DE10052526A1 - Composition for detecting nucleic acid mutations, comprises two groups of nucleic acid attached to flanking sequences that can form distinguishable hetero- and homo-hybrids - Google Patents

Abstract

Composition (A) comprising two groups of nucleic acids (NA1, NA2) each comprising linkers (L) that flank sequences characteristic of the groups and are identical for many molecules of a given group. The linker for NA1 is selected so that hybrids are formed between NA1 and NA2, that optionally after enzymatic treatment, represent a substrate for a double-strand-specific exonuclease, is new. Composition (A) comprising two groups of nucleic acids (NA1, NA2) each comprising linkers (L) that flank sequences characteristic of the groups and are identical for many molecules of a given group. The linker for NA1 is selected so that hybrids (heterohybrids; H1) are formed between NA1 and NA2, that optionally after enzymatic treatment, represent a substrate for a double-strand (ds)-specific exonuclease (exo), so that only NA1 of H1 is exonucleolytically shortened, is new. Homohybrids (H2) formed between two nucleic acids of the same group are not digested by exo. Independent claims are also included for the following: (1) analyzing sequence differences between molecules of NA1 and NA2, comprising: (a) forming H1; (b) optionally removing H2; (c) exonucleolytic digestion of NA1 in H1, using Exo; and (d) differentiation between perfectly matched H1 and those where one or more mismatches are present at the 3'-end of NA1; (2) similar composition in which NA2 contains degradation-resistant nucleotides (drnt); (3) similar composition in which NA1, of length n, contains a small proportion of drnt at random positions so that exonucleolytic digestion stops at drnt, resulting in a mixture, in equal proportions, of digestion products of all sizes between 1 and n-1 nucleotides; (4) method similar to (1) but using compositions of (2) or (3); (5) composition (B) comprising a mixture of more than two different NA from each of NA1 and NA2, containing H1, where NA1 is shortened, at either end, so that for each NA1 differently shortened variants exist; and (6) method similar to (1) but using (B).

Description

The present invention relates to a method for identifying mutation NEN or of sequence variations between nucleic acid groups to be compared, their Sequence may be known or unknown. The nucleic acid groups are on a provided certain ways. A composition comprising two such nuclei acid groups is also the subject of the invention.

The identification of sequence variations is an important discipline of the molecular Ge netics, especially human genetics. Basically, the ex divide the experimental approaches into two categories.

• 1. Method involving detection of sequence variations in known nucleotides enable cuts and
• 2. Procedure involving an investigation of unknown nucleic acids until investigation allow entire genomes for sequence variations.

The classic method for the detection of sequence variation in known nucleic acids ren is the sequencing, which today is almost exclusively after the chain breaking principle according to Sanger is carried out, but despite newer instrumental fort steps (for example, capillary sequencers, which perform parallel sequencing of 96 samples) allows only a very low throughput. cost-effective is the single strand conformation procedure that does not require sequencing. Polymorphism detection (single strand conformation polymorphism detection, SSCP, see. Orita et al, Proc. Natl. Acad. Sci. U.S.A. 1989, 86 (8): 2766-70) which the sequence dependence of the conformation and thus the mobility of a single nucleic acid strand in a denaturing polyacrylamide gel. However, the throughput is this too Procedure limited by the need for each of the nucleic acids to be examined  to provide your own gel trace, and to examine them the fragments do not exceed a certain size (100 to 200 base pairs). height The throughput allows the method of the oligonucleotide ligation method (oligonucleoti de ligation assay, OLA; Nickerson et al., Proc. Natl. Acad. Sci. U.S.A. 1990, 87 (22): 8923- 7), in which two contiguous oligonucleotides on a single-stranded a mutation at a certain position to be examined nucleic acid molecule be hybridized. Are the opposite terminal bases of the Oligonu kleotide complementary to the respective base of the nucleic acid molecule, both can Oligonucleotides are linked together by ligation. In the case of a through however, the mismatch to be detected does not find any ligati on instead. A variant of the OLA process, ligase chain reaction (ligase chain reaction, LCR; Wiedmann et al., PCR Methods Appl. 1994, 3 (4): 51-64) enables the signal ver amplification by amplification, which means detection of mutations in nucleic acids concentration (such as a single locus within the genome). As a disadvantage However, OLA states that for each position to be checked for a mutation tion is to provide its own set of two oligonucleotides, which leads to high costs can arise. Finally, a method was described in which, from one point of view known nucleic acid molecule, a Sanger sequencing reaction is carried out and the products of the primer extension are hybridized with a counter strand, which should be examined for mutation (WO 00/11222). In the case of a wrong couple At the 3 'terminus, which is caused by a sequence variation, this can mismatched nucleotide using a self-correcting polymerase (proofreading Polymerase) removed and replaced by a labeled termination nucleotide so that proof of the mismatch is possible. Since meh Further steps are necessary to make a single pair of two corresponding to each other This procedure is also used to check nucleic acid molecules for sequence differences 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 (RFLP, de Martinville et al., Am. J. Hum. Genet. Am. J. Hum 1982, 34 (2): 216-26), which is based on the occurrence or elimination of recognition sites for restriction enzymes by sequence changes and can be used, for example, to identify transpose insertion sites. However, only sequence variations can be detected that relate to the recognition sites of the respective enzyme, 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, whereby one strand from the first sample and one from the second sample, largely complementary thereto, are hybridized with one another. 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 cleava ge, Grompe et al., Proc. Natl. Acad. Sci. 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 not precluded by the unsatisfactory efficiency and specificity of the methods, for example different mismatches with different efficiency are recognized (cf., for example, Youil et al., Genomics 1996; 32 (3): 431-5 ; WO 99/42595, see here in particular Fig. 4).

