DE4129653A1 - PROCESS FOR DETECTION OF SIMILAR NUCLEIC ACIDS - Google Patents

Abstract

Method for detecting nucleic acids whose nucleotide sequences differ in a position X, wherein a set of oligonucleotides whose nucleotides corresponding to position X are complementary only to position X to one of the similar nucleic acids, but whose nucleotide sequences differ in respect in the position of another mismatch Y, as well as reagents suitable for carrying out the method and areas of application of the method. <IMAGE>

Description

The invention relates to a method for detection similar nucleic acids, a set of oligonucleotides, which are suitable, a reagent kit with which the inventive method can be carried out and various uses of the method in the Diagnosis of genes and infections.

Examination of samples for the presence of certain Nucleic acids or nucleic acid groups obtained in younger Time more and more important. This is at least for Part in that the nucleotide sequences of the Nucleic acids are a characteristic of organisms Feature. Recently, even the Tried to make differences in just one Nucleotide of the sequence for distinguishing nucleic acids exploit. Such differences can z. B. by Point mutations resulting nucleotide exchanges. Natural examples of such closely related nucleic acids are the alleles, d. H. alternative sequence variants of a Gene at a defined location in the chromosome.

From Oncogene Research 1, 235-241 (1989) and Nucl. Acids Res. 17, 8093-8099 (1989), a method is known in the optionally containing the allelic variant Area of the nucleic acid first by polymerase chain Reaction (PCR) using primers with special Design amplified and then with a Restriction enzyme is treated. That way is one  Diagnosis of alleles by a restriction fragment poly morphism (RFLP) analysis possible. The electrophoretic Separation of the cleavage products according to their length then gives Information about whether the corresponding allele in the sample was included or not. This procedure has the Disadvantage that a specific restriction digest must be made. Apart from that, this is time consuming, for any mutation that does not have already delivers a RFLP per se, also a design for one the point mutation adjacent primer be possible which allows digestion with a restriction enzyme, that cuts exactly at the said place. This can be up the technical difficulties described there bump.

EP-A-03 32 435 describes a method with which in principle one nucleic acid selectively adjacent to another Nucleic acid can be detected, which is found only in a nucleotide is different from her. This is the Effect exploited that theoretically only those to the to be detected nucleic acid hybridized oligonucleotides can be extended enzymatically, in Extension direction complementary nucleotide complementary to the corresponding nucleotide of the to be detected Nucleic acid (of one allele) is. The oligonucleotide is therefore chosen so that only with the detected Nucleic acid is such a complementarity. The on the other allele hybridized oligonucleotide becomes so theoretically not extended. It turned out that in practice to a small extent, however, a Extension of the hybridized to the other allele Oligonucleotide occurs, indicating the sensitivity and before in particular the specificity of the proof of the examined Minimizes nucleic acid. Non-specific extensions especially occur easily when on mismatch on 3'-end a T is involved or it is a CA mismatch (Kwok et al., Nucleic Acids Research 18, 999-1005  (1990)). In EP-A-03 32 435 was to increase the Specificity proposed, the nucleotide sequence of the Oligonukleotids to be chosen so that in the end another nucleotide not to the corresponding nucleotide the two nucleic acids is complementary. As proof both alleles must be carried out 2 reactions where only the presence of one of the alleles per reaction can be detected. There are two allele-specific primers and a complementary strand primer synthesized, and in 2 reactions the sample becomes once in a PCR with the opposite strand primer and one of the Allele-specific primer amplified and in one second, parallel reaction in a PCR with the Counterstrand primer and the other allele-specific primer amplified. If in one of the reactions the one concerned allele-specific PCR product is not found assumed that the relevant allele in the examined Sample is not available. Because homozygous DNA samples only contain one of the two alleles, which then each can only be demonstrated in one of the two reactions, need to control the efficiency of each for the other allele specific PCR reaction and to prove the Absence of the respective allele two more primers which are the same in all reactions, distinguishable from the specific product control product deliver. Is the control product in a PCR product before, but will not be a specific product of the examined Found alleles, it is believed that the examined Sample the allele to be detected in the prepared reaction does not contain. This methodology therefore requires two alleles detected by two separate reactions or be excluded and in every single PCR reaction a control PCR must be included.

