WO2002029095A1

WO2002029095A1 - Method for mutational analysis

Info

Publication number: WO2002029095A1
Application number: PCT/EP2001/011499
Authority: WO
Inventors: Achim Fischer
Original assignee: Axaron Bioscience Ag
Priority date: 2000-10-06
Filing date: 2001-10-05
Publication date: 2002-04-11
Also published as: AU2002223591A1

Abstract

The invention relates to a method for analysing sequence differences between nucleic acid molecules of a first and a second group. The inventive method comprises the following steps: (aa) heterohybrids are formed; (bb) homohybrids are optionally separated; (cc) nucleic acid molecules of the first group of a heterohybrid are exonucleolytically reduced by means of the double strand specific exonuclease; (dd) heterohybrids comprising a single or multi-base mismatch on the 3' end of the nucleic acid molecule of the first group, i.e. the mismatched heterohybrids, are differentiated from the perfectly matched heterohybrids.

Description

Mutation analysis method

The present invention relates to a method for identifying mutations or sequence variations between nucleic acid groups to be compared, the sequence of which may be known or unknown. The nucleic acid groups are provided in a certain way. A composition comprising two such nucleic acid groups is also the subject of the invention.

The identification of sequence variations is an important discipline in molecular genetics, especially human genetics. Basically, the experimental approaches pursued so far can be divided into two categories.

1. Methods which enable the detection of sequence variations in known nucleic acid sections and

2. Methods that allow the investigation of unknown nucleic acids up to the examination of entire genomes for sequence variations.

The classic method for recognizing sequence variations in known nucleic acids is sequencing, which today is practically carried out almost exclusively according to the Sanger chain termination principle, but despite recent instrumental advances (e.g. capillary sequencing machines that allow the parallel sequencing of 96 samples) only enables a very low throughput. The single strand cohformation polymorphism detection (SSCP, see Orita et al, Proc. Nati. Acad. Sci. USA 1989, 86 (8): 2766-70), which manages the sequence, is more cost-effective without sequencing - depending on the conformation and thus the mobility of a single nucleic acid strand in a denaturing polyacrylamide gel. However, the throughput of this method is also limited by the need to provide a separate gel trace for each of the nucleic acid samples to be examined, and the fragments to be examined must not exceed a certain size (100 to 200 base pairs). The method of the oligonucleotide ligation method (oligonucleotide ligation assay, OLA; Nickerson et al, Proc. Nati. Acad. Sci. USA 1990, 87 (22): 8923- 1), in which two adjoining oligonucleotides are hybridized to a single-stranded nucleic acid molecule to be examined for a mutation at a specific position. If the terminal bases of the oligonucleotides lying opposite one another are complementary to the respective base of the nucleic acid molecule, both oligonucleotides can be connected to one another by a ligation. In the case of a mismatch caused by the mutation to be detected, however, no ligation takes place. A variant of the OLA method, ligase chain reaction (LCR; Wϊedmann et al, PCR Methods Appl. 1994, 3 (4): 51-64), enables signal amplification by amplification, which means detection of mutations in nucleic acids low concentration (such as a single locus within the genome). A disadvantage of OLA, however, is that a separate set of two oligonucleotides must be provided for each position to be checked for a mutation, which can result in high costs. Finally, a method was described in which a Sanger sequencing reaction is carried out on a nucleic acid molecule known per se and the products of the primer extension are hybridized with a counter strand which is to be examined for mutation (WO 00/11222). In the event of a mismatch at the 3 'terminus, which is caused by a sequence variation, the mismatched nucleotide can be removed by means of a self-correcting polymerase (proofreading polymerase) and replaced by a labeled termination nucleotide, so that detection of the mismatch is possible. Since several steps are necessary to carry out this method in order to check a single pair of two mutually corresponding nucleic acid molecules for sequence differences, this method is also associated with high costs.

Probably the oldest method for finding sequence variations in unknown nucleic acids, which can be used to investigate genomes, is the detection of restriction fragment length polymorphisms (restriction fragment length polymorphisms, RFLP, de Martinville et al, Ar% J. Hum. Genet. 1982, 34 (2): 216-26), which is based on the occurrence or the elimination of recognition sites for restriction enzymes by sequence changes and can be used, for example, for the identification of transposon insertion sites. However, only sequence variations that relate to the recognition sites of the respective enzyme can be detected, so that in each RFLP experiment only a small part of all possible sequence variations can be recognized. A newer principle for identifying sequence variations between nucleic acid molecules originating from two different samples is based on the generation of heterohybrid double strands, one from the first sample and one from each from the second sample, largely complementary strand to be hybridized with each other. Sequence differences between the two strands, for example single base exchanges (single nucleotide polymorphisms, SNPs), lead to mismatches within the double strand, since non-complementary bases are opposed to one another. Such internal mismatches can be chemical (chemical mismatch cleavage, Grompe et al, Proc. Nati. Acad. Sei. USA 1989; 86 (15): 5888-92) or enzymatic (Burdon and Lees, Biosci. Rep. 1985, 5 ( 8): 627-32; Biswas and Hsieh, J. Biol. Chem., 1996, 271 (9): 5040-8) so that sequence variations can be detected. However, the widespread use of these mismatch detection methods is opposed by the unsatisfactory efficiency and specificity of the methods, for example different mismatches with different efficiency are recognized (see e.g. Youil et al, Genomics 1996; 32 (3): 431-5; WO 99/42595, see here in particular Fig. 4).

With regard to the prior art, the object of the present invention is to provide a method which combines the following advantages:

1. Detection of sequence variations between nucleic acid groups, which can be both known and unknown;

2. Can also be used with complex nucleic acid groups, for example with cDNA, genomic DNA or representations thereof;

3. reliable detection of individual sequence variations with an excellent signal-to-noise ratio;

very high throughput.

The object of the invention is achieved by a method for analyzing sequence differences between nucleic acid molecules of a first group and a second group, wherein

(a) the nucleic acid molecules of the first group and the second group flanking sequences which are characteristic of the group and have linkers which are sequence-identical for a large number of nucleic acid molecules of a group; (b) the linkers of the nucleic acid molecules of the first group are to be chosen so that at

Formation of hybrids between nucleic acid molecules of the first group and Nucleic acid molecules of the second group, from heterohybrids, starting from nucleic acids of the same group hybridized with one another, from homohybrids, the heterohybridized nucleic acid molecules of both groups, optionally after enzymatic treatment, are a substrate for a double-stranded exonuclease and essentially only the nucleic acid molecules of the first group of a heterohybride be shortened exonucleolytically; with the following steps: (aa) formation of heterohybrids;

(bb) if appropriate, separation of homohybrids if these represent a substrate for the double-stranded exonuclease;

(cc) exonucleolytic shortening of nucleic acid molecules of the first group of a heterohybrid using the double-strand-specific exonuclease; (dd) Differentiation of those heterohybrids which have a mono- or polybasic at the 3 'terminus of the nucleic acid molecule of the first group

Have mismatched, the mismatched heterohybrids, of the heterohybrids paired at their 3 'terminus according to the known base pairing rules, the perfectly paired heterohybrids.

Another object of the invention is a composition comprising nucleic acid molecules of a first group and a second group with the aforementioned features.

The nucleic acid molecules of a first group or a second group are nucleic acid mixtures, preferably nucleic acid mixtures of different origins, in particular nucleic acid mixtures, the sequence information of which can be traced back to genomic DNA, mRNA or cDNA, which was obtained from any different biological samples the term biological sample biological material, which was obtained from one or more individuals. For example, the sequence information of the nucleic acid molecules of the first and the second group can come from tumor biopsies or other biopsies from different patients or patient groups. Such patient groups can, for example, create groups of patients selected according to certain criteria or certain ethnic groups, such as the North American Amish People or the population of Iceland. Furthermore, the nucleic acid molecules of the first and the second group can be derived from the genome of organisms modified by mutagenesis or by spontaneous mutation Kind come from. Finally, the nucleic acid molecules of the first and second groups can be derived from bacterial strains of the same or closely related species that have different properties. Production strains used in biotechnology for the large-scale production of fine chemicals, such as amino acids, are of great economic importance. The determination of the sequence variations between different organism populations can be used to optimize the production processes.

The nucleic acid molecules of the first and the second group have flanking sequences, linkers, which are generally characteristic of the group. In this context, accompanying means that the characteristic sequences, linkers, occur at both ends of a double-stranded nucleic acid molecule and terminate the nucleic acid molecule towards the ends. The linkers are preferably characteristic of the respective group to which the nucleic acid molecules in question belong. This means that the sequence of the linkers of a single nucleic acid molecule and / or a possible labeling group bound to the linker or another modification, whether the nucleic acid molecule is now double-stranded or single-stranded, can be used to conclude that it belongs to a specific group. The linkers are also sequence-identical for a large number of nucleic acid molecules in a group. This means that in the case of a large number of double-stranded nucleic acid molecules (nucleic acid double strands), the sequences of the linkers match if the sequence of the linker is at the 5 'terminus or at the 3' terminus of the (+) strand or the (-) - Strand of one nucleic acid duplex with the sequence of the linker of the (+) strand or of the (-) strand of the other nucleic acid duplex at the same terminus (at the 5 'terminus or at the 3' terminus) or at the other terminus (on the 3rd 'Term or at the 5' term). A nucleic acid duplex has four linker sequences (at the 5 'terminus and at the 3' terminus of the (+) - and the (-) strand). Two nucleic acid double strands have sequence-identical linkers if the sequence of one of the four linker sequences of the one nucleic acid molecule is identical to one of the four linker sequences of the other nucleic acid molecule. Corresponding considerations also apply to the comparison of the linkers of single-stranded nucleic acid molecules. In this case, the complementary strand is mentally supplemented for the purpose of comparison. On the other hand, not all nucleic acid molecules in a group have to have sequence-identical linkers. As a rule, the nucleic acid molecules of a group each have a total of no more than 2-4 ⁵ , preferably no more than 2-4 ⁴ , 2-4 ³ , 2-4 ² in particular no more than 2-4 ³ different sequences characteristic of the group in question. In a particular embodiment, the nucleic acid molecules of a group have only one type of linker or only two types of linker, in the latter case each nucleic acid molecule carrying a linker at one end and a different linker at the opposite end.

