around the mutations and polymorphisms. The sequences are preferably chosen so that the base, in which sequence-like nucleic acid analogs differ, lies in or near the center of the sequence.

The carriers according to the invention can be used in the following fields:

Infectious diseases, simultaneous determination of different analyte parameters, but also investigation of the condition of a gene in a bacterium or virus, e.g. B. Multidrug resistance.

Oncology (detection of mutations in tumor suppressor and oncogenes and determination of the ratio of mutated to normal cells.

Examination of hereditary diseases (cystic fibrosis, sickle cell anemia, etc.).

Typing of tissues and bone marrow (MHC complex) (see Clin. Chem. 41/4, 553-5 (1995)).

In a second application, a sequence of short nucleic acid fragments can be determined using the so-called sequencing by hybridization method. For this purpose, as many different nucleic acid analogs are immobilized as there are per mutations from the selected length of the sequence. To achieve sufficient specificity, 4 ^N sites are required if N is the number of bases in each nucleic acid analog. N is preferably between 5 and 12. Correspondingly fewer sites are required for the sequencing of very short DNA fragments. The method for sequencing unknown nucleic acids by sequencing by hybridization is described in WO 92/10588.

An advantage of the present invention is that the specificity of the hybridization is largely independent of the conditions in the sample. This facilitates the simultaneous binding of nucleic acids to different areas of the surface.

Surprisingly, it has been found that nucleic acid analogs, such as PNA, have an excellent ability to discriminate between sequences on the solid phase. point. This discrimination was better than was to be expected from the melting temperatures of analog loosened connections.

Surprisingly, the carriers according to the invention are even suitable for determining nucleic acids several times in succession. After carrying out a determination, the carrier is subjected to a heat treatment in contact with a liquid. The temperature chosen is one at which the binding of the nucleic acid analog to the optionally bound nucleic acid is released. The carrier is then available for a new determination. With the aid of the method according to the invention, it is possible for the first time to determine relative amounts of very similar nucleic acids present side by side in a sample in a concentration range of at least two logs. A quantitative determination of mutants was previously z. B. possible via sequencing. The discrimination available here, however, was a maximum of 1:10.

It is also possible with the aid of the method according to the invention to bind double-stranded DNA to the immobilized nucleic acid analogs of the solid support without denaturation. Comparative studies have shown that this is not possible with immobilized DNA. It has also been shown that mismatches that are not in the center of the hybrid can also be discriminated with high selectivity.

In FIG. The sequences of the PNA molecules are shown with the help of which the method is explained by way of example. The PNAs were produced according to WO 92/20702.

In FIG. 1b shows the sequences of the DNA molecules which are homologous to the PNA sequences from FIG. la and were used for the DNA / ODN hybridization experiments.

In FIG. The sequences of the complementary oligonucleotides (ODN) used are indicated which are labeled with digoxigenin or with polynucleotide kinase and ³ P-γ-ATP 5 at the 5'-phosphate end using the 5'-DIG end labeling kit (Boehringer Mannheim) - were phosphorylated.

In FIG. The possible combinations of oligonucleotide (ODN) and PNA for the formation of hybrids are shown. Identical combinations apply to hybridizations between DNA probes (DNA, see FIG. 1b) and oligonucleotides (ODN, see FIG. 1c). In FIG. 2 shows the hybridization results from Example 4, which are intended to illustrate the selectivity of the method. The conditions were: 200 nl spot volume (100; 10; 1; 0, 1 μM PNA, one concentration per column), incubation at 45 ° C.

In FIG. 3 shows the hybridization results from Example 6, which demonstrates the detection of PCR amplicons by immobilized PNA probes. The conditions were: 200 nl spot volume (100; 10; 1; 0.1 μM PNA, one concentration per column) incubation at 45 ° C. Also mean:

I control experiment, ODN la (lpMol, InM), line 1 (PNA1), line 2 (PNA2) line 3 (PNA3)

II ss amplificate (118bp, simply DIG-labeled) 5 min. Heat denaturation at 94 ° C

(50μl PCR mixture diluted in 1ml hybridization buffer) Row 1 (PNA1), Row 2 (PNA2) Row 3 (PNA3)

III ds amplificate (118bp, simply DIG marked)

(50μl PCR mix directly diluted in 1ml hybridization buffer line 1 (PNA1), line 2 (PNA2) line 3 (PNA3)

In FIG. 4 shows the results of the qualitative and quantitative analysis of analyte mixtures by PNA arrays. The conditions were: 200 nl spot volume (100 μM PNA, one of the PNA per line, one analyte mixture per column).