In view of 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 that both be knows as well as can be unknown;
• 2. can also be used with complex nucleic acid groups, for example with cDNA, genomic DNA or representation thereof;
• 3. Reliable detection of individual sequence variations with an excellent Signal-to-noise ratio;
• 4. very high throughput.

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

• a) flanking the nucleic acid molecules of the first group and the second group, Characteristic sequences for the group, linkers, have in a variety are sequence identical 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, heterohybrids, starting from nucleic acids of the same group hybridized with each other, from homohybrids, the heterohybridized nucleic acid molecules of both Groups, optionally 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;
  with the following steps:
  • a) formation of heterohybrids;
  • b) separation of homohybrids if these represent a substrate for the double-strand-specific exonuclease;
  • c) exonucleolytic shortening of nucleic acid molecules of the first group of a heterohybrid with the aid of the double-strand-specific exonuclease;
  • d) Differentiation of those heterohybrids which 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.

Another object of the invention is a composition comprising nucleic acid remolecules of a first group and a second group with the aforementioned characteristics len.

The nucleic acid molecules of a first group or a second group are concerned are different nucleic acid mixtures, preferably nucleic acid mixtures Origin, especially around nucleic acid mixtures, the sequence information on genomi cal DNA, mRNA or cDNA, which can be derived from any different Chen biological samples was obtained. Here the term biological pro means be biological material obtained from one or more individuals. For example, the sequence information of the nucleic acid molecules of the first and the second group from tumor biopsies from different patients or patient groups come. Such patient groups can, for example, select according to certain criteria  groups of patients or certain ethnic groups, such as the North American Amish people or the population of Iceland. Furthermore, the Nu Small acid molecules of the first and second groups from the genome of Muta genetically modified organisms come from the same species. Finally, the nu small acid molecules of the first and the second group from bacterial strains of the same Originate from species or closely related species that have different properties. Production strains used in biotech are of great economic importance technology for large-scale extraction of fine chemicals, such as amino acids, be used. The determination of the sequence variations between different organizations nism populations can be used to optimize production processes.

The nucleic acid molecules of the first and second groups have flanking ones for Group of characteristic sequences, linker on. Flanking means in this together hang that the characteristic sequences, linkers, at both ends of a double-stranded gene nucleic acid molecule occur and the nucleic acid molecule towards the ends conclude. The linkers are for the respective group to which the nucleic acid in question is concerned Remolecules belong, characteristic. This means that of the sequence of the linkers one single nucleic acid molecule, whether it is double-stranded or single-stranded, belonging to a certain group can be concluded. The left are also sequence identical for a large number of nucleic acid molecules in a group. This means that with a large number of double-stranded nucleic acid molecules (Nu small acid double strands) the sequences of the linkers match if one considers the Se sequence of the linker at the 5 'terminus or at the 3' terminus of the (+) strand or of the (-) - strand of a nucleic acid duplex with the sequence of the linker of the (+) - strand or the (-) - strand of the other nucleic acid duplex on the same Terminus (at the 5'-terminus or at the 3'-terminus) or at the other their term (at the 3 'term or at the 5' term). A nucleus acid double strand has four linker sequences (at the 5'-terminus and at the 3'-terminus of the (+) strand and the (-) strand). Two nucleic acid duplexes are sequential dental linker when the sequence is one of the four linker sequences of one nucleotide acid molecule with one of the four linker sequences of the other nucleic acid molecule is identical. Corresponding considerations also apply to the comparison of linkers cell-stranded nucleic acid molecules. In this case the complementary strand to 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 which are characteristic of the group in question.