These linkers are generally added to the nucleic acid mixture or nucleic acid mixtures to be investigated by known methods. For example, genomic DNA can be cut with one or more restriction enzymes, and double-stranded oligonucleotides can be added to the fragments obtained using a ligase (Mueller and Wold, Science 1989; 246 (4931): 780-6). The two sequences flanking a fragment can be identical or different from one another. The term “oligonucleotide” is to be understood in the broadest sense and encompasses a double-stranded or single-stranded, but preferably double-stranded nucleic acid with 10 to about 100 nucleotide building blocks, which are both naturally occurring and artificially modified or artificially generated nucleotide building blocks amplification by means of PCR is now possible, by using primers which can hybridize with the known flanking sequences.An alternative to the polymerase chain reaction is to use the promoter sequence of an RNA polymerase, for example introducing T7 RNA polymerase, in one or both of the appended flanking sequences and to perform amplification in the form of an in w ^'tro transcription. both ends of a (double) Nuklemsäuremoleküls can be selectively provided with various sequences. in the simplest case this is done by cutting the starting nucleic acid molecules with at least two different restriction endonucleases which produce distinguishable ends, for example an overhanging and a smooth end or a 3 'overhanging end and 5' overhanging end. Then specific and different oligonucleotides are added for the respective end, each forming different linkers. Subsequent amplification with primers which can hybridize to different linkers, using the known PCR suppression effect (Luk'ianov et al, Bioorg. Khim. 1996; 22 (9): 686-90), makes it possible to target those of different ones Sequences flanked nucleic acid fragments amplify, in which one end was generated by one of the restriction endonucleases used and the other end was generated by the other restriction endonuclease used before the linker was added. At the same time, this leads to a reduction in the complexity of the starting nucleic acids used, which is the case, for example, in the case of genomic DNA higher organisms may be desired. It is particularly advantageous to reduce the complexity by generating representations, regarding the term: Lisitsyn et al, Science 1993; 259: 946-51, ie the extraction of subsets defined by clear criteria from the totality of the starting nucleic acids to be analyzed. Such representations can be generated, for example, by cutting genomic DNA with less frequently cutting restriction endonucleases with a recognition sequence which comprises 6, 8 or more bases, followed by a size selection, for example all those fragments whose length does not exceed 1 kb. Another way to generate representations is to use a less frequently cutting restriction endonuclease in combination with a more often cutting restriction endonuclease, followed by obtaining fragments with different ends. As a rule, however, representations are produced in such a way that a set of non-overlapping fragment populations is generated, which in its entirety comprises all or at least largely all of the sequence regions contained in the starting nucleic acids. For this purpose, it is particularly advantageous to fragment the starting nucleic acids with the aid of a restriction endonuclease which cuts sufficiently frequently or a mixture of restriction endonucleases which cuts sufficiently frequently in order to generate fragments in the size range of a few hundred base pairs. This fragment population now initially comprises the entire complexity of the starting nucleic acids. In order to reduce the complexity by dividing it into different, non-overlapping fractions, double-stranded oligonucleotides are added by ligation, the oligonucleotides inserting the sequence of the later linkers, followed by PCR amplification with primers that hybridize with the sequences of the linkers can. Although restriction endonucleases that cut within their recognition site can also be used, the use of type IIS restriction endonucleases is particularly advantageous. These restriction endonucleases cut outside their recognition site and create an overhang of defined length and position relative to the recognition site. In a subsequent step, double-stranded oligonucleotides are added on both sides to the fragments produced. For this purpose, a set of oligonucleotides is provided which contains all possible overhangs of the type specified by the respective restriction endonuclease (i.e. 5 'overhangs or 3' overhangs of a defined length), the sequence of the oligonucleotides, i.e. the later linker sequences, being so different from one another distinguishes that they do not form heterohybrids with each other. This results in a clear assignment of the oligonucleotide, which forms the later linker sequence, and the fragment overhang to which the oligonucleotide in question can be added. The Li gation takes place under conditions which suppress a linkage of overhangs which are not completely complementary to one another (Shaw-Smith et al, Biotechniques 2000, 28 (5): 958-64). By appropriate selection of the primers in the context of a PCR reaction, different representations of nucleic acid molecules of one group can be formed, which representations can be compared with nucleic acid molecules of another group in the context of the method according to the invention.

A further possibility for generating representations consists in amplifying left-flanked nucleic acid fragments by means of primers, so-called selective primers, which are extended at their 3 'end beyond the sequence specified by the linkers, and which allow amplification only of those nucleic acid fragments with which they in particular perfectly base pair can enter at their 3 'end formed by the selective bases.

In addition, representations of genomic DNA can be generated by generating chromosome-specific nucleic acids in a known manner (e.g. with Fluss-Karyotypie, Harris et al, Hum. Genet. 1985; 70 (l): 59-65). The nucleic acids obtained in this way are provided with linkers, for example as described above by restriction with type IIS restriction endonucleases and subsequent ligation with overhang-specific oligonucleotides which contribute the later linker sequences.

The nucleic acid molecules of the first and second groups are thus generally obtained in the same way, but preferably differ by the sequence of their linkers, which according to the invention is characteristic of the group in question, and by the sequence variations, the detection of which is the subject of the method according to the invention , The linkers of the nucleic acid molecules of both groups are generally to be chosen such that when hybrids are formed between nucleic acid molecules of the first group and nucleic acid molecules of the second group, starting from heterohybrids, starting from hybridized nucleic acids of the same group, homohybrids, the heterohybridized linkers of the nucleic acid molecules of both Groups are sequence complementary over at least one sub-area. In the context of the invention, the term hybrid is understood to mean a dimer of nucleic acids with the formation of base pairings. Hybrids between nucleic acid molecules of the first group and nucleic acid molecules of the second group (heterohybrids) are generally formed by melting the double strands of nucleic acids of the same group hybridized with one another (from homohybrids) and subsequent cooling, whereby not only nucleic acid molecules and recombine the same group, but also heterohybrids are formed. Depending on the conditions under which this recombination process is carried out, that is, whether the kinetic or thermodynamic control of the reaction predominates, the proportion of hybrids that have mismatches varies. Optimal conditions consist in a maximum yield of heterohybrids with a minimal proportion of by-products, i.e. hybrids from nucleic acid molecules that are not homologous. In this context, homology means a close relationship between the nucleic acids in question, so that the sequence-complementary region of the two nucleic acid molecules is considerably more extensive than the region occupied by the sequence variations. A nucleic acid molecule (or a region thereof) is sequence complementary to another nucleic acid molecule (or a region thereof) if it can form base pairings with the other molecule (or a region thereof) without mismatches, that is to say perfect base pairings.

The linkers of both groups, which are opposed to each other in the heterohybrid, can generally form base pairing at least over a sub-area. The linkers which have a homohybrid at both ends need not necessarily be attributed to the same oligonucleotides. The partial range of the sequence complement generally comprises at least 4 bases, preferably at least 10 bases, in particular at least 20 bases.

The linkers of the nucleic acid molecules of both groups are also to be chosen such that the heterohybrids, if appropriate after enzymatic treatment, represent a substrate for a double-stranded exonuclease and essentially only the nucleic acid molecules of the first group of a heterohybride are exonucleolytically shortened. Within the scope of the invention, a double-strand-specific exonuclease is an enzyme which degrades a nucleic acid molecule or both nucleic acid molecules of a nucleic acid double-strand in the region of the double-strand which is double-stranded from a terminus of the respective nucleic acid molecule. Degradation in the middle of a nucleic acid molecule (endonucleolytic degradation) is said to be negligible. The use of exonuclease III as double-strand-specific exonuclease is preferred (for the term exonuclase III see bioanalytics, F. Lottsspeich, H. Zorbas, ed., Spektrum Akademischer Verlag, Heidelberg, Berlin, 1998, ISBN 3-4274-0041-4, p. 759). Exonuclase III shortens one or both nucleic acid molecule (s) of a double strand from the 3 'terminus. The 3 'terminus of a nucleic acid molecule is only shortened if the 3' Terminus together with the other nucleic acid molecule forms a smooth end or a 3'- recessed end (i.e. a 5 'overhang).

The heterohybrids do not have to be a substrate for the double-stranded exonuclease in the state in which they are formed. It is possible to incubate the heterohybrids with another enzyme such as a restriction endonuclease (enzymatic treatment) before they are reacted with the double-strand-specific exonuclease. Then, as a rule, the heterohybrids only become a substrate for the double-strand-specific exonuclease as a result of this enzymatic treatment. For example, the heterohybrids can be incubated with a restriction endonuclease or with restriction endonucleases which cut in the region of the linkers, that is to say in the regions of the nucleic acid molecules. The linkers can be selected so that there is a restriction site for the restriction enzyme or the restriction enzymes with which the enzymatic treatment is carried out in the case of homohybrids but not in the case of heterohybrids or vice versa. This ensures, among other things, that the heterohybrids are a substrate for a double-stranded exonuclease and that essentially only the nucleic acid molecules of the first group of a heterohybride (that is to say are the constituents of a heterohybrid) and not the nucleic acid molecules of the second group of a heterohybride are exonucleolytically shortened. This does not mean that homohybrids, which are present in addition to the heterohybrids, should not form a substrate for the double-stranded exonuclease. Within a heterohybrid, however, essentially only the nucleic acid molecules of the first group of a heterohybrid are shortened exonucleolytically. As far as there is an exonucleolytic shortening of the nucleic acid molecules of the second group, this reaction is only a negligible side reaction.