FIG. 5 shows the influence of linker length on hybridization. The conditions were: 1 μl spot volume (100; 40; 20; 10; 5; 1 μM PNA, one concentration per column, (Ado) ₃ - PNA in row 1, (Ado) β-PNA in row 2, (Ado) o-PNA in row 3). In FIG. 5 mean:

A. Prehybridization / hybridization in 5mM sodium phosphate, 0.1% SDS, pH 7.0

B. Prehybridization / Hybridization in 10 mM sodium phosphate, 0.1% SDS, pH 7.0

C. Prehybridization / Hybridization in 25mM sodium phosphate, 0.1% SDS, pH 7.0

The FIG. 6a-6c demonstrate the possibility of using PNA-derivatized membranes several times after regeneration. The conditions were: 1 μl spot volume (100; 40; 20; 10; 5, 1 μM PNA, one concentration per column). In FIG. 6 mean:

6a: signal intensities after the first hybridization

6b: Signal intensities after the regeneration procedure

Membrane 1: no regeneration (control)

Membrane 2: regeneration with 0. IM sodium hydroxide solution, RT In, 2x 10min. bidest. Water RT membrane 3: regeneration with IM sodium hydroxide solution, RT lh, 2x 10min. bidest. Water RT membrane 4: regeneration with dist. Water, 70 ° C lh, 2x 10min. bidest. Water RT membrane 5: regeneration with 0. IM sodium hydroxide solution, 70 ° C lh, 2x 10min. bidest. Water RT

6c: signal intensities after rehybridization

The present invention is further illustrated by the following examples:

Examples

General:

The nucleic acid analogs used were prepared in accordance with WO 92/20702. Unless otherwise specified, chemicals and reagents were from Boehnnger Mannheim GmbH.

Example 1:

Covalent derivatization of nylon membranes

200 nl of a solution which contains PNA in the desired concentration in 0.5 M sodium carbonate pH 9.0 are applied to an Immunodyne ABC membrane (Pall) with a pipette. After the spots have dried, any remaining reactive functional surface groups are deactivated by washing the membrane with 0.1 M sodium hydroxide solution. It is washed with water and then the membrane is dried.

Example 2:

Detection of a hybridization event by luminescence

The membrane is derivatized using 100 μM, 10 μM 1 μM and 0.1 μM PNA solutions as described in Example 1. It is then prehybridized in a 50 ml hybridization vessel with 10 ml hybridization buffer (10 mM sodium phosphate pH 7.2, 0.1% SDS (sodium dodecyl sulfate)) in the hybridization oven at 45 ° C. After 30 min. 10 μl of a solution which contains DIG-labeled oligonucleotide in a concentration of 1 μM are added, and the mixture is stirred for a further 60 min. hybridizes. Then 2 x 10 min. washed with 25 ml wash buffer (5 mM sodium phosphate pH 7.2, 0.05% SDS) at 45 ° C. The detection reaction is carried out in accordance with the digoxigenin detection protocol (DIG detection kit, Boehringer Mannheim GmbH, FRG). Anti-DIG-AP conjugate is diluted with 1: 10000 used. CDP-Star ™ is used as a substrate for the alkaline phosphatase in a dilution of 1: 10000.

Example 3:

Detection of a hybridization event by fluorescence

The membrane is derivatized using 100 μM, 10 μM and 1 μM PNA solution as described in example 1. The membrane is prehybridized in a 50 ml screw vessel with 10 ml hybridization buffer (cf. Example 2) in the oven at 45 ° C. After 30 min. 10 μl of a solution which contains a fluorescence-labeled oligonucleotide in a concentration of 1 μM are added, and there are a further 60 min. hybridizes. The membrane is then 2 x 10 min. washed with 25 ml wash buffer (see Example 2) at 45 ° C. The fluorescence intensities are measured after the membrane has dried.