These linkers are usually attached to the Nu to be examined by known methods added small acid mixture or the nucleic acid mixtures to be examined. at for example, genomic DNA can contain one or more restriction enzymes cut, and to the fragments obtained using a ligase double-stranded oligo nucleotides are added (Mueller and Wold, Science 1989; 246 (4931): 780-6). there the two sequences flanking a fragment can be identical or ver be divorced. The term "oligonucleotide" is to be understood in the broadest sense and around summarizes a double-stranded or single-stranded, but preferably double-stranded nu small acid with 10 to about 100 nucleotide building blocks. Following this is an Am plication by means of PCR possible by using primers which are compatible with the known can hybridize flanking sequences. An alternative to polymerase Chain reaction is, for example, the promoter sequence of an RNA polymerase T7 RNA polymerase into one or both of the attached flanking sequences perform and perform an amplification in the form of an in vitro transcription. Both The ends of a (double-stranded) nucleic acid molecule can be targeted with different Sequences are provided. In the simplest case, this is done by cutting the Starting nucleic acid molecules with at least two different restriction endonu cleases which produce distinguishable ends, for example an overhanging and a smooth end or a 3 'overhang and 5' overhang. It will then specific and different oligonucleotides for each end added that each form different linkers. By subsequent amplification with primers that can hybridize to different linkers can be exploited the known PCR suppression effect (Luk'ianov et al., Bioorg. Khim. 1996; 22 (9): 686-90) specifically target the nucleic acid fragments flanked by different sequences amplify. This also leads to a reduction in the complexity of the deployments gang nucleic acids, which, for example, in the case of genomic DNA of higher organ nisms may be desired. A reduction in complexity is particularly advantageous by creating representations, to the term: Lisitsyn et al., Science 1993; 259: 946-51, i.e. the extraction of parts defined by clear criteria quantities from the total of the starting nucleic acids to be analyzed. Such representations For example, sentations can be generated by using genomic DNA with less frequently cutting restriction endonucleases with a recognition sequence which is 6, 8  or more bases is cut, followed by size selection, at for example all those fragments whose length does not exceed 1 kb. Another Representations can be generated by using a less frequently cutting restriction endonuclease in combination with a frequently cutting Restriction endonuclease followed by obtaining fragments with different End up. As a rule, however, representations are made in such a way that a sentence does not overlapping fragment populations is generated, which in turn in its entirety all or at least largely all of the sequence contained in the starting nucleic acids areas include. For this purpose, it is particularly advantageous to remove the starting nucleic acid ren to fragmentation with the help of a sufficiently frequently cutting restriction nuclease to generate fragments in the size range of a few hundred base pairs. This Fragment population now initially covers the entire complexity of the starting nu small acids. To achieve the desired reduction in complexity by dividing it into various The non-overlapping fractions are double-stranded oli gonucleotides added by ligation, the oligonucleotides being the sequence of the later ones Insert linker followed by PCR amplification using primers matching the sequences the linker can hybridize. Although restriction endonucleases are also used who can cut within their recognition site is the use of Type IIS Restriction endonucleases are particularly advantageous. These restriction endonucleases cut the outside of their detection point and create an overhang of defined length and Position relative to the recognition site. In a subsequent step, he will be sent to generated fragments double-stranded oligonucleotides attached on both sides. For this, a Set of oligonucleotides provided that cover all possible overhangs depending on the certain restriction endonuclease of a given type (i.e. 5'-overhangs or 3'- Overhangs of a defined length), the sequence of the oligonucleotides, that is, the later linker sequences, so different from each other that they do not match ne heterohybrids form. This results in a clear assignment of oligonucleo tid, which forms the later linker sequence, and fragment overhang on which this relates oligonucleotide can be added. The ligation is done under conditions that suppress a linkage of overhangs that are not completely complementary to each other (Shaw-Smith et al., Biotechniques 2000, 28 (5): 958-64). By appropriate choice of Primers in a PCR reaction can have different representations of Nu small acid molecules of a group are formed, which with nucleic acid molecules another group can be compared in the context of the inventive method can.  

In addition, representations of genomic DNA can be generated by clicking on known manner generates chromosome-specific nucleic acids (e.g. with flux Karyotype, Harris et al., Hum. Genet. , 1985; 70 (1): 59-65). The nucleic acid thus obtained Linkers are provided, for example by restriction as described above with type IIS restriction endonucleases and subsequent ligation with overhang-specific oligonucleotides that contribute the later linker sequences.

The nucleic acid molecules of the first and second group are usually on same way, but differ in the sequence of their linkers, which according to the invention are characteristic of the group in question, and by the Se 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 usually to be chosen so that at Formation of hybrids between nucleic acid molecules of the first group and nuclein acid molecules of the second group, of heterohybrids, starting from hy with each other bridized nucleic acids of the same group, homohybrids, which heterohybridized Linker of the nucleic acid molecules of both groups over a partial sequence sequence are mental. In the context of the invention, the term hybrid is a dimer of Nu small acids understood under the formation of base pairings. Hybrid between nu small acid molecules of the first group and nucleic acid molecules of the second group (Heterohybrids) are usually by melting the double strands together other hybridized nucleic acids of the same group (from homohybrids) and subsequently eats cooling formed, and not only nucleic acid molecules one and the same Recombine group, but also heterohybrids are formed. Depending on which, under what conditions this recombination process is carried out, whether the kineti or the thermodynamic control of the reaction predominates, the proportion of Hy brides that have mismatches of different sizes. Optimal conditions best hen 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 be matching nucleic acids, so that the sequence complementary region of both nucleic acids remolecules is considerably more extensive than that of the sequence variations my area. A nucleic acid molecule (or a region thereof) is sequence-complete mentally to another nucleic acid molecule (or a region thereof) if it is with the other molecule (or a region thereof) base pairings without mismatches, can form perfect base pairings.  