Incidentally, it would also be conceivable to protect the nucleic acid molecules of the second group by a high proportion of degradation-resistant nucleotides, for example one or more of the natural nucleotides could be completely replaced by a degradation-resistant derivative. In this case, it would of course also be possible to attach linkers serving as binding sites for amplification primers only after the formation of heterohybrids from unabridged nucleic acid molecules or of heterohybrids from truncated nucleic acid molecules of the first group and essentially unabridged nucleic acid molecules of the second group. A composition comprising nucleic acid molecules of a first group and a second group as described above is an object of the invention.

Another object is a method for analyzing sequence differences between nucleic acid molecules of a first group and a second group, as described above, with the following steps: (aa) formation of heterohybrids;

(bb) if appropriate, separation of homohybrids if these represent a substrate for the double-stranded exonuclease; (cc) exonucleolytic shortening of nucleic acid molecules of the first group of a heterohybrid using the double-strand-specific exonuclease; (dd) Differentiation of those heterohybrids that have a single- or polybasic mismatch at the 3 'terminus of the nucleic acid molecule of the first group, the mismatched heterohybrids, from the perfectly paired heterohybrids.

The method according to the invention thus initially comprises the formation of heterohybrids, as already described. If the homohybrids represent a substrate for the double-stranded exonuclease in addition to the heterohybrids, which is not excluded according to the invention, then these homohybrids can be separated. This can be done by a variety of measures. A suitable measure would be the separation of the homohybrids from the heterohybrids by the different electrophoretic mobility of homo- and heterohybrids. Furthermore, after the formation of heterohybrids and before the exonucleolytic shortening, an enzymatic treatment can take place, for example a restriction cut in the region of the linkers, which takes place either only in homohybrids or only in heterohybrids, and the separation of homohybrids from selective groups which can be immobilized the heterohybrids. Such a separation is not necessary, for example, if the heterohybrids are converted into a substrate for the double-strand-specific exonuclease by the action of the restriction endonuclease ^), while the homohybrids do not become a substrate for the double-strand-specific endonuclease due to the restriction cut which is not carried out. This can be done in that the linkers carry nucleotides which exclude degradation by a double-stranded exonuclease and which are separated from the heterohybrids as a result of a restriction cut, but not from the homohybrids. This is followed by an exonucleolytic shortening of nucleic acid molecules of the first group of heterohybrids with the aid of the double-strand-specific exonuclease, preferably from the 3 'terminus of the nucleic acid molecule to be degraded in the direction of the 5' terminus. Within a heterohybrid, the nucleic acid molecules of the first group are essentially exclusively exonucleolytically shortened, while the nucleic acid molecules of the second group remain unaffected. In this context essentially means that the exonucleolytic shortening of nucleic acid molecules of the second group of heterohybrids, if it takes place, remains a negligible side reaction. The exonucleolytic truncation of nucleic acid molecules in the first group preferably takes place in such a way that a large number of heterohybrids are formed in which the nucleic acid molecule in question in the first group has been truncated to different extents, and the truncation products are present in as many copies as possible. Accordingly, the collection of the truncated variants of a given nucleic acid molecule with a length of 100 bp, for example, ideally contained all truncated molecules with a length of 1 bp to 99 bp at their 3 'end, the linker being ignored here. In order to achieve a uniform distribution of all possible shortening variants of a heterohybrid species by means of exonucleolytic degradation, the reaction conditions, in particular the reaction time, temperature and amount of the exonuclease used, are chosen such that complete strand degradation does not take place. A very important factor is the processivity of the exonuclease. If the processivity is very high, complete strand degradation is preferred. If the processivity is very low, the shortening products show essentially the same degree of shortening. Using genetic engineering methods, the processivity of the exonuclease must be adjusted so that it lies between these extremes. The term processivity encompasses the number of those nucleotide building blocks that are broken down on a statistical average before the enzyme dissociates from the double-stranded nucleic acid. A particularly uniform distribution of the shortening products can also be achieved in the nucleic acid molecule to be degraded by the statistical incorporation of nucleotides which are resistant to degradation by double-strand-specific exonucleases. These can be, for example, αS thionucleotides (Schreiber et al, Nucleic Acid Res. 1985; 13 (21): 7663-72). If such degradation-resistant nucleotides are statically distributed in the appropriate frequency in the nucleic acid molecule to be degraded, then the double-strand-specific exonuclease can be used in excess, so that complete exonucleolytic degradation takes place up to the respective degradation-resistant nucleotide. An alternative procedure for the production of heterohybrids is to shorten the nucleic acid molecules of the first group by treatment with an exonuclease and only then to produce heterohybrids from shortened nucleic acid molecules of the first group and non-shortened nucleic acid molecules of the second group in a second step , Heterohybrids can also be obtained by generating the shortened nucleic acid molecules of the first group as described in WO 00/11222 by a Sanger sequencing reaction. Such generation of heterohybrids by hybridization already (shortened by exonucleolytic degradation or by strand formation with incorporation of termination nucleotides) shortened nucleic acid molecules of the first group with unabridged nucleic acid molecules of the second group is not preferred in the context of this invention, since it differs Distinguish long nucleic acid molecules in their hybridization kinetics and also very short nucleic acid molecules tend to non-specific hybridization (“cross hybridization”), which could impair the efficiency of the method.

Finally, a distinction is made between those heterohybrids which have a single- or polybasic mismatch at the 3 'terminus of the nuclear acid molecule of the first group, the mismatched heterohybrids, from the perfectly paired heterohybrids. The distinction is usually based on the known ability of certain nucleic acid polymerases to recognize mismatches at the 3 'terminus of a nucleic acid molecule and to exclude such molecules from strand extension.

A DNA or RNA polymerase can be used as the polymerase, which is able to extend set back 3 'terms, but not to extend mismatched terms, but not polymerases which can reduce terminal mismatches with exonuclease activity (proofreading polymerases). Examples of suitable polymerases are, for example, genetically modified T7 DNA polymerases (Δ28T7 DNA polymerase), Taq polymerase or reverse transcriptase. In contrast, native T7 DNA polymerase or Pfu polymerase are not suitable. However, if, as described above, degradation-resistant nucleotides such as thionucleotides are used, proofreading polymerases can of course also be used. In a preferred embodiment of the method according to the invention, the distinction is made

- by filling in the perfectly paired heterohybrids while maintaining a continuous double strand; - separation of the filled heterohybrids from the unfilled, mismatched heterohybrids; and

- Identification of the unfilled heterohybrids.

The perfectly paired heterohybrids are filled in by a DNA polymerase, which extends the shortened nucleic acid molecule in the 5 '-3' direction, i.e. fills in until a continuous double strand with a mostly smooth end is obtained. The filled heterohybrids are separated from the unfilled, mismatched heterohybrids. This can be done by using nucleotide monomers which carry immobilizable groups in the replenishment reaction in the previous step, so that the products of the replenishment reaction themselves can be immobilized and thus differentiated from the hetero-hybrids that have not been replenished, the mismatched heterohybrids can be. The non-filled heterohybrids are identified by known methods, for example by hybridization with an arrangement of single-stranded nucleic acids on a surface. This can be followed by known sequencing methods, such as the Sanger sequencing method (strand-lengthening) or Brenner (strand-shortening, US Pat. No. 5,846,719).

In a further embodiment of the method according to the invention, the distinction is made:

by filling in the perfectly paired heterohybrids to obtain a continuous double strand with nucleotides of a first type;

Removal of the mismatched base (s) of the mismatched heterohybrids, so that the mismatched heterohybrids become perfectly paired heterohybrids;

Filling in the now perfectly paired heterohybrids formed with nucleotides of a second type; the nucleotides of a first type have an immobilizable group that do not have the nucleotides of the second type, or the nucleotides of the first type do not have an immobilizable group that have the nucleotides of the second type;

Separation of the originally mismatched heterohybrids from the originally perfectly paired heterohybrids by immobilizing either the originally mismatched heterohybrids or the originally perfectly paired heterohybrids; - Identification of the originally mismatched heterohybrids.

The removal of the mismatched base (s) of the mismatched heterohybrids, followed by the replenishment of the perfectly paired heterohybrids, can of course be replaced by replenishment of mismatched heterohybrids if the polymerization conditions set for replenishment are sufficiently permissive, i.e. the most complete possible extension of mismatched heterohybrids by allow the chosen polymerase. For example, the Klenow fragment of DNA polymerase I from E. coli is able to efficiently replenish mismatched heterohybrids at high nucleotide and magnesium concentrations (for example 200 μM dNTPs / 2 mM MgCl ₂ ).

The immobilizable group is preferably biotin, which enables immobilization via streptavidin. Other examples of immobilizable groups are sugars that can be recognized by lectins, or molecular groups that allow immobilization by means of antibodies binding the molecular groups. Examples of the latter would be digoxigenin or dinitrophenol derivatives. Such immobilizable groups can of course also be used as so-called caged compounds (Methods Enzymol. 1998; 291: 415-30), in which the immobilizable group is only released as a result of a photo reaction.

In a further embodiment of the method according to the invention, the distinction is made by

- Filling the perfectly paired heterohybrids while maintaining a continuous double strand; - Isolation of the mismatched heterohybrids via hybridization with an immobilized or immobilizable oligonucleotide and subsequent immobilization of the hybrid of heterohybrid and oligonucleotide;

- Identification of the originally mismatched heterohybrids.

The perfectly paired heterohybrids are filled in while maintaining a continuous double strand as described above. This means that only the mismatched heterohybrids have a non-continuous double strand. This enables hybridization with an immobilized or immobilizable oligonucleotide and the subsequent immobilization of the hybrid of heterohybrid and oligonucleotide to a solid phase, as a result of which the mismatched heterohybrids can be separated from the originally perfectly paired heterohybrids. The isolated, originally mismatched heterohybrids are identified by known methods such as the sequencing methods described above.