Example 4:

Selectivity of the method

Three membrane strips are each with three (AdoVPNA molecules that differ in their base sequence at one or two positions (see FIG. La, SEQ.ID.NOS. 1, 2, 3)) using PNA solutions in the Concentration range between 100 μM and 0.1 μM derivatized analogously to example 1. The membrane strips are prehybridized in 50 ml screw-top containers with 10 ml hybridization buffer for 30 min. Then one of the three DIG-labeled oligonucleotides (FIG. 1b, SEQ.ID.NOS 4, 5, 6) After 60 minutes of hybridization, 2 × 10 minutes are washed with 25 ml of washing buffer each, and the hybridization events are demonstrated as described in Example 2.

In FIG. All possible double-stranded hybrids between the PNA molecules involved and the oligonucleotides are shown. From FIG. 2 it can be seen that practically only the oligonucleotide which is immobilized is detected Nucleic acid analogs (PNA 1, PNA 2, PNA 3) is exactly complementary. The signal-to-noise ratios (S / N) can also be estimated from the figure. They were evaluated quantitatively, the results are shown in Table 1.

Table 1

Hybrid (PNA / ODN) S / N signal (hybrid) / signal (match)

1/1 655.2 100.0%

2/1 20.7 3.2%

3/1 10.3 1.6%

1/2 23.1 2.6%

2/2 871.8 100.0%

3/2 6.4 0.7%

1/3 109.4 22.7%

2/3 12.3 2.5%

3/3 481.1 100.0%

Example 5:

quantification

Membrane strips are derivatized with three (AdoVPNA molecules, which differ in their base sequence (FIG. La, SEQ.ID.NOS. 1, 2, 3), in a concentration of 100 μM analogously to Example 1. They are then in 20 ml Hybridization tubes with 10 ml hybridization buffer (cf. Example 2) prehybridized at 45 ° C. The buffer is changed after 30 min .. In experiments 1 to 7, the added buffer differs in the analyte concentrations of the DIG-labeled components oligonucleotide 1, 2 and 3, SEQ.ID.NOS. 4, 5, 6. After 60 minutes of hybridization at 45 ° C., 2 × 10 minutes are washed with 10 ml of washing buffer and the detection of a hybridization event is carried out as in Example 2. The luminescence signal is also monitored recorded by a luminescence imager and then evaluated (FIG. 4).

The signal intensities found enable both a qualitative and semi-quantitative statement about the composition of the analyte mixture. Absolute quantitative statements are possible after calibration of the signal intensities.

Example 6:

Detection of PCR amplicons

A. Obtaining a suitable analyte (amplificate)

A double-stranded DNA fragment is ligated into a pUC19 plasmid, the sequence of which is complementary to the PNA probe PNA1. The plasmid is transformed into E. coli, cloned and then sequenced. For the subsequent hybridization experiments, a section of the plasmid sequence is amplified and DIG- marked during the amplification reaction. The amplification is carried out in a total volume of 50 μl. The amplification mixture consists of 1 μl plasmid (1 ng / μl), 1 μl primer Fl (10 μM), 1 μl DIG primer R1 (10 μM), 5 μl 10 × PCR buffer (100 mM Tris / HCl, 15 mM MgCl _2, 500 mM KC1, pH 8.3), 2 μl dNTP solution (10 mM dATP, 10 mM dCTP, 10 mM dGTP, 10 mM dTTP in distilled water pH 7.0), 0.5 μl Taq polymerase (5 unit / μl) and 38.5 μl water .

Primer Fl: 5'-GTA AAA CGA CGG CCA GT-3 '(SEQ.ID.NO. 12)

Primer Rl: 5'-DIG-AAC AGC TAT GAC CAT GA-3 '(SEQ.ID.NO. 13)

Each reaction batch is 3 min. heated to 96 ° C and then 30 rounds of a 3-step PCR cycle are carried out (45 sec. 96 ° C, 30 sec. 48 ° C, 1 min. 72 ° C). In the last cycle, the elongation step is extended by 5 minutes at 72 ° C.