The linkers of both groups, which are opposed to each other in the heterohybrid, can thus Rule base pairing over a sub-area. The linkers who have a homohybrid has both ends, do not necessarily have to refer to the same oligonu have kleotide returned. The part of the sequence complementarity includes in the Usually at least 4 bases, preferably at least 10 bases, especially 15 bases.

The linkers of the nucleic acid molecules of both groups are also to be chosen so that the Heterohybrids, optionally after enzymatic treatment, a substrate for a dop represent pel strand-specific exonuclease and essentially only the nucleic acid molecules of the first group of a heterohybrid are exonucleolytically shortened. A In the context of the invention, double-stranded exonuclease is an enzyme which is a nu small acid molecule or both nucleic acid molecules of a nucleic acid duplex in the area of the double strand, which is double-stranded, from a term of each degrades from nucleic acid molecule. A breakdown in the middle of a nucleic acid mole küls (endonucleolytic degradation) should be negligible. The use of Exonu clease III as a double-stranded exonuclease is preferred (for the term exonuclease III see Bioanalytics, F. Lottsspeich, H. Zorbas, ed., Spektrum Akademischer Verlag, Heidelberg, Berlin, 1998, ISBN 3-4274-0041-4, p. 759). Exonuclease III shortens one or both Nucleic acid molecule (s) of a double strand from the 3'-terminus. The 3'- Term of a nucleic acid molecule is only shortened if the 3'-terminus is together men with the other nucleic acid molecule a smooth end or a 3'-set back End (so a 5 'overhang) forms.

The heterohybrids do not have to be in the state in which they are after be a substrate for the double-stranded exonuclease. It is possible the hey terohybrids with a double strand specific exonuclease prior to their implementation incubate another enzyme such as a restriction endonuclease (enzymatic treatment lung). Then the heterohybrids are usually only due to this enzymatic loading act as a substrate for the double-stranded exonuclease. For example the heterohybrids with a restriction endonuclease or with restriction endonucleases are incubated, which intersect in the area of the linker, i.e. the areas of Nu small acid molecules. Here, the linker can be chosen so that a restriction interface for the restriction enzyme or the restriction enzymes with which the en zymatic treatment is carried out on homohybrids, but not on heterohy briden is present or vice versa. This ensures, among other things, that the He terohybrids represent a substrate for a double-stranded endonuclease and in  essentially only the nucleic acid molecules of the first group of a heterohybrid (the means the components of a heterohybrid) and not the nucleic acid molecules of the second group of a heterohybrid can be shortened exonucleolytically. this means not that homohybrids, which exist alongside the heterohybrids, are not a substrate for the double strand-specific exonuclease should form. But within a hetero hybrid essentially only the nucleic acid molecules of the first group of a heterohy exonucleolytically shortened brids. As far as an exonucleolytic shortening of the If nucleic acid molecules of the second group come, this reaction only neglects one acceptable secondary reaction.

A composition comprising nucleic acid molecules of a first group and one second group, as described above, is an object of the invention.

Another subject 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:

• a) formation of heterohybrids;
• b) separation of homohybrids, if these are a substrate for the double-stranded spec represent exonuclease;
• c) exonucleolytic shortening of nucleic acid molecules of the first group of a Heterohybrids with the help of the double-strand-specific exonuclease;
• d) Differentiation of those heterohybrids that are at the 3'-terminus of the nucleic acid remolecule of the first group have a single or multi-base mismatch, the mismatched heterohybrids, of the perfectly paired heterohybrids there.

The method according to the invention thus initially comprises the formation of heterohybrids, as already described. If the homo-hybrid is a substrate in addition to the hetero-hybrid represent for the double-stranded exonuclease, which is not sufficient according to the invention is closed, then these homohybrids must be separated. This can be done through a Plenty of measures are done. A suitable measure would be to separate the Homohybrids from heterohybrids due to the different electrophoretic mo bility of homo- and heterohybrids. Furthermore, after the formation of heterohybrids and an enzymatic treatment takes place before the exonucleolytic shortening, for example a restriction cut in the area of the linker, which is either only in Homohy briden or only in heterohybrids, and immobilisierba by selective cleavage groups enables the separation of homohybrids from the heterohybrids. A  such separation is not necessary if by the action of the restriction end nuclease (s) the heterohybrids into a substrate for the double-stranded exonuclease are transferred while the homohybrids due to the non-restriction cut does not become a substrate for the double-strand-specific endonuclease. This can by the fact that the linkers carry nucleotides which break down by a double exclude strand-specific exonuclease, and which due to a restriction cut of be separated from the heterohybrids, but not from the homohybrids.