In a further embodiment of the method according to the invention, the distinction is made by:

- Extension of the 3 'terminus of the perfectly paired heterohybrids by a nucleotide leading to chain termination;

- if necessary, removal of unincorporated nucleotides leading to chain termination;

- Filling in the perfectly paired heterohybrids formed in the previous step to form a continuous double strand; Isolation of the heterohybrids formed in the form of a double strand and formed in the previous step;

- Identification of the isolated heterohybrids.

For the replacement of the removal of the mismatched base (s) of the mismatched heterohybrids, followed by the replenishment of the perfectly paired heterohybrids, by replenishment of mismatched heterohybrids, the above applies analogously.

With the help of an appropriate DNA polymerase, the 3 'terminus of the perfectly paired heterohybrids is extended by a nucleotide leading to chain termination. A preferred nucleotide leading to chain termination is a dideoxynucleotide such as ddATP, ddCTP, ddTTP and ddGTP. Thereafter, the nucleotides which are not incorporated and which lead to chain termination are optionally removed (optional). The mismatched base (s) of the mismatched heterohybrids are preferably removed with the participation of a self-correcting DNA polymerase (proofreading polymerase). The perfectly paired heterohybrids formed in the previous step are then filled in to form a continuous double strand, no nucleotides leading to chain termination being used. The heterohybrids, which are in the form of a double strand, are then isolated, for example on the basis of their electrophoretic mobility or by cloning. If the originally mismatched heterohybrids are isolated by cloning, the heterohybrids present in the form of a continuous double strand become at both ends with a restriction enzyme, preferably in the region of the linker, cut and ligated with a correspondingly prepared vector and then cloned. Linker or restriction endonucleases are to be selected in such a way that double strands which are not continuous cannot be cut on both sides by the restriction endonucleases. The isolated heterohybrids are identified by known measures.

In a further embodiment of the method according to the invention, the nucleotide leading to chain termination carries an immobilizable grappe and the heterohybrids present in the form of a double strand are isolated by immobilization and separation of the heterohybrids extended by an immobilizable group. The immobilizable group can be, for example, a biotin group, which can be immobilized by binding to streptavidin.

In a further embodiment of the method according to the invention, the homohybrids of step (bb) are cut in the region of the linkers, the restriction site being present only in the case of homohybrids, but not in the case of heterohybrids. In an alternative embodiment of the method according to the invention, the reverse is the case. The restriction interface is only available for heterohybrids, but not for homohybrids. As a result, either homohybrids or heterohybrids have cut ends which enable the homohybrids to be separated from the heterohybrids. If the linkers have nucleotides which are resistant to exonucleolytic degradation and which are separated in heterohybrids but not in homohybrids as a result of the restriction, then only the heterohybrids are a substrate for the double-strand-specific exonuclease, so that the homohybrids are separated in step (bb ) can be omitted. In this case, the linker of the nucleic acid molecule of the second group should carry a nucleotide resistant to exonucleolytic degradation in the vicinity of the 3 'terminus, that is to say in the region of the linker, in order to ensure that essentially only the nucleic acid molecules of the first group of a heterohybrid shorten exonucleolytically ): become.

In a further embodiment of the method according to the invention, the linkers of the nucleic acid molecules of the first Grappe are chosen so that when heterohybrids are formed, the linkers of the nucleic acid molecules of both groups form a 5 'overhang and a 3' overhang and both overhangs each by the nucleic acid molecules of the second Group are formed. The easiest way to achieve this is that the linkers of the nucleic acid molecules of the second group of a heterohybrid are longer than the linkers of the nucleic acid molecules of the first group of the same heterohybride. In a further embodiment of the method according to the invention, the homohybrids have 3 'overhangs on both sides, so that a separation can be dispensed with.

In a further embodiment of the method according to the invention, the mismatched heterohybrids are distinguished from the perfectly paired heterohybrids by amplification. For this purpose, for example, the filled-in, perfectly paired heterohybrids can be cut in the region of one of the two linkers by means of one or more specifically double-stranded DNA-cutting restriction endonuclease (s), so that the complete linker concerned or a partial region of this linker is removed. Since the corresponding area is single-stranded in the mismatched, unfilled heterohybrids, it is not separated from them in the restriction step. If an amplification is subsequently carried out using primers whose primer binding site is in the region of the linkers flanking the hybrids, only those molecules can be amplified whose linkers have been retained in the restriction step. Before the restriction step, care must of course be taken that only one of the two linkers of a heterohybrid, which was single-stranded after the exonucleolytic truncation, can be cut, but not the other, which remains in the double-stranded state. This can be achieved, for example, by the linker strands in the heterohybrid containing non-functional, “masked” restriction sites, that is to say interfaces which are only present in a homohybrid but not in a heterohybrid linker in a fractional state. The reaction sequence “exonucleolytic shortening - Replenishment of the perfectly paired heterohybrids ", the linker strand, which has become single-stranded after shortening, is supplemented by its reverse complement to the double strand, which now contains an" unmasked ", ie functional interface. The linker at the opposite end of the same heterohybride, however, remains non-functional, so that only shortened and then replenished heterohybrids can be cut at one end in the linker (in the "unmasked" interface): NNN GGATCC GGTACCNNN NNN CCATGG CCTAGGNNN

shortening

NNN GGATCC NNN CCATGG CCTAGGNNN

filling

NNN GGATCC GGATCC NNN NNN CCATGG CCTAGGNNN

Cut with Bamül (recognition site GGATCC)

NNN GGATCC G NNN CCATGG CCTAG

If this procedure is performed after cutting the filled heterohybrids and before amplification, the mismatched heterohybrids are also filled (see Fig. 15), the amplification product contains a mixture of both sequence variants of the corresponding nucleic acid molecule. If, on the other hand, the mismatched heterohybrids are not filled in, only the strand which has remained unabridged and which comes from nucleic acid molecules of the second group is amplified.

A procedure analogous to the result for distinguishing mismatched from perfectly paired heterohybrids is, after successful exonucleolytic shortening and extension of the perfectly paired heterohybrids, by a nucleotide leading to chain termination and subsequent filling of the mismatched heterohybrids under permissive conditions or using a proofreading polymerase complete double strand to remove single-stranded overhangs. Since the perfectly paired heterohybrids, which are extended by a kettan-terminating nucleotide, have a single-stranded overhang encompassing the region of one of the linkers, can also be removed in this way, primer binding sites not to amplify desired nucleic acid molecules (see Fig. 14)

Also possible, although not preferred, would be the generation of heterohybrids from already shortened nucleic acid molecules of the first Grappe and not shortened nucleic acid molecules of the second group.

Furthermore, the attachment of linkers as binding sites for amplification primers could also only be carried out after shortening and differentiation of mismatched heterohybrids from perfectly paired heterohybrids.

In a likewise preferred embodiment of the method according to the invention for identifying sequence differences, which are represented differently frequently in two different groups of individuals, the procedure is as follows (cf. FIG. 12): The nucleic acid molecules isolated from the respective individuals of a patient group become nucleic acid molecules of the first Grappe first Origin combined and the nucleic acid molecules isolated from the respective individuals of a second patient group are combined to form nucleic acid molecules of the first Grappe of second origin. Furthermore, nucleic acid molecules of a further origin are provided as a "reference", which are obtained, for example, from an individual who does not belong to either of the above two groups or from a cell culture. In this context, patient group is understood not only as an individual group of human patients, but also as a group of individual animals, plants, cells or tissues. According to one of the embodiments explained in more detail above, the method according to the invention is then carried out in parallel batches, the nucleic acids obtained from one patient group (first origin) in one of the batches and the nucleic acids obtained from the other patient group (second batch) in the other batch ) are used as nucleic acid molecules of the first group. In both approaches, the reference nucleic acid molecules serve as nucleic acids of the second group. The method according to the invention is then used to isolate nucleic acid molecules which, in the respective batch, have sequence differences from the reference nucleic acid molecules provided. The procedure is preferably such that the isolated nucleotides Acid molecules of the two patient groups to be compared (first and second origin) obtained markings distinguishable from each other in both approaches. For example, when carrying out the method according to the second embodiment described above, the unfilled heterohybrids obtained in step (6b) could be labeled with the fluorescent dye Cy3 in the case of the first patient group and with the fluorescent dye Cy5 in the case of the second patient group. An arrangement of reference nucleic acid molecules would then be provided, for example in the form of an array on a solid surface, which can take place in a conventional manner by depositing nucleic acid clones in a regular arrangement or by surface amplification following a random arrangement, or in the form of particles loaded with identical nucleic acid molecules. In any case, the arrangement should have the properties already described above that identical and hybridization-capable nucleic acid molecules are localized at one location. Location can mean both an area on a mostly planar surface and the surface of a particle. In any case, the differently labeled nucleic acid molecules described above, which each represent sequence differences between nucleic acids of the first patient group (first origin) and the reference or between nucleic acids of the second patient group (second origin) and the reference, would be hybridized with said arrangement. After evaluation, four different situations can be distinguished: (1) a fragment from the first patient group is identical to the sequence both with the corresponding fragment from the second patient group and with the respective reference fragment. There will be no heterohybrids whose double-stranded region ends in a mismatch, so no probe molecules representing this fragment will be obtained. After hybridization, no hybridization signal is obtained from the locations of the arrangement belonging to this fragment. (2) a fragment from the first patient group has a sequence difference compared to the respective reference fragment, while the corresponding fragment from the second patient group is sequence-identical with the respective reference fragment. Therefore, only probe molecules are obtained that carry a label that identifies a sequence difference between the reference and the first group of patients, in the example above a Cy3 label. After hybridization, the locations of the arrangement belonging to this fragment become only one of these (Cy3) labels received signal. (3) a fragment from the second patient group has a sequence difference compared to the respective reference fragment, while the corresponding fragment from the first patient group is sequence-identical with the respective reference fragment. Therefore, only probe molecules are obtained that carry a label that identifies a sequence difference between the reference and the second patient group, in the example above, that is, a Cy5 label. After hybridization, only a signal originating from this (Cy5) label is obtained from the locations of the arrangement belonging to this fragment. (4) The fragments from the first and second patient groups have a sequence difference compared to the respective reference fragment. Therefore, both probe molecules are obtained which have a marking which distinguishes a sequence difference between the reference and the first group of patients, and probe molecules which have a marking which distinguishes a sequence difference between the reference and the second group of patients. After hybridization, a signal originating from both labels is therefore obtained from the locations of the arrangement belonging to this fragment. To identify those fragments which are distinguished by a sequence difference between the first and second patient group, nucleic acid molecules located at those locations are therefore identified which are distinguished by a hybridization signal obtained only or predominantly from one of the two probe preparations. This isolation can be carried out as described above by using the respective clones in the case of an ordered array, by photolytically releasing the nucleic acid molecules of interest in the case of a random arrangement generated by surface amplification, or by means of particles in the case of particles loaded with nucleic acids the relevant particles are sorted out in a sorting apparatus. ^J