B. Hybridization reaction

The membranes are each with three (AdoVPNA sequences, which differ in their base sequence at one or two positions (see FIG. La, SEQ.ID.NOS. 1, 2, 3), using PNA solutions in Concentration range between 100 μM and 0.1 μM derivatized analogously to Example 1. The membrane is pretreated in a 20 ml hybridization vessel with 5 ml hybridization buffer at 45 ° C. After 30 minutes, the buffer is changed and the analyte solution is added of the analyte solution, the amplification mixture is diluted directly (ds Amplicon) or after 5 min heat denaturation (ss Amplicon) in 1 ml hybridization buffer, after 1 h, 2 h 30 min or 4 h hybridization at 45 ° C, 2 x 10 min. washed with 5 ml of washing buffer each Hybridization events are detected as described in Example 2 (FIG. 3).

In FIG. 3 9 fields can be seen. The rows differ in the length of the incubation period (4 h, 2 ^l Λ h and 1 h). The columns differ in the type of nucleic acid to be detected. In each of the 3 fields of column I there are 3 rows of spots on top of each other. The rows differ in the sequence of the PNAs, while the columns of each field differ in concentration. Both the specificity and the quantifiability in the case of oligonucleotides as the detecting nucleic acid are shown in column I. The influence of the incubation time can be seen in the figure. It is clear that an excellent sequence discrimination for ODN la and the amplificates is achieved even with one hour's hybridization. Columns II and in FIG. 3 differ in that in one case a previously made single-stranded amplificate and in column m a previously not made single-stranded amplificate are used as the nucleic acid to be detected. It can be seen from the signals that it is not necessary to denature double-stranded nucleic acids before application to the solid support. This leads to a saving of work steps (heat step, single strand separation, washing step) and thereby reduces the risk of contamination, so that PNA probes in connection with low salt conditions show clear advantages over DNA probes.

Example 7

Comparison of PNA / DNA hybridization

Membrane strips are each with three (Ado) ₆ -PNA sequences (SEQ.ID.NOS. 1, 2, 3) and three DNA molecules (SEQ.ID.NOS. 8, 10, 11), which differ in their base sequence differentiate at one or two positions (see FIG. la and lb), derivatized using 50 μM solutions analogous to Example 1. In contrast to Example 1, the spot volume is 400 nl instead of 200 nl. The membrane strips are placed in 20 ml hybridization vessels with either 5 ml of low salt buffer (see example 2) or high salt buffer (6 x SSC: 0.9 M NaCl, 90 mM sodium citrate, 0.1% SDS, pH 7.0) at 37 ° C or alternatively 45 ° C 30 min. pre-hybridized. Then one of the three dig-labeled oligonucleotides (FIG. 1c, SEQ.ID.NOS. 4, 5, 6) is added. After 60 min. Hybridization time is washed 2 x 10 min with 5 ml wash buffer at 37 ° C and 45 ° C, respectively. The wash buffer from Example 2 is used for the low salt experiments and a 1 x SSC buffer with 0.02% SDS, pH 7.0 for the high salt experiments. The hybridization events are detected as in Example 2. The evaluation was carried out quantitatively and is shown in Table 2.

Both the DNA and the PNA probes are able to distinguish completely complementary single-stranded target sequences from single and double mismatched sequences. There are clear advantages of the PNA probes over the DNA probes for certain types of mismatch, especially if it is not in the middle of the sequence, but is shifted terminally. This becomes particularly clear with the example of a decentralized G / T mismatch (probe 1 / ODN 3) that is more tolerated by the DNA probe than by the sequence-identical PNA probe.

TabeUe 2

PNAhzv »DNA1 ₁ PNA or - DNA 2ς,

ODN1 PNA 45'C, low salt 100.0% 1.2% 1.5% DNA 37 ^* C, high salt 100.0% 1.2% 6.4%

ODN 2 PNA 45 ^P C, low salt 1.1% 100.0% 2.9% DNA 37'C, high salt <2% * 100.0% <2% ^*

ODN 3 PNA 45 ^* C. low salt 26.0% 1.5% 100.0% DNA 37 ^β C, high salt 66.5% <2% ^* 100.0%

* exact value cannot be determined, because spot intensity is less than standard deviation of the background signal