This is followed by an exonucleolytic shortening of the nucleic acid molecules first group of heterohybrids with the help of the double strand specific exonuclease and preferably from the 3'-terminus of the nucleic acid molecule to be degraded in Towards the 5 'terminus. Within a heterohybrid are essentially excluded Lich exonucleolytically truncates the nucleic acid molecules of the first group, while the Nucleic acid molecules of the second group remain unaffected. 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 shortening of nucleic acid molecules of the first group is found preferably so that a variety of heterohybrids is formed, in which the be relevant nucleic acid molecule of the first group has been shortened to different extents and the abbreviation products are present in as many copies as possible. Accordingly, the collection of shortened versions included one given nucleic acid molecule a length of 100 bp, ideally all at their 3'- End shortened molecules with a length of 1 bp to 99 bp, the linker here unbe remains considered. For an even distribution of all possible shortening options to achieve a heterohybrid species by exonucleolytic degradation, the Reaction conditions, especially reaction time, temperature and amount of the set exonuclease, chosen so that there is no complete strand degradation. A very the processivity of the exonuclease is an important factor. If the processivity is very high, so complete strand degradation is preferred. If the processivity is very low, show the shortening products have essentially the same degree of shortening. By means of gen technological methods, the processivity of the exonuclease should be adjusted so that it lies between these extremes. The concept of processivity includes the number of those nucleotide building blocks that are broken down on a statistical average before the En zym dissociated from nucleic acid duplex. A particularly even distribution The shortening products are also due to the statistical incorporation of nucleoti those that are resistant to degradation by double-stranded exonucleases, in  to reach the nucleic acid molecule to be degraded. This can be, for example Act αS thionucleotides (Schreiber et al., Nucleic Acid Res. 1985; 13 (21): 7663-72). If such degradation-resistant nucleotides are statically distributed in the nucleic acid to be degraded are present in the appropriate frequency, then the double-stranded exonu Klease be used in excess, so that a complete exonucleolytic Ab construction takes place up to the respective degradation-resistant nucleotide.

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

A DNA or RNA polymerase can be used as the polymerase is able to extend set 3'-terms, but not to extend it of mismatched terms, but not polymerases that are treated with an exonuclease Activity can reduce terminal mismatches (proofreading polymerases). Examples for suitable polymerases there are, for example, genetically modified T7 DNA polymerases (Δ28T7 DNA polymerase), Taq polymerase or reverse transcriptase. Not suitable in contrast, native T7 DNA polymerase or Pfu polymerase.

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 consistent one Duplex;
• - Separation of the filled heterohybrids from the non-filled, mismatched ones Straight hybrids; and
• - Identification of the unfilled heterohybrids.

The perfectly paired heterohybrids are filled in by a DNA polymerase, which is not capable of self-correction (no proofreading polymerase). In this way the shortened nucleic acid molecule is extended in the 5'-3 'direction as long as H. up fills until a continuous double strand with a smooth end is obtained. The up Filled heterohybrids are made up of the unfilled, mismatched heterohybrids Cut. This can be done by the previous replenishment Step nucleotide monomers are used which carry immobilizable groups, so  that the products of the replenishment reaction themselves can be immobilized and thus by the He terohybrids that were not replenished, mismatched the mismatched heterohybrids can be. The non-filled heterohybrids are identified by known methods, for example by hybridization with a two-dimensional approach order of single-stranded nucleic acids. Known sequencers can be used here connect methods such as the Sanger sequencing method (strangver longer) or according to Brenner (strand shortening, US-A 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 while maintaining a consistent one Duplex with nucleotides of a first kind;
• - Removal of the mismatched base (s) of the mismatched heterohybrids, so that from the mismatched heterohybrids become perfectly paired heterohybrids;
• - Filling the perfectly paired heterohybrids formed in the last step with nu second type clothing;
• - The nucleotides of a first type having an immobilizable group which Do not have nucleotides of the second type, or the nucleotides of the first type one do not have immobilizable group which has the nucleotides of the second type sen;
• - Separation of the originally mismatched heterohybrids from the originally perfect ones paired heterohybrids by immobilizing either the originally failed mated heterohybrids or the originally perfectly paired heterohybrids;
• - Identification of the originally mismatched heterohybrids.

The immobilizable group is preferably biotin, which has an Im mobilization via streptavidin enabled. Further examples of immobilizable groups Pen are sugars that can be recognized by lecithins or by groups of molecules that enable immobilization by the molecular groups binding antibodies. On Examples of the latter would be, for example, dinitrophenol derivatives. Such immobilisable Right groups can of course also be known as caged compounds (Methods Enzymol 1998; 291: 415-30) are used, in which the immobilizable group only as a result of a Photo reaction is released.

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

• - Filling the perfectly paired heterohybrids while maintaining a consistent dop pelstrangs;
• - Isolation of the mismatched heterohybrids by hybridization with an immobili based or immobilizable oligonucleotide and subsequent immobilization of the Hybrids of heterohybrid and oligonucleotide;
• - Identification of the originally mismatched heterohybrids.