While the classification described above only distinguishes cases in which the nucleic acid molecules from the first and from the second patient group are identical to each other, it is of course also conceivable that sequence differences exist within a patient group. This will usually be the case, for example, if nucleic acid molecules are obtained from different, non-clonal individuals and interact with one another. who are united. For example, a certain allele of a given gene within a patient group can occupy all relative frequencies between 0% and 100%. If the relative frequency of this allele differs between the two individual patient groups, probe molecules derived from the corresponding fragment and representing the first or the second patient group are obtained in different amounts, so the hybridization leads to mixed signals at the corresponding locations of the arrangement. The method according to the invention therefore not only allows the identification of sequence differences that occur in a first group of patients and does not occur in a second patient group, but also the identification of sequence differences that occur in different patient groups at different frequencies.

In a further modification of the method according to the invention, the shortened heterohybrids can also be produced using an exonuclease with 5 '→ 3' exonuclease activity. Here, the exonuclease with 5'— 3'-exonuclease activity - just like the exonuclease with 3 '→ 5'-exonuclease activity - again degrades only one strand of the double-stranded heterohybrids (the nucleic acid molecule of the first group). Similar to the embodiments described above, the exonucleolytic degradation of only one strand of nucleic acid can preferably be achieved using the exonuclease with 5 '->3'-exonuclease activity in such a way that one of the linkers has incorporated α-thio-nucleotides which have an exonucleolytic degradation of the strand prevent who has said linker sequence at its 5 'end. This further preferred embodiment of the method according to the invention consists in that the nucleic acid molecules of the first group are shortened by means of an exonuclease working in the 5 '→ 3' direction, for example T7 exonuclease or lambda exonuclease, followed by an extension of a linker primer by means of a polymerase without strand displacement activity and a ligation of the extension products to the shortening products to distinguish perfectly paired heterohybrids from heterohybrids, which are characterized by a terminal mismatch. Such a modification would include in step (dd) a distinction between those heterohybrids that were at the 5 'terminus of the nucleic acid molecule of the first grappe have a single- or multi-base mismatch, the mismatched heterohybrids, of the perfectly paired heterohybrids. In this case, a distinction would be the ability of ligases, in the case of interruptions of one of the two sugar-phosphate chains, single-strand breaks, present in a nucleic acid double strand, to differentiate between perfectly paired 5 'terms and mismatched 5' terms by perfectly paired 5 'terms with the neighboring 3' -Termini are connected by ligation, while mismatched 5 '-terminini are not connected to the neighboring 3'-termini. The subsequent separation of perfectly paired ligated heterohybrids from unmatched mismatched heterohybrids could be done, for example, by one of the following measures:

- Incorporation of immobilizable nucleotides during the extension of a primer hybridized to the truncated heterohybrids, after pairing perfectly paired

Heterohybrids the non-ligated extension products are obtained by immobilization,

Incorporation of immobilizable nucleotides into the nucleic acid molecules of the first and / or the second grappe, the ligated extension products being obtained by immobilization after ligation of perfectly paired heterohybrids,

- Incorporation of immobilizable nucleotides into the extension products which have remained unligated and contain no immobilizable nucleotides, in that the extension products after the ligation step have been carried out in the presence of said immobilizable nucleotides by means of a polymerase, which is distinguished by strand displacement activity, with displacement of the shortened to an am 5 'end-lying mismatch of the nucleic acid molecule from the first grappe, followed by immobilization of the extension products thus extended,

- Melting of the extension products which have remained unligated from the shortened heterohybrid, followed by a re-hybridization of the nucleic acid molecules of the first and the second group previously hybridized with one another, followed by a separation of the primer binding site belonging to the respective extension product by means of a restriction endonuclease which is only present in a double-stranded linker region, but cannot cut in a single-stranded linker region, followed by amplification only of those nucleic acid molecules in which no restriction endonucleolytic separation of a linker region has taken place, the shortened nucleic acid molecule of the first group and the unabridged nucleic acid molecule of the second group during the melting and re-hybridization, optionally by the use of hαirpin-ilάenάen linkers, preferably covalently linked to one another. The invention is described in more detail below by the figures. It shows:

1 the production of heterohybrids, which enable a selective shortening of only one strand,

2 shows the production of heterohybrids with a shortened strand, FIG. 3 shows the isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second group, FIG. 4 shows an alternative isolation of a fragment from a mixture from nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second group, FIG. 5 shows a further alternative isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second group,

6 shows a further alternative isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second group, FIG. 7 shows a further alternative isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between 8 shows a further alternative isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second group, FIG. 9 shows a further alternative isolation of a fragment from a Mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and second grappa, FIG. 10 shows the selective restriction of homohybrids in the region of the linkers, FIG. 11 shows the selective restriction of homohybrids in the region of the link he followed by isolation of a heterohybride carrying a sequence variation and preparation of a hybridization probe (Fig. 11 comprises FIGS. 1 a and 1 lb, which form a coherent FIG. 11), FIG. 12 shows the use of the method according to the invention for the identification of sequence variations between nucleic acids originating from two different samples using reference nucleic acids (FIG 12 includes Fig. 12a,

12b and 12c which form a coherent Fig. 12), 13 shows the selective restriction of heterohybrids in the region of the linkers, FIG. 14 shows an alternative isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second grappa (FIG. 14 includes FIGS. 14a and FIG 14b, which form a coherent Fig. 14),

15 shows a further alternative isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second grappa (FIG. 15 comprises FIGS. 15a and 15b, which form a coherent FIG. 15), 16 shows a further alternative isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second group.

Fig. 1 shows the production of heterohybrids, which enable a selective shortening of only one strand. It shows in detail

1 Production of heterohybrids from nucleic acid molecules of a first group (linkers marked by open rectangles) and a second group (linkers marked by hatched rectangles) Grapples, which are characterized by differently long flanking linkers. A sequence variation (SNP) of the nucleic acid molecules of the first origin compared to the nucleic acid molecules of the second origin is identified by a dot.

2 Exonucleolytic shortening of those strands which are distinguished by a recessed 3 'end in the 3' → 5 'direction. "Exo" denotes the exonuclease acting on it. For each heterohybrid double strand, only one possible shortening variant from a mixture of shortening variants is shown here.

Fig. 2 shows the production of heterohybrids with a shortened strand, shows in the eggshell

1 the production of heterohybrids from nucleic acid molecules of two groups, which are characterized by linkers flanking for different lengths, 2 the selective exonucleolytic shortening of those strands which are distinguished by a recessed 3 'end. Three shortening variants from the obtained collection of differently shortened molecular variants are shown. In the middle of this, the last base of the double-stranded region coincides with the position of the sequence variation mentioned in FIG. 1, so that there is a terminal mismatch.

3 shows the isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second group. The mixture here consists of two fragments, of which the shorter one between the first and second origin is sequence-identical, while the longer one has a sequence variation between the first and second origin. 1 shows the extraction of heterohybrids,

2 the selective exonucleolytic shortening of the nucleic acid molecules of the first group of the heterohybrids from (1), made possible by protecting the nucleic acid molecule of the second group of the heterohybrids against exonucleolytic degradation. Two fragments with shortenings of different strengths are shown, the longer of the two fragments being one

Shortening product represents, whose last nucleotide of the double-stranded region represents the sequence variation between nucleic acid molecules of both grappas (point). This shortening product is therefore characterized by a mismatch at the end of the double-stranded area,

3 Filling in those heterohybrids which have no mismatch at the end of the double-stranded region with the addition of biotin-modified nucleotide building blocks. Biotin grapples are represented by "B", 4 separation of biotin-containing heterohybrids from unextended, non-biotin-containing heterohybrids, which represent fragments containing sequence variations.

4 shows an alternative isolation of a fragment from a mixture of nucleic acid molecules, which shows a sequence variation between the nucleic acid molecules of the first and the second group in detail 1 the extraction of heterohybrids,

2 the selective exonucleolytic shortening of nucleic acid molecules of the first group in the heterohybrids from (1),

3 replenishment of those heterohybrids which have no mismatch at the end of the double-stranded area,

4 removal of the mismatched terminal base or bases in unfilled heterohybrids, followed by filling with addition of biotin-modified nucleotide building blocks,

5 Isolation of biotin-containing heterohybrids filled in (4), which represent fragments containing sequence variations, with separation in (3) filled-in, non-biotin-containing heterohybrids.

5 shows a further alternative isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second grappa. In detail shows

1 the extraction of heterohybrids,

2 the selective exonucleolytic shortening of the nucleic acid molecules of the first Grappe in the heterohybrids from (1),

4 Isolation of unfilled heterohybrids, which represent fragments containing sequence variations, by hybridization with an immobilized or immobilizable oligonucleotide which is complementary to the unfilled single-stranded linker section of the nucleic acid molecule of the second grappe.