Promoting selectivity by lowering the dNTP Concentration was in Biochem. Biophys. Res. Commun. 160 441-447 (1989). Even if this  Additional measure was taken, but it can in the Try the alleles in separate reactions prove to come to unspecific products.

In the ligase chain reaction (LCR; WO 89/09 835) is a thermostable ligase used to specifically two to link adjacent oligonucleotides, only then when at stringent hybridization temperature are hybridized to a complementary target and a complete base pairing at the point of attachment is present. Are two alleles different? Mutation at the position of the point of attachment, so is the said condition of complete base pairing only fulfilled for one of the alleles. Two more Oligonucleotides complementary to the first two additionally needed in the ligase chain reaction the To amplify ligation product. For the detection of two Alleles have so far been 2 reactions with at least a total of 6 oligonucleotides performed, detection of the amplification product was carried out by a radioactive label (Proc Natl Acad Sci., USA 88, 189-193 (1991)).

From Proc. Natl. Acad. Sci. USA 1985, Vol. 82, 1585-1588 and New England Journal of Medicine 317, 985 (1987) an allelic evidence known on the differential Hybridization of "allele-specific" oligonucleotides (ASO) is based on the alleles. For this 2 Oligonucleotides of z. B. each synthesized 20 bases in length, one suitable for one of each different alleles, but with a roughly in the middle the oligonucleotide sequence located mismatch to each other allele. By differential hybridization with labeled oligonucleotides is thus a distinction of Alleles both in analysis of human genomic DNA, as well as analysis of PCR products.  

Although this process is also a direct and discrete analysis of genomic DNA possible that is without PCR, but then there are additional digestive and Electrophoresis steps necessary.

In Nucleic Acids Research 17, 2437-2448 (1989) and EP-A-03 33 465 describes a method which it allowed, pre-amplified human genomic DNA by Competition of allelic selective primers in a few further PCR cycles on the presence of various alleles to investigate (competitive oligonucleotide priming = COP). The ASO technique described above becomes one Transfer PCR technology. In the original ASO technique is an error of z. B. 5% by cross-hybridization yet tolerable, since then by intensity comparisons of Signals at appropriate controls an absolute clear interpretation of the results is possible. at a PCR reaction in which allele-specific Oligonucleotides used as primers would be one such error in a reaction approach in which the However, amplification of both alleles is possible after 10 Cycles already lead to an error of about 12% when a sample contains only one of the alleles. Although could Gibbs et al. show that the competition of the primers the Selectivity increased, nevertheless, became the interesting one Range of genomic DNA first by PCR amplified and then the analysis for the different ones Alleles performed in 10 more cycles, with two alleleselective primer and a complementary strand primer in these 10 cycles were used, and for two to be performed Reactions each one of the allelic selective primer was radioactively labeled. Selective detection of different alleles could be used for oligonucleotides with a Lengths from 12 to 16 bases are shown, with longer ones Oligonucleotides were under the conditions described but also received unspecific product.  

Those based on differential hybridization Procedures are so far limited to very specific Situations and also very complicated in terms of the Execution and susceptible to interference. In addition, the Pre-amplification in the COP a step, the you would like to save.

The object of the invention was to provide a simple method for specific detection of similar nucleic acids to provide, which no restriction digestion needed, and where it is possible, the similar Amplify nucleic acids in a reaction mixture.