Example 8

Influence of the length of the linker between the membrane and PNA probes

Membrane strips are made with PNA molecules (see FIG. La, SEQ.ID.NOS. 7, 1, 9), which differ in the length of the linker (Ado ₃ , Ado ₆ and Ado ₉ ), using PNA - Derivatized solutions in the concentration range between 100 uM and 1 uM analogous to Example 1. In contrast to Example 1, the spot volume is 1 μl instead of 200 nl. The membrane strips are in hybridization vessels with 10 ml hybridization buffer (5, 10 or 25 mM sodium phosphate, 0.1% SDS, pH 7.0) for 30 min. Prehybridized at 35 ° C. Then 10 pmol ³ P-labeled oligonucleotide (see FIG. 1c: ODN 1b, SEQ.ID.NO. 4) are added and 60 min. hybridized at 50 ° C. The membranes are 2 x 10 min. washed with 50 ml washing buffer (5 mM sodium phosphate, 0.1% SDS, pH 7.0) at 50 ° C. Hybridization events are detected by autoradiography (FIG. 5). The figure shows that hybridization can be significantly improved with a longer linker.

Example 9

Reuse of PNA membranes

A membrane is derivatized with (Ado) ₆ -PNA molecules (see FIG. La: PNA lb, SEQ.ID.NO. 1) using PNA solutions in the concentration range between 100 μM and 1 μM analogously to Example 1. The PNA is applied in five identical concentration series. As in Example 8, the spot volume is 1 μl. The membrane is in a hybridization vessel with 10 ml hybridization buffer (10 mM sodium phosphate, 0.1% SDS, pH 7.0) for 30 min. Prehybridized at 35 ° C. Then 10 pmol ³ P-labeled oligonucleotide (see FIG. 1c: ODN 1b, SEQ.ID.NO. 4) are added and 60 min. hybridized at 50 ° C. The membrane is 2 x 10 min. washed with 50 ml washing buffer (5 mM sodium phosphate, 0.1% SDS, pH 7.0) at 50 ° C. Hybridization events are detected by autoradiography. (FIG. 6a). After autoradiography, the membrane is cut into five identical strips. These membrane strips are treated differently in the course of the further experiment. Membrane 1 is not incubated and serves as a control membrane, membrane 2 becomes 60 min. incubated at room temperature with 50 ml of 0.1 M sodium hydroxide solution, membrane 3 for the same period with 50 ml of 1 M sodium hydroxide solution, membrane 4 becomes 60 min. at 70 ° C with 50 ml dist. Incubated water and membrane 5 is 60 min. incubated at 70 ° C with 50ml 0.1 N sodium hydroxide solution. Then all membranes are 2 x 10 min. with dist. Washed water at room temperature. After this procedure, an autoradiography is carried out again (FIG. 6b). Finally, these membrane strips are used again in a hybridization reaction as described and the hybridization events are detected by autoradiography (FIG. 6c).

As FIG. 6b shows, very different results are obtained through the different treatment methods. Treatment with bidest water at 70 ° C (membrane 4) causes the membrane to regenerate almost completely. Unexpectedly, the success of the regeneration is worse if conditions are used that i.a. are common for denaturing nucleic acids. Incubation of membrane 3 with 1 M sodium hydroxide solution at room temperature thus has hardly any regeneration effect. Lowering the sodium hydroxide concentration from 1 M to 0.1 M increases the degree of regeneration both at room temperature (membrane 2) and 70 ° C (membrane 5). However, none of these conditions leads to an approximately good degree of regeneration, as is the case with bidest. Water is the case (membrane 4). The example shows that these conditions are important parameters for the efficient denaturation of membrane-bound PNA / DNA double strands.

Regardless of the regeneration method, all membranes can be used for renewed hybridization (FIG. 6c) without a significant deterioration in the signal / noise ratio being observed. Up to 6 re-hybridizations could be carried out without any noticeable influence on the regenerability or the re-hybridization ability of the PNA membranes. Sequence protocol oll

(1. GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Boehringer Mannheim GmbH

(B) STREET: Sandhoferstr. 116

(C) LOCATION: Mannheim

(E) COUNTRY: DE

(F) POSTAL NUMBER: 68298

(G) TELEPHONE: 0621 759 4348 (H) TELEFAX: 0621 759 4457

(ii) DESCRIPTION OF THE INVENTION: Sequence-specific detection of

Nucleic acids

(in) NUMBER OF SEQUENCES: 13

(iv) COMPUTER READABLE VERSION:

(A) DISK: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentin Release # 1.0, Version # 1.30 (EPA)