6 shows a further alternative isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second group. 1 shows the extraction of heterohybrids,

3 Extension of those heterohybrids which have no mismatch at the end of the double-stranded region by a nucleotide leading to chain termination, 4 removal of the mismatched terminal base or bases in hetero-hybrids that have not been extended, followed by filling with the addition of biotin-modified nucleotide building blocks,

5 Isolation of biotin-containing heterohybrids filled in (4), which represent fragments containing sequence variations, with separation of the non-biotin-containing heterohybrids extended in (3).

7 shows a further alternative isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second grappa. In detail shows

1 the extraction of heterohybrids,

3 extension of those heterohybrids which have no mismatch at the end of the double-stranded region by a nucleotide leading to chain termination,

4 removal of the mismatched terminal base or bases in unextended heterohybrids, followed by replenishment,

5 Separation of unfilled heterohybrids, which do not represent fragments containing sequence variations, by hybridization with an immobilized or immobilizable oligonucleotide which is complementary to the unfilled single-stranded linker section of the nucleic acid molecule of the second group.

8 shows a further alternative isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second group. In detail shows

1 the extraction of heterohybrids,

2 the selective exonucleolytic shortening of the nucleic acid molecules of the first group in the heterohybrids from (1),

3 Extension of those heterohybrids which have no mismatch at the end of the double-stranded region by a nucleotide coupled to biotin and leading to chain termination,

4 Separation in (3) extended, biotin-containing heterohybrids. 9 shows a further alternative isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second group. 1 shows the extraction of heterohybrids, 2 shows the selective exonucleolytic shortening of the nucleic acid molecules of the first group in the heterohybrids from (1), 3 extension of those heterohybrids which have no mismatches at the end of the double-stranded region by a nucleotide leading to chain termination, 4 Removal of the mismatched terminal base or bases in unfilled heterohybrids, followed by an extension with biotin-modified, chain terminating nucleotide building blocks, 5 isolation of biotin-containing, in (4) elongated heterohybrids, which represent fragments containing sequence variations, with separation in ( 3) prolonged, non-biotin-containing heterohybrids.

10 shows the selective restriction of homohybrids in the region of the linkers. Linkers were attached to nucleic acid fragments (gray) generated using the restriction endonuclease HiήPll (recognition sequence GCGC). Linkers of the fragments of the first group (open rectangles) contain a recognition site for the restriction endonuclease Hαell (recognition sequence AGCGCT), linkers of the fragments of the second group (hatched) an alternative recognition site for the same restriction endonuclease GGCGCC. In detail shows

1 the denaturation of the nucleic acid fragments flanked by linkers of both Grappes, followed by a rehybridization of strands that are partially complementary to one another to form homo- and heterohybrids,

2 the treatment of the reaction mixture with Haell, so that the linkers of the Ηomohybrids formed in step (1) by “reannealing” are shortened, while the Ηeterohybrids formed remain flanked by intact linkers. Since the recognition site of the restriction endonuclease used for the initial fragment generation, H PlI, which is part of the recognition sites used in step (2), prevents undesired, fragment-internal cuts in step (2). The 3 ′ overhanging ends produced by Hαell are not a substrate for exonuclease III (“Exo”), while the 3 ′ -Returned ends of the heterohybrids of a strand-specific shortening according to FIG. 1 are accessible. There are de ends of a heterohybrid shown; only one of them can be shortened exonucleolytically.

11 shows the selective restriction of homohybrids in the region of the linkers, followed by the isolation of a heterohybride representing a sequence variation and the

Manufacture of a hybridization probe. In detail shows

1. the denaturation of the nucleic acid fragments flanked by linkers of both groups, followed by a re-hybridization of at least partially complementary strands to homo- and heterohybrids, 2 the treatment of the reaction mixture with two restriction endonucleases (recognition sites shown as black or dotted rectangles), so that the Linkers of the homohybrids formed in step (1) by “reannealing” are separated off, but the heterohybrids formed remain flanked by linkers, 3 exonucleolytic shortening of the nucleic acid molecules of the first

Grappe in the heterohybrids. As an example, only a shortening product is shown, the last base of the double-stranded region is located at the position of a sequence variation,

4 replenishment of all shortening products whose last base of the double-stranded region does not form a mismatch, followed by solid-phase immobilization of the shortening products that have not been filled by hybridization with an immobilized oligonucleotide (corresponding to steps 3 and 4 from FIG. 5),

5 washing away all non-immobilized nucleic acids, 6 denaturing detachment of the immobilized nucleic acids,

7 linear amplification of the nucleic acid molecules of second origin representing a sequence variation (corresponding to the “long” fragments from FIGS. 3 to 9) in the presence of labeled nucleotide building blocks, so that hybridization probes representing sequence variation are produced. Labeled probe molecules are identified by an asterisk.

12 shows an application of the method according to the invention for the identification of

Sequence variations between nucleic acids from two different samples using reference nucleic acids. A certain nucleic acid fragment flanked by linker molecules is shown, which consists of the first sample originating from short hatched linkers, from the second sample originating from short dotted linkers and from the reference sample originating from long rectangles represented as open rectangles. The position of a sequence variation (SNP) between the fragment originating from the first sample and the fragments originating from the second sample or the reference sample is identified by a dot. The fragments from the reference sample used for the described implementation with fragments from the first sample are provided with a marker group, which is symbolized by an asterisk. The fragments used for the described implementation with fragments from the second sample and originating from the reference sample are provided with a marking group which can be distinguished from the first marking range, which is symbolized by "#"

1 Production of heterohybrids from nucleic acid molecules from the first sample and from the reference sample or from the second sample and the

Reference sample, which are characterized by flanking linkers of different lengths,

2 Exonucleolytic shortening of those strands which are distinguished by a recessed 3 'end, so that heterohybrids are formed with single-stranded 5' overhangs which vary in length,

3 Filling in those partially single-stranded heterohybrids whose transition from double-stranded area to single-stranded area is characterized by perfect base pairing in the presence of biotin-modified nucleotide building blocks (symbolized by “B”), 4 separation of the biotinylated heterohybrids filled in (3) by

Immobilization on a streptavidm-coated surface, 5 recovery of the labeled reference fragment strands hybridized on strands which remained unfilled in (4) by complete exonucleolytic degradation of the already shortened strand, 6 combination of the strands in (5) by hybridization of strands from the first

Sample with strands of the reference sample as well as labeled reference fragment strands obtained from strands from the second sample with strands of the reference sample, 7 hybridization of the labeled reference fragment strands from (6) with an arrangement of nucleic acid molecules bound to particles, or alternatively : 8 hybridization of the labeled reference fragment strands from (6) with an arrangement of nucleic acid molecules bound to a planar surface,

9 steps 1-7 or alternatively steps 1-6 followed by step 8; then detection of the hybridization signals obtained. 10 means: no signal, since there is no sequence variation between the first and second sample and reference sample; 11: signal indicates sequence variation between first sample and reference sample; 12: signal indicates sequence variation between second sample and reference sample; 13; Signal indicates sequence variation, both between the first sample and reference sample and between the second sample and reference sample.

13 shows the selective restriction of heterohybrids in the region of the linkers. Biotinylated linkers are attached to nucleic acid fragments generated by means of the restriction endonuclease Dpril (recognition sequence GATC). The linkers contain masked recognition sequences for the restriction endonuclease Bamlϊl (recognition sequence GGATCC). The linkers attached to the nucleic acid molecules of the second group have a thionucleotide in the vicinity of the recognition sequence (denoted by “S”). In detail, 1. shows the denaturation of the nucleic acid fragments flanked by linkers of both grappas, followed by a re-hybridization of strands that are partially complementary to one another to homo and hetero hybrids,

2. the treatment of the reaction mixture with Bamlϊl, so that the linkers of the heterohybrids formed in step (1) by “reannealing” are shortened, while the resulting heterohybrids remain flanked by intact linkers,

3. Separation of the biotinylated homohybrids by immobilization on a streptavidin-coated solid phase. Only the nucleic acid molecule of the first group of a fragment can be shortened exonucleolytically, that of the second group is protected by degradation-resistant nucleotides. Both ends of the nucleic acid duplex are shown.

14 shows an alternative isolation of a fragment from a mixture of nucleic acid molecules, which has a sequence variation between the nucleic acid molecules of the first and the second group. In detail shows 1 the extraction of heterohybrids,

4 Replenishment of the shortened heterohybrids that have remained unfilled in (3) and ends in a mismatch under “permissive” conditions under which the selected polymerase terminally mismatched nucleic acid ranks are accepted as a substrate (alternatively, a proofreading polymerase could also be used, which terminal mismatch initially removed),

5 Breakdown of single-stranded overhangs, which characterize the heterohybrids filled in (3) with a nucleotide leading to chain termination, by means of a single-strand-specific nuclease such as Mung

Bean nuclease

6 the amplification of the nucleic acid molecules filled in (4) into double strands with blunt ends by means of amplification primers which can bind to the linker regions of the nucleic acid molecules.

15 shows a further alternative isolation of a fragment from a mixture of nucleic acid molecules, which has a sequence variation between the nucleic acid molecules of the first and the second group. 1 shows the extraction of heterohybrids,

3 extension of those heterohybrids which have no mismatch at the end of the double-stranded region to nucleic acid double strands with smooth ends,

4 cutting the filled nucleic acid molecules in the area marked by the linkers so that the linker which has become double-stranded again by filling is completely or partially separated from it, 5 optionally filling the heterohybrids which have remained unfilled in (3) and which are characterized by a terminal mismatch , under "Permissive" conditions under which the granted polymerase terminal accepts mismatched nucleic acid strands as a substrate (alternative

_ A proofreading polymerase could also be used, which first removes the terminal mismatch), 6 the amplification of the double strands flanked in (5) to both sides by linkers by means of amplification primers, which can bind to the linker regions of the nucleic acid molecules, which essentially match those in the in (3) filled heterohybrids in (4) distant areas.