The invention therefore provides a method for Detection of similar nucleic acids, their nucleotide sequences differ in at least one position (X), which contains at least the following sub-steps:

• Implementation of a set of oligonucleotides, whose nucleotide sequences are in the Position X corresponding position distinguish, with the provable Nucleic acids under hybridization conditions;
• - Extension of the hybridized Oligonucleotides using the to be detected nucleic acids as a template; and
• - proof of extension products, wherein the oligonucleotides of the sentence also occur in at least one further position (Y) from each other differ.

Likewise subject of the invention are a set of Oligonucleotides, one reagent kit and several Uses of the method.  

Similar or closely related nucleic acids in the sense of Invention are nucleic acids whose nucleotide sequences in are substantially identical, but at least a position of the nucleotide sequence makes a difference respectively. This position will be described below with X designated. If several differences to the subject of These are with X1, ... XN designated. Such differences come for example by point mutations (eg substitutions by others Bases or by deletions or insertions of a or more bases. The similar ones Nucleic acids are then commonly called alleles designated.

Significant point mutations are found, for example, in the LDL receptor gene (Arteriosclerosis 9, 1-8 to 1-13 (1989)) or in the apolipoprotein B gene (CGG → CAG mutation of codon 3500; Proc. Natl. Acad. Of Sciences USA 86, pp. 587-591 (1989)), in the gene of the virus-specific reverse transcriptase of HIV (A → T mutation in codon 215 , Science 246 , pp 1155-1158 (1989)) or in the β-globin gene the A → T exchange in codon 6 in sickle cell anemia. In addition, similar nucleic acids are found in many closely related organisms, such as bacteria and viruses (serovars). In particular, nucleic acids of individual bacterial strains are very similar in the sequence of their ribosomal genes or rRNA. However, similar nucleic acids within the meaning of the invention are also control nucleic acids which resemble a nucleic acid to be detected. The position X is also called a polymorphic place. The nucleic acids to be detected may be RNA or DNA. In bacterial diagnostics, rRNA has proven to be particularly useful.

The original target is the nucleic acid which contained in the sample. The later with her Use resulting polynucleotides, which also be used again as a target nucleic acid and the usually shorter than the original target, are called Product target called.

If, in the following, the detection of two nucleic acids or Alleles are mentioned, these embodiments may be in easy way to detect nucleic acids with all conceivable differences in X (eg all conceivable Alleles) by using appropriate oligonucleotides be transmitted.

The position is a defined position on a Nucleic acid is called. This position can for example, be occupied by a nucleotide.

Complementary nucleotides are called which a ensure regular, Watson-Crick base pairing (eg. G-C, A-T and AU, respectively). These lead to so-called matches, non-complementary to mismatches.

If other bases or base analogues such So allow base pairing (7-deaza-dGTP, dITP) as well which is called complementary.

For carrying out the detection method according to the invention the nucleic acids to be detected must be in for in vitro Reak tion of suitable form. This is one too examining sample of, for example, a tissue, single cells or a cell-containing fluid first, in a known way, open-minded to the Cell walls break up (eg thermal, enzymatic or chemical lysis or combinations thereof).  

If the nucleic acids are not in single-stranded form they are in such a known manner transferred (eg thermal or alkaline denaturation, enzymatic strand separation or destabilization of the Double strands under certain salt conditions).

Subsequently, the sample is filled with a set of Oligonucleotides brought into contact. It will be Conditions selected in which the inventive Oligonucleotides with the associated areas of hybridize to be detected nucleic acids. These Hybridization is also commonly called annealing designated. Hybridization with non-detectable Nucleic acids can be selected by selecting the nucleotide sequence, length, Temperature, auxiliaries, etc. are avoided.

The set of oligonucleotides according to the invention consists of at least as many different oligonucleotides as different nucleic acids are to be detected. When detecting two different nucleic acids therefore at least two different oligonucleotides (and optionally Gegenstrangoligonukleotide) required. The used oligonucleotides preferably have a length of less than 100, more preferably from 5 to 50 nt. Each oligonucleotide has a nucleotide sequence corresponding to the in the vicinity of the nucleotide X lying area so is highly complementary that the oligonucleotide with the can hybridize to be detected nucleic acids. The oligonucleotides of a set according to the invention however, they differ completely in their sequence certain way.