Filling the perfectly paired heterohybrids while maintaining a consistent dop pelstrangs takes place as described above. Thus only the mismatched heterohybri point de a non-continuous double strand. This enables hybridization with an immobilized or immobilizable oligonucleotide and the subsequent Im mobilization of the hybrid of heterohybrid and oligonucleotide to a solid phase, where due to the mismatched hetero-hybrids from the originally perfectly paired hetero-hybrids briden can be separated. The isolated, originally mismatched heterohybrids are by known methods such as the sequencing method described above identified.

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 one to the ket leading nucleotide;
• - Removal of the mismatched base (s) of the mismatched heterohybrids, so that from the mismatched heterohybrids become perfectly paired heterohybrids;
• - Filling the perfectly paired heterohybrids formed in the previous step forming a continuous double strand;
• - Isolation of those formed in the previous step, in the form of a double strand present heterohybrids;
• - Identification of the isolated heterohybrids.

With the help of an appropriate DNA polymerase, the 3 'terminus is the perfect ge mated heterohybrids extended by a nucleotide leading to chain termination. On preferred nucleotide leading to chain termination is a dideoxynucleotide, like ddATP, ddCTP, ddTTP and ddGTP. The removal of the mismatched base (s) of the Mismatched heterohybrids are preferably carried out with the help of one for self-correction capable DNA polymerase (proofreading polymerase). Then the filling takes place  of the perfectly paired heterohybrids formed in Bil end of a continuous double strand, with no Nu leading to chain termination kleotide can be used. Then the isolation takes place in the form of a double heterohybrids present in a strand, for example due to their electrophoretic mo bility or by cloning. Are the originally mismatched heterohybrids through Cloning isolated, they will be in the form of a continuous double strand the heterohybride at both ends with a restriction enzyme, preferably in Be rich the linker, cut and ligated with an appropriately prepared vector and then cloned. Linker or restriction endonucleases should be chosen so that not continuous double strands of the restriction endonucleases not on both sides can be cut. The isolated heterohybrids are identified by known measures.

In a further embodiment of the method according to the invention, this contributes to the ket nucleotide leading an immobilizable group and the isolation of the in Double stranded heterohybrids are formed by immobilization and Separation of the heterohybrids extended by an immobilizable group. The immo bilisable group can for example be a biotin group, which by binding to Streptavidin can be immobilized.

In a further embodiment of the method according to the invention, the ho mohybride cut from step (bb) in the area of the linker, the restriction interface is only available for homohybrids, but not for heterohybrids. In an old one native embodiment of the method according to the invention, the reverse is the case. The Re The restriction interface is only available for heterohybrids, but not for homohybrids. As a result, either homohybrids or heterohybrids carry restricted ends, the one Allow separation of the homohybrids from the heterohybrids. Assign the left Nucleotides that are resistant to exonucleolytic degradation and that in Heterohy brid, but not separated in homohybrids due to the restriction, then stel len only the heterohybrids represent a substrate for the double-strand-specific exonuclease, so that the separation of the homohybrids in step (bb) can be omitted. In this case should the linker of the nucleic acid molecule of the second group at the 3 'terminus, that is in Area of the linker, to carry a nucleotide resistant to exonucleolytic degradation ensure that essentially only the nucleic acid molecules of the first group ei Nes heterohybride are linked exonucleolytically.  

In a further embodiment of the method according to the invention, the linkers of the nucleic acid molecules of the first group so chosen that when heterohy briden, the linker of the nucleic acid molecules of both groups, a 5 'overhang and egg form a 3 'overhang and both overhangs each through the nucleic acid molecules of the second group. The easiest way to achieve this is that the linkers of the nucleic acid molecules of the second group of a heterohybrid are longer as the linkers of the nucleic acid molecules of the first group of the same heterohybrid.

In a further embodiment of the method according to the invention, the ho mohybride on both sides 3 'overhangs, so that there is no separation can.

The invention is described in more detail below by the figures. It shows:

Fig. 1 shows the preparation of heterohybrids which enable selective shortening of only one strand,

Fig. 2 shows the preparation of heterohybrids with a shortened strand,

Fig. 3 shows the isolation of a fragment from a mixture of nucleic acid molecules having a sequence variation between nucleic acid molecules of the first and the second group,

Fig. 4 shows an alternative isolation molecules of a fragment from a mixture of nucleic acid having a sequence variation between nucleic acid molecules of the first and the second group,

5 shows another alternative insulation small acid molecules. A fragment of a mixture of Nu, which acid molecules, a sequence variation between nucleic the first and the second group comprises,

6 shows another alternative insulation small acid molecules. A fragment of a mixture of Nu, which acid molecules, a sequence variation between nucleic the first and the second group comprises,

Fig. 7 shows another alternative insulation small acid molecules of a fragment from a mixture of Nu, which acid molecules, a sequence variation between nucleic the first and the second group comprises,

8 shows another alternative insulation small acid molecules. A fragment of a mixture of Nu, which acid molecules, a sequence variation between nucleic the first and the second group comprises,

Figure 9 shows another alternative insulation small acid molecules. A fragment of a mixture of Nu, which acid molecules, a sequence variation between nucleic the first and the second group comprises,

Fig. 10, the selective restriction of homohybrids in the linker,

Fig. 11, the selective restriction of homohybrids in the linker, followed by the isolation of a sequence variation bearing hetero Hybrids and forth position of a hybridization probe (Fig. 11 comprises FIGS. 11a and FIG. 11b, the egg ne contiguous Fig. 11) form .