16 shows a further alternative isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second group. 1 shows the production of heterohybrids, 2 shows the selective exonucleolytic shortening of the nucleic acid molecules of the first group in the heterohybrids from (1) in the 5 '→ 3' direction,

3 shows the hybridization of an oligonucleotide primer to the single-stranded linker region, followed by an extension by means of a polymerase having no beach displacement activity, wherein nucleotide building blocks containing biotin are incorporated,

4 the ligation of those heterohybrids in which the 5 'terminus of the nucleic acid molecule of the first grappe is perfectly paired, those heterohybrids in which the 5' terminus of the nucleic acid molecule of the first group is characterized by a mismatch remain unligated,

5 the further extension of the prime extension products from step (3) which remained unligated in step (4) by means of a polymerase with strand displacement activity, followed by immobilization by binding to a streptavidin-coated solid phase, followed by the removal of the nucleic acid molecules of the second group hybridized to the extension products and a subsequent amplification of the immobilized nucleic acid strands by means of amplification primers which hybridize specifically with the linker regions. The invention is described below by examples:

Example 1: Production of linkers

For the linker production, oligonucleotides DL20 (5'-GCA TCA CAA GAA TCG ACG CT-3 '), LD24 (5'-phosphate-GAT CAG CGT CGA TTC TTG TGA TGC-3'), ML25BGL (5'-TCA CAT) were supplied lyophilized GCT AAG TGA CGT AGA TCT T-3 '), LM33BGL (5'-Phosphate-GAT CAA GAT CTA CGT CAC TTA GCA TGT GAC AAT-3'), ML33BAM (5'-GCT CAT TGT CAC ATG CTA AGT GAC GTG GAT CCT-3 ') and LM41BAM (5'-phosphate-GAT CAG GAT CCA CGT CAC TTA GCA TGT GAC AAT GAG CAT CG-3') to a final concentration of 100 pmol / μl in water. In three separate batches, 30 ul DL20 and 30 ul LD24 (".Dp / iII-Linker"), 30 ul ML25BGL and 30 ul LM33BGL ("Rg / II-Linker") or 30 ul ML33BAM and 30 ul LM41BAM ( “JSαmHI linker”) was mixed with 11 μl lOx ligation buffer (Röche Molecular Biochemicals AG, Mannheim) and 39 μl water. The mixtures were placed in a thermal cycler preheated to 90 ° C. (Personal Cycler, Biometra GmbH, Göttingen) and cooled at a cooling rate of 0.05 ° C / sec .. The finished linkers DL2024 (from DL20 and LD24), ML2533BGL (from ML25BGL and LM33BGL) and ML3341BAM (from ML33BAM and LM41BAM) were frozen at -20 ° C until use stored.

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Example 2: Production of left-flanked genomic DNA

Two different strains of Corynebacterium glutamicum, to be examined for sequence variations, were cultivated and used to inoculate liquid cultures (“preparation 1” and “preparation 2”). 30 ml on an OD ₆₀ o = 2 grown overnight cultures in LB medium supplemented with 2% glucose were centrifuged off; the cell pellets were washed with 5 ml TE buffer (10 mM Tris pH 8.0 / 1 mM EDTA). The cells were resuspended in 1.5 ml TE buffer, which had previously been adjusted to 50 mM glucose. After adding 15 mg of lysozyme (Sigma-Aldrich GmbH, Taufkirchen) and 1.5 mg of RNase (Promega GmbH, Mannheim), the mixture was incubated at 37 ° C. for 2 h. A further 1.1 ml of TE / 50 mM glucose were added and 1 ml of 10% SDS (Sigma) and 15 μl of an aqueous 10 mg / ml Proteinase K solution (Promega) were added, mixed and incubated at 55 ° C. for 1 hour. The reaction batches were cooled on ice, extracted with 1.5 ml of TE-saturated phenol and then with 1.5 ml of chloroform, and 1/10 part by volume of 3M sodium acetate solution pH 5.2 was added. After addition of 2.5 volume parts of ethanol, DNA precipitated was fished out of the solution using a Pasteur pipette melted into a tick, washed in a fresh reaction vessel with 70% ethanol, air-dried and finally dissolved in TE buffer. 2 μg each of the genomic C. glutamicum DNA were taken up in 40 μl NEBuffer Dpnϊl (New England Biolabs GmbH, Frankfurt), mixed with 5 U Dpnll (New England Biolabs) and incubated for 1.5 h at 37 ° C. The samples were extracted with phenol, then with chloroform and precipitated with ethanol. It was taken up in 10 μl of a ligation mixture containing 0.6 μl lOx ligation buffer (Röche), 1 μl 10 mM ATP (Röche), 4 μl 0.5 μg / μl linker DL2024, 3.9 μl H ₂ O and 0, 5 ul T4 DNA ligase (Röche). The ligation took place at 16 ° C. overnight. Unligated and self-ligated linkers were separated by centrifugation through ChromaSpin 100 columns (Clontech Inc., Palo Alto, CA, USA).

Example 3: Amplification of left-flanked geήomic DNA and linker exchange

One-tenth of the left-flanked fragments of genomic DNA obtained in Example 2 were used as templates in a 100 μl amplification mixture containing 10 μl 10 × AmpHTaq buffer (PE Applied Biosystems, Foster City, CA, USA), 2 mM MgCl ₂ , 0, 4 mM oligonucleotide DL20, 200 μM dNTPs, and 2.5 U AmpHTaq DNA polymerase (PE Applied Biosystems). One of the preparations (preparation 1) was 10% of each nucleotide replaced by its respective α-thioanalog (Amersham-Pharmacia GmbH, Freiburg). An amplification was carried out with the following temperature program: 1 min 95 ° C, then 25 cycles consisting of 30 sec 95 ° C, 30 sec 60 ° C, 1 min 72 ° C. The amplification was checked using agarose gel electrophoresis, then the reactions were purified using QiaQuick columns (Qiagen GmbH, Hilden). The eluted amplification products were taken up in 50 μl NEBuffer Dpήll, mixed with 5 U Dpnϊl and incubated for 1.5 h at 37 ° C. The cut linkers were removed using ChromaSpin 100 columns and the eluted DNA fragments were precipitated with ethanol. The pellets were taken up in 10 μl ligation batches, containing 0.6 μl 10 × ligation buffer, 1 μl 10 mM ATP, 4 μl 0.5 μg / μl linker, 3.9 μl H ₂ O and 0.5 μl T4 DNA - ligase. Linker ML2533BGL was used for the fragments of preparation 1 and linker ML3341BAM for preparation 2. The ligation took place at 16 ° C. overnight. Unligated and self-ligated linkers were separated by centrifugation through ChromaSpin 200 columns (Clontech).

Example 4: Extraction and replenishment of exonucleolytically shortened heterohybrids

A mixture of 1 ug of the obtained in Example 3 linkerflankierten DNA fragments from Preparation 1 and 1 ug of the linkerflankierten DNA fragments from Preparation 2 was dissolved in 30 .mu.l of hybridization buffer (Sambrook et al., Molecular Cloning., 2 ^nd Edition, 1989) was added , After overlaying with paraffm oil and denaturing for 2 min at 95 ° C, hybridization was carried out at 70 ° C for 24 h. It was made up to 100 μl with water and precipitated with ethanol. The precipitated nucleic acids, which now form homohybrids and heterohybrids, were taken up in 50 μl of ExoIII buffer (New England Biolabs). After addition of 10 units (U) of exonuclease III (New England Biolabs), the mixture was incubated at 37 ° C. for 30 min, then the enzyme was denatured at 70 ° C. for 20 min. The nucleic acids were extracted with phenol, then with chloroform and precipitated with ethanol. The pellet was dissolved in 50 μl TY z-PCR buffer (Röche), which contained all four dNTPs in a concentration of 100 μM. After adding 2 U Tt / VPolymerase (Röche) for 5 incubated min at 75 ° C, so that the shortened, perfectly paired heterohybrids were filled. It was purified using QiaQuick, and the nucleic acids were taken up in 100 μl RαmHI buffer (New England Biolabs). After adding 50 U each of BamBI and Bgal (New England Biolabs), the mixture was incubated at 37 ° C. for 2 h. The linkers separated from the homohybrids and from the filled heterohybrids were removed by centrifugation through ChromaSpin 100 columns. 1 μl of the eluate, which contained the sequence variation fragments of preparation 2 in unabridged form, was mixed with 200 μM dNTPs, 1.5 mM MgCl ₂ , 0.5 μM oligonucleotide primer ML 18 (5′-TCA CAT GCT AAG TGA CGT-3 ') and 2.5 U AmpHTaq DNA polymerase amplified in 50 μl AmpHTaq buffer; the amplification conditions were: 25 cycles consisting of 30 sec 95 ° C, 30 sec 55 ° C, 1 min 72 ° C. The amplification products obtained were checked by means of agarose gel electrophoresis and used for cloning in a T / A vector (pCR2.1 TOPO, Invitrogen, Groningen, NL). The clones obtained were sequenced and used to design PCR primers for verifying the identified regions, which were identified as having sequence variations between genomic DNA of preparation 1 and preparation 2.

Claims

claims

1. A composition comprising nucleic acid molecules of a first grappe and a second grappe, wherein

(a) the nucleic acid molecules of the first group and the second group flanking sequences which are characteristic of the group and have linkers which are sequence-identical for a large number of nucleic acid molecules of a group;

(b) the linkers of the nucleic acid molecules of the first group are to be selected such that when hybrids are formed between nucleic acid molecules of the first group and nucleic acid molecules of the second group, from heterohybrids, starting from nucleic acids of the same group that are hybridized with each other, from homohybrids, the heterohybridized nucleic acid molecules of both groups , optionally after enzymatic treatment, constitute a substrate for a double-stranded exonuclease and essentially only the nucleic acid molecules of the first group of a heterohybrid are exonucleolytically shortened.