A difference in the nucleotide sequence of Oligonucleotides of a sentence is located in the Position, which in the anhybridized state of the position X corresponds to the nucleic acid. This difference can  by substitution with another base or Base analogue or deletion / insertion.

For the purposes of the invention, the oligonucleotide as refers to "match" oligonucleotide, which at position X has a nucleotide which corresponds to the nucleotide of the Position X of the nucleic acid to be detected complementary is. The matching nucleic acid to the match oligonucleotide (with which an extension takes place) is used as a target- Nucleic acid is called. As a "mismatch" oligonucleotide is an oligonucleotide referred to, the nucleotide in Position X is a nucleotide other than the nucleotide in position X for consideration nucleic acid to be detected is complementary.

Depending on the selected variant of the invention Method, nucleotide X is at a particular Place the oligonucleotides, for example in the middle or at one end of the oligonucleotides. In the first case is the inventive method an improvement of Procedure based on competitive priming (in Following variant 1 called) and in the second case of the Mismatch priming method of EP-A-03 32 435 and related method (hereinafter called variant 2). In In any case, however, will be proven for each of the Nucleic acids used an oligonucleotide whose Nucleotide in position X of the nucleic acid to be detected corresponding nucleotide to this nucleotide complementary is. This means that this oligonucleotide is in position X contains a nucleotide which corresponds to the corresponding Nucleotides of the remaining ones present in the sample different with regard to heading X Nucleic acids is not complementary.

The oligonucleotides according to the invention differ in at least one other position Y from each other. This can be achieved through substitutions by others  Bases or base analogs and / or deletions / insertions in the sequence of the oligonucleotide. At least one, however, preferably each of the invention Oligonucleotides of a sentence indicates a Nucleotide position Y preferably has a nucleotide which to the corresponding nucleotide of the to be detected Nucleic acids is not complementary. Essential for the optimal implementation of the method according to the invention is that for the oligonucleotides of a sentence this Positions Y in different relative position to X are located. For variant 1 the position of Y is on the Oligonucleotides not critical. It can therefore be in essentially freely chosen.

Position Y is preferred in variant 2 in each case in the vicinity of the nucleotide position X in the 3'-end of the Oligonucleotides. Preferably, the position Y 1-8, preferably 1-3 nucleotides away from X. For example it has proved to be favorable if Y of the one Oligonucleotide targeting the final nucleotide (position X) following nucleotide and Y of the other oligonucleotide the two or three nucleotides from the end of the nucleotide is. In another oligonucleotide (for the detection of three similar nucleic acids) could then be, for example the non-complementary nucleotide three or two Nucleotides removed from the final nucleotide, etc.

In the following, the positions Y of the 1, 2. , , N oligonucleotides of a set denoted by Y1, Y2 ... YN. Each oligonucleotide may also be in multiple positions Nucleotides corresponding to the definition of the nucleotide Correspond to position Y Preferably contains Oligonucleotide 1-5 such nucleotides.  

For use therefore preferably a set of Oligonucleotides which at least two inventive Contains oligonucleotides, these at least in their nucleotides corresponding to X and two in each case different nucleotides. This arrangement of non-complementary nucleotides in the oligonucleotides has the consequence that in any phase of the subsequent carried out extension reaction, such as a PCR according to EP-A-02 00 362, an extension product is formed, which with another oligonucleotide of the Sentence only one difference in the nucleotide in Position X has. Maintaining a second However, different nucleotide position is for the Sensitivity of the detection is very important, as from the EP-A-03 32 435 is known. Oligonucleotides of a sentence is at least one nucleotide sequence area in common, the except in position Y to a common sequence of is complementary to nucleic acids to be detected. These Areas preferably comprise at least 50% of the length of the Oligonucleotides.