Fig. 12 shows the application of the inventive method for the identification of Se quenzvariationen between originating from two different samples of nucleic acids using reference nucleic acids (Fig. 12 comprises FIGS. 12a,
Fig. 12b and Fig. 12c which form a coherent Fig. 12),

Fig. 13, the selective restriction of heterohybrids in the linker.

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 (linker characterized by open rectangles) and a second group (linker characterized by hatched rectangles) groups 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. "Exo" means the exonuclease acting. Only one possible shortening variant from a mixture of shortening variants is shown here for each heterohybrid double strand.

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

Fig. 3 shows the isolation of a fragment from a mixture of nucleic acid molecules having a sequence variation between 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 identical to the sequence, while the longer one between the first and second origin has a sequence variation. In detail shows
1 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 protection of 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 representing a shortening product, the last nucleotide of the double-stranded region representing the sequence variation between nucleic acid molecules in the groups (point). This shortening product is therefore characterized by a mismatch at the end of the double-stranded range,
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 groups are represented by "B",
4 Separation of biotin-containing heterohybrids from unextended, non-biotin-containing heterohybrids which represent fragments containing sequence variations.

Fig. 4 shows an alternative isolation molecules of a fragment from a mixture of nucleic acid having a sequence variation between nucleic acid molecules of the first and the second group. In detail shows
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 another alternative insulation small acid molecules. A fragment of a mixture of Nu, which acid molecules, a sequence variation between nucleic the first and the second group comprises. 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 replenishment of those heterohybrids which have no mismatch at the end of the double-stranded area,
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 group.

6 shows another alternative insulation small acid molecules. A fragment of a mixture of Nu, which acid molecules, a sequence variation between nucleic the first and the second group comprises. 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 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 extended, non-biotin-containing heterohybrids in (3).

Figure 7 shows another alternative insulation small acid molecules. A fragment of a mixture of Nu, which acid molecules, a sequence variation between nucleic the first and the second group comprises. 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 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 another alternative insulation small acid molecules. A fragment of a mixture of Nu, which acid molecules, a sequence variation between nucleic the first and the second group comprises. 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.

Figure 9 shows another alternative insulation small acid molecules. A fragment of a mixture of Nu, which acid molecules, a sequence variation between nucleic the first and the second group comprises. 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 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 heterohybrids extended in (4), which represent fragments containing sequence variations, with separation in (3) elongated, non-biotin-containing heterohybrids.

Fig. 10, the selective restriction of homohybrids in the linker. Linkers were ligated to the nucleic acid fragments (gray) generated by means of the restriction endonuclease HinP1I (recognition sequence GCGC ). Linkers of the fragments of the first group (open rectangles) contain a recognition site for the restriction sendonuclease HaeII (recognition sequence A GCGC T), linkers of the fragments of the second group (hatched) an alternative recognition site for the same restriction endonuclease G GCGC C. In detail shows
1 denaturing the nucleic acid fragments of both groups flanked by linkers, followed by re-hybridization of strands which are partially complementary to one another to form homo- and heterohybrids,
2 the treatment of the reaction mixture with HaeII, so that the linkers of the homohybrids formed in step (1) by "reannealing" are trimmed, while the resulting heterohybrids remain flanked by intact linkers. Since the recognition site of the restriction endonuclease used to generate the original fragments is part of the recognition sites used in step (2), undesired, fragment-internal cuts are avoided in step (2). The 3'-overhanging ends produced by HaeII are substrate for exonuclease III ("Exo"), while the 3'-recessed ends of the heterohybrids are amenable to strand-specific shortening according to FIG. 1. Both ends of a hybrid hybrid are shown; only one of them can be shortened exonucleolytically.

Fig. 11, the selective restriction of homohybrids in the linker, followed by the isolation of a sequence variation representing hetero hybrid, and the production of a hybridization probe. In detail shows
1 denaturation of the nucleic acid fragments flanked by Linkem from both groups, followed by rehybridization of at least partially complementary strands to form homo- and heterohybrides,
2 the treatment of the reaction mixture with two restriction endonu cleases (recognition sites shown as black or dotted rectangles) so that the linkers of the homohybrids formed in step (1) are separated by "reannealing", while the heterohybrids formed remain flanked by linkers,
3 Exonucleolytic shortening of the nucleic acid molecules of the first group 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 which have not been filled in 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 components, so that hybridization probes representing a sequence variation arise. Marked probe molecules are marked with an asterisk.