(2) INFORMATION ON SEQ ID NO: 1:

(i) SEQUENCE LABEL:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ü) TYPE OF MOLECULE: peptide

(iii) HYPOTHETICAL: NO (ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: / product = "OTHER"

/ note = "Xaa is

N - ((1-thyminyl) acetyl) -N- (2-amino (N'-hexa (8-amino-3, 6-dioxa-octano-1-yl)) ethyl) beta alanine "

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: / product = "OTHER" / note = "Xaa is N - ((1-guaninyl) acetyl) -N- (2-aminoethyl) -beta-alanine"

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: / product = "OTHER" / note = "Xaa is N - ((1-thyminyl) acetyl) -N- (2-aminoethyl) -beta-alanine"

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: / product = "OTHER" / note = "Xaa is N - ((1-adenynyl) acetyl) -N- (2-aminoethyl) -beta- alanine"

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: / product = "OTHER" / note = "Xaa is N - ((1-cytosyl) acetyl) -N- (2-aminoethyl) -beta-alanine "

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 6

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: / product = "OTHER" / note = "Xaa is N - ((1-thyminyl) acetyl) -N- (2-aminoethyl) -beta-alanine ^M

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 8

(D) OTHER INFORMATION: / product = "OTHER" / note = "Xaa is N - ((1 -cytosyl) acetyl) -N- (2-aminoethyl) -beta-alanine"

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 9

(D) OTHER INFORMATION: / product = "OTHER" / note = "Xaa is N - ((1-adeninyl) acetyl) -N- (2-aminoethyl) -beta-alanine"

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 10

(D) OTHER INFORMATION: / product = "OTHER" / note = "Xaa is - 32 -

N - ((1-cytosyl) acetyl) -N- (2-aminoethyl) -beta-alanine "

(ix) FEATURE:

(A) NAME KEY: Modified-site

(B) LOCATION: December 11th

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 13

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 14

(D) OTHER INFORMATION: / product = "OTHER" / note = "Xaa is N - ((1-thyminyl) acetyl) -N- (2-aminoethyl) -beta-alanine ''

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 15

(D) OTHER INFORMATION: / product = "OTHER" / note = "Xaa is N- (1-adeninyl) acetyl) -N- (2-aminoethyl) -beta-alanine"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly 1 5 10 15 (2) INFORMATION ON SEQ ID NO: 2:

(i) SEQUENCE LABEL:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Peptide

(iü) HYPOTHETICAL: NO

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: / product = "OTHER"

/ note = "Xaa is

N - ((1-thyminyl) acetyl) -N- (2 - uτϋno (N-hexa (8-amino-3, 6-dioxa-octano-1-yl)) - ethyl) -beta-alanine "

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 2

(ix) FEATURE:

(A) NAME KEY: Modified-site

(B) LOCATION: 3

(ix) FEATURE: (A) NAME / KEY: Modified-site

(B) LOCATION: 4

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 5

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 6

(ix) FEATURE:

(A) NAME KEY: Modified-site

(B) LOCATION: 7

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 8

(D) OTHER INFORMATION: / product = "OTHER" / note = "Xaa is N - ((1-guaninyl) acetyl) -N- (2-aminoethyl) -beta-alanine" (ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 9

(D) OTHER INFORMATION: / product = "OTHER" / note = "Xaa is N - ((1-adenynyl) acetyl) -N- (2 - uτ-inoethyl) -beta-alanine"

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 10

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: December 11th

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 13

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 14

(D) OTHER INFORMATION: / product = "OTHER" / note = "Xaa is N - ((1-thyminyl) acetyl) -N- (2-aminoethyl) -beta-alanine" (ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 15

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly 1 5 10 15

(2) INFORMATION QEQIDNO: 3:

(i) SEQUENCE LABEL:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ü) TYPE OF MOLECULE: peptide

(iii) HYPOTHETICAL: NO

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: / product ^ "OTHER"

/ note = "Xaa is

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 2 (D) OTHER INFORMATION: / product = "OTHER" / note = "Xaa is N - ((1-guaninyl) acetyl) -N- (2-aminoethyl) -beta-alanine"

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 3

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 4

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 5

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: / product = "OTHER" / note = "Xaa is N - ((1-adeninyl) acetyl) -N- (2-aminoethyl) -beta-ala.nine ''