After hybridization of the oligonucleotides to the Nucleic acids to be detected are the resulting Hybrid subjected to an extension reaction. A such extension reaction is preferably attached of mononucleotides which correspond to the corresponding Nucleotides of the original target nucleic acid complementary are to the oligonucleotide in the 3'-5 'direction. This happens preferably then in the extension direction terminal nucleotide of the hybridized oligonucleotide to the corresponding nucleotide of the one nucleic acid is complementary (match) and of the chemical structure is extendable ago. The reaction conditions for a Such chain extension by means of a polymerase and Mononucleotides are known, for example, from EP-A-02 01 184 or EP-A-03 32 435.  

In addition, the extension by tying a further oligonucleotide to the already anhybridized Match oligonucleotide be accomplished, the to be attached oligonucleotide substantially complementary must be to the single-stranded region of the nucleic acid, which adjoins the area to which the match oligonucleotide is already hybridized. This kind of Extension can be achieved with a ligase reaction become. Such an extension reaction is for example in Proceedings of the Natl. Acad. of Sciences USA 88, pp. 189-193, (1991) or WO 89/09 835 described. Further amplification methods on which The invention can be applied are Repair Chain Reaction (WO 90/01 069), the method of DE-A-40 10 465 and the first step of the process of WO 91/03 573.

In variant 1, all nucleic acids hybridized to nucleic acids to be detected are lengthened. However, since the mismatch oligonucleotide P 1 of the one nucleic acid has at least two mismatch nucleotides (X, Y 1 ), it does not hybridize well with this nucleic acid under competitive conditions, as does the match oligonucleotide P 2 , which at the X position Matchnukleotid, it being assumed that the 7-positions in the two respectively competitive oligonucleotides are chosen so that the competition of the oligonucleotides is nevertheless decided by the respective base at position X (and not by the differences in the Y-positions) , The oligonucleotides can also be hybridized offset to each other on variant variant 1 on the original target, but the positions X and preferably also the positions Y lie in the common area. The main product of the extension reaction is therefore a nucleic acid based on the extension of the match oligonucleotide.

Although variant 2 can also mismatch oligonucleotides hybridize to the particular nucleic acid to be detected, an extension is only for the Match oligonucleotide instead.

Prerequisite for the specificity of the extension reaction in variant 2, the enzyme containing the Extension reaction performs, not completely independent of mismatches, an extension of the Catalysed oligonucleotides. These requirements are however, for example, in Thermus aquaticus DNA polymerase (EP-A-02 58 017), or E. coli ligase.

For amplification of the area which is between the outer ends of the oligonucleotides attached to the be detected hybridized nucleic acids are the terminated extension products, for example, with Oligonucleotides reacted to the Elongaten in are essentially complementary (Counterstrand oligonucleotides) and for hybridization brought. These oligonucleotides are used the extension products produced in the first step extended as a template, where the position X usually lies in the area of the extension. The Counterstrand oligonucleotides can be detected for all Nucleic acids be the same. You can, however, too be allelic selective.

This extension can also be done by attaching Mononucleotides or oligonucleotides are performed.

When using the oligonucleotides according to the invention, the except in position X still in at least two others Distinguish positions, arises in the second Extension reaction (referred to as cycle 2 below) Product which is identical to all other oligonucleotides except due to its hybridization with the  to be detected nucleic acid produced the product itself is (the original match oligonucleotide), at least 3 (ie more than originally available) not having complementary nucleotides. This is one Extension of mismatch oligonucleotides at this Product difficult. In an analogous way can by additional y positions the false annealing to the Amplification product of the other allele difficult become. This will otherwise block the Amplification product for the appropriate oligonucleotide, and an associated reduction in the yield avoided.

This improvement of the known methods is explained below with reference to FIGS. 1 to 4.