2. Method for analyzing sequence differences between nucleic acid molecules of a first and a second grappa, wherein

(a) the nucleic acid molecules of the first Grappe and the second group have flanking sequences, linkers, which are characteristic of the Grappe and which are sequence-identical in a large number of nucleic acid molecules of a Grappe;

(b) the linkers of the nucleic acid molecules of the first Grappe are to be chosen so that when hybrids are formed between nucleic acid molecules of the first Grappe and nucleic acid molecules of the second Grappe, heterohybrids, starting from nucleic acids of the same group hybridized with each other

Homohybrids, which are heterohybridized nucleic acid molecules of both groups, optionally after enzymatic treatment, a substrate for a double-stranded exonuclease and essentially only the nucleic acid molecules of the first group of a heterohybrid are shortened exonucleolytically; with the following steps: (aa) formation of heterohybrids;

(bb) optionally separating homohybrids;

(cc) exonucleolytic shortening of nucleic acid molecules of the first

Group of a heterohybrid using the double-strand-specific exonuclease; (dd) Differentiation of those heterohybrids that are at the 3 'terminus of the

Nucleic acid molecule of the first group is a single or multi-base

Show mismatch, the mismatched heterohybrids, of the perfectly paired heterohybrids.

3. The method according to claim 2, characterized in that the distinction is made by

- Filling the perfectly paired heterohybrids while maintaining a continuous double strand;

Separation of the filled heterohybrids from the unfilled, mismatched heterohybrids; and

- Identification of the unfilled heterohybrids.

4. The method according spoke 2, characterized in that the distinction is made by

- Filling the perfectly paired heterohybrids to obtain a continuous double strand with nucleotides of a first type;

Filling the perfectly paired heterohybrids formed in the last step with nucleotides of a second type;

- The nucleotides of a first type have an immobilizable group that the nucleotides of the second type do not have, or the nucleotides of the first type do not have an immobilizable group that the nucleotides of the second

Have kind;

Separation of the originally mismatched heterohybrids from the originally perfectly paired heterohybrids by immobilization of either the originally mismatched heterohybrids or the originally perfectly paired heterohybrids;

- Identification of the originally mismatched heterohybrids. Method according to claim 2, characterized in that the distinction is made by:

- Isolation of the mismatched heterohybrids via hybridization with an immobilized or immobilizable oligonucleotide and subsequent immobilization of the hybrid of heterohybrid and oligonucleotide;

- Identification of the originally mismatched heterohybrids.

6. The method according to claim 2, characterized in that the distinction is made by:

- Extension of the 3 'terminus of the perfectly paired heterohybrids by a nucleotide leading to chain termination; - If necessary, separation of non-installed, leading to chain break

nucleotides;

- Filling in the perfectly paired heterohybrids formed in the previous step to form a continuous double strand;

Isolation of the heterohybrids formed in the form of a double strand and formed in the previous step;

- Identification of the isolated heterohybrids.

7. The method according to claim 6, characterized in that the nucleotide leading to chain termination carries an immobilizable group and the isolation of the heterohybrids present in the form of a double strand is carried out by immobilization and separation of the heterohybrids extended by the immobilizable group.

8. The method according to claim 2, characterized in that the homohybrids of step (bb) are cut in the region of the linker and the restriction interface is only present in homohybrids, but not in heterohybrids.

9. The method according to claim 2, characterized in that the heterohybrids of step (aa) are cut in the region of the linker and the restriction interface is only present for heterohybrids, but not for homohybrids.

10. The method according to any one of claims 2 to 9, characterized in that in feature (b) the linkers of the nucleic acid molecules of the second grappa have at least one nucleotide building block resistant to exonucleolytic degradation.

11. The composition according to claim 1, characterized in that the heterohybridized linkers of the nucleic acid molecules of both groups form a 5 'overhang and a 3' overhang and both overhangs are each formed by the nucleic acid molecules of the second group.

12. The method according to any one of claims 2 to 8, characterized in that in feature (b) the linker of the nucleic acid molecules of the first Grappe are to be selected such that when heterohybrids are formed, the linker of the nucleic acid molecules of both groups is a 5 'and a Form 3 'overhang and both overhangs are each formed by the nucleic acid molecules of the second group.

13. The method according to claim 12, wherein in step (bb) the homohybrids are provided in a form which is characterized by 3 'overhangs on both sides and the separation is dispensed with.

14. The composition according to claim 1, characterized in that the linkers of the unabridged heterohybrids comprise a recognition sequence of at least one restriction endonuclease in the form that the linker strand forming the 5 'end of the respective strand contains said recognition sequence, during which the 3' end of the linker strand forming the respective strand does not contain the reverse complement of this sequence.

15. The method according to any one of claims 2 to 8, characterized in that in feature (b) the linkers of the nucleic acid molecules of the first group are to be selected so that when heterohybrids are formed, the linkers of the unabridged heterohybrids have a recognition sequence of at least one restriction endonuclease in the Form include that the linker strand forming the 5 'end of the respective strand contains said recognition sequence, while the linker strand forming the 3' end of the respective strand does not contain the reverse complement of this sequence.

16. The method according spoke 15, characterized in that the distinction is made by:

- Filling the perfectly paired heterohybrids while maintaining a continuous double strand; Cutting the filled heterohybrids in the region of the linker by means of at least one restriction endonuclease, which can cut the linker which has become double-stranded again due to filling, but cannot cut the single-stranded linker or the opposite linker,

Amplification of the heterohybrids, which were not cut in the region of the linker in the previous step, by means of amplification primers which can only bind to uncut, but not to cut, linker regions of the heterohybrids,

- Identification of the originally mismatched heterohybrids.

17. The method according to claim 4 or 6, characterized in that the removal of the mismatched base (s) of the mismatched heterohybrids is dispensed with and the subsequent replenishment takes place under conditions under which the polymerase used also accepts terminally mismatched heterohybrids as a substrate.

18. A composition comprising nucleic acid molecules of a first group and a second group, wherein

(a) the nucleic acid molecules of the first group and the second group flanking sequences which are characteristic of the Grappe and have linkers which are sequence-identical in a large number of nucleic acid molecules of a Grappe;

(b) the nucleic acid molecules of the second group contain degradation-resistant nucleotides, so that the nucleic acid molecules are also essentially degradation-resistant

19. A composition comprising nucleic acid molecules of a first group and a second group, wherein

(a) the nucleic acid molecules of the first group and the second group flanking sequences which are characteristic of the group and have linkers which are sequence-identical for a large number of nucleic acid molecules of a group; (b) the nucleic acid molecules of the first grappe with the length n contain a small proportion of degradation-resistant nucleotides at a random position, so that an exonucleolytic strand degradation also terminates at a random position, the position of a degradation-resistant nucleotide, and the mixture of shortening products obtained essentially all contains possible shortening products of a length between 1 and n-1 nucleotides in essentially equal proportions.

20. A method for analyzing sequence differences between nucleic acid molecules of a first and a second Grappe, wherein

(a) the nucleic acid molecules of the first Grappe and the second group have flanking sequences, linkers, which are characteristic of the group and which are sequence-identical for a large number of nucleic acid molecules of a group; (b) the nucleic acid molecules of the second grappe contain degradation-resistant nucleotides, so that the nucleic acid molecules are also essentially degradation-resistant; with the following steps: (aa) formation of heterohybrids; (bb) optionally separating homohybrids;

(cc) exonucleolytic shortening of nucleic acid molecules of the first

Group of a heterohybrid using a double-stranded exonuclease;

(dd) Differentiation of those heterohybrids which have a mono- or polybasic at the 3 'terminus of the nucleic acid molecule of the first group

21. A method for analyzing sequence differences between nucleic acid molecules of a first and a second group, wherein

(a) the nucleic acid molecules of the first group and the second group flanking sequences which are characteristic of the Grappe and have linkers which are sequence-identical in a large number of nucleic acid molecules of a Grappe; (b) the nucleic acid molecules of the first group with the length n contain a small proportion of degradation-resistant nucleotides at a random position, so that an exonucleolytic strand degradation also terminates at a random position, the position of a degradation-resistant nucleotide, and the mixture of shortening products obtained essentially all contains possible shortening products of a length between 1 and n-1 nucleotides in essentially equal proportions; with the following steps: (aa) formation of heterohybrids; (bb) optionally separating homohybrids;

(cc) exonucleolytic shortening of nucleic acid molecules of the first

Group of a heterohybrid using a double-stranded exonuclease;

(dd) Differentiation of those heterohybrids that have a mono- or polybasic at the 3 'terminus of the nucleic acid molecule of the first Grappe

22. A composition comprising a mixture of more than two different nucleic acid molecules each containing a first grappe and a second group

Heterohybrids each consisting of a nucleic acid molecule from the first grappe and, forming a double strand with the latter, an at least partially complementary nucleic acid molecule of the second grappe, the nucleic acid molecules of the first group at their 3 'end or their 5' end in such a shortened form exist that there are different shortened variants of each nucleic acid molecule of the first group.

23. A method for analyzing sequences different between nucleic acid molecules of a first group and a second grappe, comprising the following steps: (a) providing a composition comprising a mixture of at least two different nucleic acid molecules, each of a first grappe and a second grappe, containing heterohybrids from each a nucleic acid molecule from the first group and, forming a double strand with the latter, an at least partially complementary nucleic acid molecule of the second group, the nucleic acid molecules of the first Grappe at their 3 'end or at their 5 'ends are present in such a shortened form that there are different shortened variants of each nucleic acid molecule of the first Grappe, (b) extension at their 3' terminus of perfectly paired shortened nucleic acid molecules of the first Grappe to a primer extension product extending up to this term , wherein mismatched shortened nucleic acid molecules remain essentially unpaired at their 3 'terminus, or ligation at their 5' terminus of perfectly paired shortened nucleic acid molecules, whereby mismatched shortened nucleic acid molecules remain essentially unligated at their 5 'terminus, (c) separation of the heterohybrids from each other, which on the one hand contain an extended and on the other hand an unextended nucleic acid molecule of the first group, or separation of the heterohybrids from one another which contain on the one hand a ligated and on the other hand an unligated nucleic acid molecule of the first group.