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 7 (D) OTHER INFORMATION: / product = "OTHER" / note = "Xaa is N - ((1-thyminyl) acetyl) -N- (2-aminoethyl) -beta-alanine"

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 8

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 9

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 10

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: December 11th

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 13 (D) OTHER INFORMATION: / product = "OTHER" / note = "Xaa is N - ((1 -cytosyl) acetyl) -N- (2-aminoethyl) -beta-alanine"

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 14

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 15

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly

1 5 10 15

(2) INFORMATION: QIDNO: 4:

(i) SEQUENCE LABEL:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide"

(iii) HYPOTHETICAL: NO (ix) FEATURE:

(A) NAME / KEY: misc_feature

(B) LOCATION: 15

(D) OTHER INFORMATION: / note = "marked on 5'-phosphate with digoxigenin via amino linker (Boehringer Mannheim GmbH, FRG) or 32-P"

(xi) SEQUENCE DESCRIPTION: SEQIDNO: 4:

TAGTTGTGACGTACA 15

(2) SPECIFICATIONS QIDNO: 5:

(i) SEQUENCE LABEL:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide"

(üi) HYPOTHETICAL: NO

(ix) FEATURE:

(A) NAME KEY: misc_feature

(B) LOCATION: 15

(D) OTHER INFORMATION: / note = "marked on 5'-phosphate with digoxigenin via amino linker (Boehringer Mannheim GmbH, FRG)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

TAGTTGTCAC GTACA 15 (2) SPECIFICATIONS QIDNO: 6:

(i) SEQUENCE LABEL:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE. Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide"

(iii) HYPOTHETICAL: NO

(ix) FEATURE:

(A) NAME / KEY: misc eature

(B) LOCATION: 15

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

TAGTTGTGAT GTACA 15

(2) INFORMATION ON SEQ ID NO: 7:

(i) SEQUENCE LABEL:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ü) TYPE OF MOLECULE: peptide

(iii) HYPOTHETICAL: NO (iv) ANTISENSE: YES

(ix) FEATURE:

(A) NAME KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: / product = "OTHER"

/ note = "Xaa is

N - ((1-thyminyl) acetyl) -N- (2-amino (N'-tri (8-amino-3, 6-dioxa-octano-1-y 1)) - ethyl) -beta-alanine "

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 2

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 3

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: / product = "OTHER" / note = "Xaa is N - ((l-adeninyl) acetyl) -N- (2-aminoethyl) -beta-alanine"

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: / product = "OTHER" / note = "Xaa is

N - ((1-cytosyl) acetyl) -N- (2-aminoethyl) -beta-alanine "

(ix) FEATURE:

(A) NAME KEY: Modified-site

(B) LOCATION: 6

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 7

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 8

(ix) FEATURE:

(A) NAME KEY: Modified-site

(B) LOCATION: 9

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION. 10

(D) OTHER INFORMATION: / product = "OTHER" / note = "Xaa is

N - ((1-cytosyl) acetyl) -N- (2-aminoethyl) -beta-alanine "

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: December 11th

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 13

(ix) FEATURE:

(A) NAME KEY: Modified-site

(B) LOCATION: 14

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 15

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly

1 5 10 15 (2) SPECIFICATIONS QIDNO: 8:

(i) SEQUENCE LABEL:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

TTTTTTTTTT TTTTTTGTAC GTCACAACTA 30

(2) INFORMATION ON SEQ ID NO: 9:

(i) SEQUENCE LABEL:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ü) TYPE OF MOLECULE: peptide

(iii) HYPOTHETICAL: NO

(ix) FEATURE:

(A) NAME KEY: Modified-site (B) LOCATION: 1

(D) OTHER INFORMATION: / product = "OTHER" / note = "Xaa is N - ((1-thyminyl) acetyl) -N- (2-amino (N'-nona (8-amino-3, 6-dioxa-octano-1-yl)) ethyl) beta alanine "

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 2

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 3

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 4

(ix) FEATURE.