The main product of the extension reaction in variant 1 is a nucleic acid which is based on the extension of the match oligonucleotide. Hybridization of the mismatch oligonucleotide to an extension product of cycle 2 from hybridization with the match oligonucleotide is virtually impossible since there are already three mismatches (X, Y 1 , Y 2 ). This situation is shown by way of example in FIG . Practically, the success is achieved in particular by skillful choice of the annealing temperature (if desired, other reaction parameters, such as concentration of the oligonucleotides).

By heating to z. B. 90 ° C, cooling to a temperature just above the T _M value of both primers on the original target and then slowly cooling to a temperature just above the T _M value of both primers on the original target and then slowly cooling to a temperature well below the T _M values of both primers are then achieved so that the allelic selective primers hybridize to the better-matched target, respectively. The T _M value is defined as the temperature at which 50% of a target sequence is hybridized with a primer. At this temperature, a bound matching oligonucleotide can also be rapidly released (equilibrium). However, for a less well-fitting oligonucleotide, the T _M value at the same target will be lower, that is, at this lower T _M value, the less suitable oligonucleotide will hardly bind to said target but will be displaced by the more than 50% binding oligonucleotide , This annealing occurs at the appropriate temperatures for both alleles. In the amplification of the PCR product, the T _M values for the respectively perfectly complementary target combination ((E), (H), FIG. 1) are markedly increased in comparison to the T _M values of the oligonucleotide / target combinations (( FIG. A), (B), (C), (D) Fig. 1), since there the additional mutations had lowered the respective T _M value by Mismatchbildung. In contrast, for the oligonucleotide / PCR product target combination in which an oligonucleotide hybridizes with the non-matching allele ((F), (G), Fig. 1), the T _M value again significantly lower than the original target, since now the according to the present invention accumulate additional mutations selected from both oligonucleotides in such a pair. As a result, a further increase in the specificity of the amplification of the PCR products is achieved. If, for annealing in the PCR, a temperature is chosen which is just below the T _M values of the perfectly complementary combinations, the incorrect annealing on PCR products (F), (G) or on the original template (B), (C ) practically impossible. Compared with the known COP process variant 1 has the advantage that a differentiation by temperature selection in the subsequent cycles must be coarsely or not at all. In addition, the direct detection of genomic nucleic acids without pre-amplification is possible.

The variant described here is particularly suitable for multiplex analyzes, since not only the T _M values for a mutation and the corresponding primers can be achieved in said annealing step carried out prior to the PCR, but also by slow cooling over a range from the highest T _M Value below the lowest T _M value, all oligonucleotides are allowed to selectively anneal.

The second variant will be explained with reference to FIGS. 2-4. This case, in which the 3 'end nucleotide of the oligonucleotides corresponds to position X, is preferred because of its higher selectivity. This is due to the fact that it is often of no practical importance for the selectivity whether the mismatch oligonucleotide is hybridized or not.

To illustrate the principle z. B. a PCR reaction in a single reaction vessel considered simultaneously in which two alleles of a gene can be detected, which are in a base position distinguish (an allele there have the base M (utant) that other N (ormal)). Three primers are used, one antisense primer and one primer each for each one of the alleles. The primers are selective in that the base at the 3 'end of the one selective primer to M, the of the other primer to N is complementary. The two alleleselective primers are now but z. B. by two different substitutions from each other, where in each primer each a mismatch to the template arises, so that each of the two primers a mismatch to the each selected allele has and two mismatches to other allele. For easy distinction of resulting PCR products can also (eg) different long alleleselective primer can be selected. These changes are made in the respective PCR products  installed so that in the further course of the PCR by each one of the allelic selective primer resulting PCR product a better (namely completely complementary) Target for this primer represents as the original target, and also for the other alleleselective Primer is no longer a suitable target because of this now even has three mismatches to the modified target. This is the fault-determining selection of alleles by the primers substantially in annealing to the Original target or / and in the following extension of the primer; the further amplification of the existing ones PCR products are separated by the additional changes.