FIG. 12 shows an application 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. A certain nucleic acid fragment flanked by linker molecules is shown, which is characterized by short hatched linkers from the first sample, short dotted linkers from the second sample and long linkers shown as open rectangles from the reference sample , 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 used for the described implementation with fragments from the first sample and originating from the reference sample are provided with a marking 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 marker group which can be distinguished from the first marker group and which is symbolized by "#". It shows in detail
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 linkers flanking for different lengths,
2 Exonucleolytic shortening of those strands which are distinguished by a recessed 3 'end, so that heterohybrids with single-stranded 5' overhangs varying in length are formed,
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 streptavidin-coated surface,
5 Obtaining the labeled reference fragment strands hybridized on strands which have remained unfilled in (4) by complete exonucleolytic degradation of the already shortened strand,
6 combination of the labeled reference fragment strands obtained in (5) by hybridization of strands from the first sample with strands of the reference sample and of 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.

Fig. 13, the selective restriction of heterohybrids in the linker. Biotinylated linkers were ligated to nucleic acid fragments generated by means of the restriction endonuclease DpnI (recognition sequence GATC). The linkers contain masked recognition sequences for the restriction endonuclease BamHI (recognition sequence G GATC C). The linkers attached to the nucleic acid molecules of the second group have a thionucleotide in the vicinity of the recognition sequence (identified by "S"). In detail shows

• 1. the denaturation of the nucleic acid fragments flanked by Linkem both groups, followed by partial re-hybridization to each other complementary strands to homo- and heterohybrids,
• 2. the treatment of the reaction mixture with BamHI, so that the linker in step (1) trimmed heterohybrids formed by "reannealing" the resulting heterohybrids, however, from intact linkers stay flanked,
• 3. Separation of the biotinylated homohybrids by immobilization on egg ne streptavidin-coated solid phase. Only the nucleic acid mole The first group of a fragment can be exonucleolytically shortened be that of the second group by degradation-resistant nucleotides protects. Both ends of the nucleic acid duplex are shown.

Claims (13)

1. 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 have flanking sequences which are characteristic of the group, linkers which are sequence-identical in a large number of nucleic acid molecules in 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 hybridized nucleic acids of the same group, from homohybrids, the heterohybridized nucleic acid molecules of both groups optionally represent a substrate for a double strand-specific exonuclease after enzymatic treatment 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 group, wherein

• a) the nucleic acid molecules of the first group and the second group have flanking sequences which are characteristic of the group, linkers which are sequence-identical in a large number of nucleic acid molecules in 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 hybridized nucleic acids of the same group, from homohybrids, the heterohybridized nucleic acid molecules of both groups optionally represent a substrate for a double strand-specific exonuclease after enzymatic treatment and essentially only the nucleic acid molecules of the first group of a heterohybride are shortened exonucleolytically;
  with the following steps:
  • a) formation of heterohybrids;
  • b) separation of homohybrids if these represent a substrate for the double-strand-specific exonuclease;
  • c) exonucleolytic shortening of nucleic acid molecules of the first group of a heterohybrid with the aid of the double-strand-specific exonuclease;
  • d) Differentiation of those heterohybrids which 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.

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 to claim 2, characterized in that the distinction is made by
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 the perfectly paired heterohybrids formed in the last step with nucleotides of a second type;
wherein 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 have the nucleotides of the second type;
Separating 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. 5. 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;
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;
Removal of the mismatched base (s) of the mismatched heterohybrids so that the mismatched heterohybrids become perfectly paired heterohybrids;
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 formed in the previous step;
Identification of the isolated heterohybrids. 7. The method according to claim 6, characterized in that the chain termination leading nucleotide carries an immobilizable group and the isolation of the in the form of a double strand heterohybrids by immobilization and Ab Separation of the heterohybrids extended by the immobilizable group takes place. 8. The method according to claim 2, characterized in that the homohybrids of Step (bb) are cut in the area of the linker and the restriction cut place only with homohybrids, but not with heterohybrids.   9. The method according to claim 2, characterized in that the heterohybrids of Step (aa) are cut in the area of the linker and the restriction cut place only with heterohybrids, but not with homohybrids. 10. The method according to any one of claims 2 to 9, characterized in that in Feature (b2) the linkers of the nucleic acid molecules of the second group have nucleotide building block resistant to exonucleolytic degradation. 11. The composition according to claim 1, characterized in that the heterohybridi based linker of the nucleic acid molecules of both groups a 5 'overhang and egg Form a 3 'overhang and both overhangs by the nucleic acid molecules of the second group are formed. 12. The method according to any one of claims 2 to 8, characterized in that in Feature (b2) to choose the linkers of the nucleic acid molecules of the first group are that when heterohybrids are formed, the linkers of the nucleic acid molecules form a 5 'and a 3' overhang of both groups and both overhangs because are formed by the nucleic acid molecules of the second group. 13. The method of claim 12, wherein in step (bb) the homohybrids in one form be provided, which is characterized by bilateral 3 'overhangs and on the separation is waived.