(A) NAME / KEY: Modified-site

(B) LOCATION: 5

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: / product = "OTHER" / note = "Xaa is

N - ((1-guaninyl) acetyl) -N- (2-aminoethyl) -beta-alanine "

(ix) FEATURE:

(A) NAME KEY: Modified-site

(B) LOCATION: 7

(ix) FEATURE:

(A) NAME KEY: Modified-site

(B) LOCATION: 8

(ix) FEATURE:

(A) NAME KEY: Modified-site

(B) LOCATION: 9

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 10

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 11.12

(D) OTHER INFORMATION: / product = "OTHER" / note = "Xaa is

N - ((1-adeninyl) acetyl) -N- (2-aminoethyl) -beta-alanine "

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 13

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 14

(ix) FEATURE:

(A) NAME / KEY: Modified-site

(B) LOCATION: 15

(D) OTHER INFORMATION: / product = "OTHER" / note = "Xaa is N- (l-adeninyl) acetyl) -N- (2-aminoethyl) -beta-alanine"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly

1 5 10 15

(2) INFORMATION QEQIDNO: 10:

(i) SEQUENCE LABEL:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonicleotide"

(iü) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

TTTTTTTTTT TTTTTTGTAC GTGACAACTA 30

(2) INFORMATION ON SEQ ID NO: 11:

(i) SEQUENCE LABEL:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

TTTTTTTTTT TTTTTTGTAC ATCACAACTA 30

(2) INFORMATION ON SEQ ID NO: 12:

(i) SEQUENCE LABEL:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

GTAAAACGAC GGCCAGT 17th

(2) INFORMATION ON SEQ ID NO: 13:

(i) SEQUENCE LABEL:

(A) LENGTH: 17 base pairs

(B) ART. Nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) TYPE OF MOLEC δL: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide"

(üi) HYPOTHETICAL: NO

(ix) FEATURE:

(A) NAME / KEY: misc_feature

(B) LOCATION: 1

CD) OTHER INFORMATION: / note = "A at the 5 'terminus is with amino modifier

(Boehringer Mannheim GmbH) bound to digoxigenin "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

AACAGCTATG ACCATGA 17

Claims

claims

1. Solid support, on the surface of which two or more nucleic acid analogs of different base sequence are bound at different points.

2. Carrier according to claim 1, characterized in that the nucleic acid analogs are covalently bound.

3. Carrier according to one of claims 1 or 2, characterized in that the nucleic acid analogs are bound via a linker of more than 15 atoms and less than 200 atoms in length.

4. Carrier according to one of the preceding claims, characterized in that the solid carrier has an uncharged and / or hydrophilic surface.

5. Method for the sequence-specific detection of a nucleic acid

Bringing a sample containing nucleic acid into contact with the sites on the surface of a support according to any one of claims 1 to 4, wherein at least one nucleic acid analog has a base sequence that is complementary to a base sequence of the nucleic acid to be detected and at least one further nucleic acid analog has a base sequence, which is not complementary to a base sequence of the nucleic acid to be detected, under conditions in which the nucleic acid to be detected binds to the nucleic acid analogue;

- Determination of the binding that has taken place at the predetermined location as a sign of the presence of the nucleic acid to be detected.

6. The method according to claim 5, characterized in that the conditions under which the nucleic acid to be detected binds to the nucleic acid analogs mean the presence of less than 100 mM salts.

7. The method according to any one of claims 5 or 6, characterized in that the nucleic acid to be detected is the result of a nucleic acid amplification reaction.

8. The method according to any one of claims 5 to 7, characterized in that the nucleic acid is detectably marked.

9. The method according to any one of claims 5 to 7, characterized in that the binding takes place is detected using an intercalating agent.

10. The method according to any one of claims 5 to 7, characterized in that the binding takes place is detected with the help of a detectably labeled antibody against the binding product.

11. A method for the selective detection of mutants of nucleic acids in the presence of a large excess of non-mutant nucleic acids, characterized gekenn¬ characterized in that the nucleic acid-containing sample is brought into contact with a carrier according to claim 1 and the binding of the mutant nucleic acids or / and non-mutant nucleic acids are determined at different sites.

12. A method for determining the relative amount of a mutant and a normal nucleic acid by contacting a sample containing nucleic acid with a solid support according to claim 1 and deriving information for the amount of mutant nucleic acids by comparing the bound mutant nucleic acid and non-mutant nucleic acid.

13. Use of a solid support according to claim 1 for the quantitative determination of nucleic acids.