FIG. 2 shows the relative position of the oligonucleotides used in the examples (position 1 at the 3 'end). This is the detection of two alleles of the apolipoprotein B gene (Proc. Natl. Acad. Of Sciences USA 86, pp. 587-591, 1989).

For additional separation of the amplification of the individual alleles in one reaction, the oligonucleotides P 1 and P 2 were distinguished by further Y positions at pos. 28 (Y3: C, mismatch) and 29 (Y4: C, mismatch) from P2. In the following, the statement match or mismatch is related to the sequence of the normal allele.

Oligonucleotide P 1 :
Asa short (29 bases): SEQ ID no. 1
Nucleotide in position X: T (mutant selectively)
Nucleotide in position 2 (Y 1 ): G (mismatch)
Nucleotide in position 4 (Y 2 ): G (match)

Oligonucleotide P 2 :
Asa lang (49 bases): SEQ ID no. 2
Nucleotide in position X: C (selective normal)
Nucleotide in position 2 (Y 1 ): C (match)
Nucleotide in position 4 (Y 2 ): T (mismatch)

Oligonucleotide P 3 :
Counterstrand primer: SEQ ID no. 3 completely complementary to the strand

Oligonucleotide P 2 ':
Asa lang '(49 bases): SEQ ID no. 4
Nucleotide in position X: C (selective normal)
Nucleotide in position 2 (Y 1 ): G (mismatch)
Nucleotide in position 4 (Y 2 ): G (match)

FIG. 3 shows the results of the first three cycles of a PCR when the oligonucleotides P 1 and P 2 according to the invention are used in an amplification approach. The last base at the 3 'end of the oligonucleotide P 1 matches the mutant allele, the last base at the 3' end of the oligonucleotide P 2 matches the normal allele, so that extension of the primer at those shown in Figure 3 as (B) and (C) marked primer / template combinations. Products of the reaction are the nucleic acid hybrids (F) and (G). The differences (mismatchs) marked with _* in (B) and (C) do not completely prevent the extension of the primers. In the second PCR cycle exactly complementary complementary strands (I) or (K), upper sequence form to the extension products. Although these newly formed products can hybridize with the other oligonucleotide to the products (O) or (L), an extension reaction does not take place, since 3 mismatches are present in these hybrids. In contrast, an extension of the corresponding associated oligonucleotides from the products (M) and (N) takes place.

In Fig. 4 it is shown that the positions Y of the non-complementary nucleotides in different oligonucleotides P 1 and P 2 may not be identical, since otherwise a side reaction greatly reduces the sensitivity of the test. In Fig. 4, the case is shown that the two oligonucleotides P 1 and P 2 'each have a mismatch in the first two positions of the extension direction in the end. It is clear that in the second cycle of the PCR products are formed which have only one single mismatch with the other oligonucleotide (products O and L). These products may undergo a specificity-reducing extension reaction because the products have only one mismatch each. It would therefore not be possible with the known method to determine the two alleles with sufficient specificity in a reaction mixture.

The specificity of the detection in variant 2 can be increased by additional differential hybridization of the oligonucleotides to the nucleic acids to be detected in the manner of COP. In the previously described embodiments of variant 2, the oligonucleotides hybridized with all the nucleic acids to be detected approximately with the same efficiency. If, however, the oligonucleotides have further differences in position Y (for example mismatchs), then, by judicious choice of the temperature of the hybridization of the oligonucleotides to the nucleic acids to be detected, eg. For example, in the case of PCR (just below the T _M value of the hybrid of nucleic acid to be detected and the corresponding oligonucleotide (match oligonucleotide), a further increase in the specificity and yield of the amplification can be achieved.These additional Y positions are not specific to one, for example The oligonucleotides differ in these positions after definition of the Y positions.The changes of each oligonucleotide are incorporated into the respective match product and the corresponding complementary strand product and thus make it more difficult if appropriate The annealing of the match oligonucleotide can therefore hardly be hindered by an annealing of the mismatch oligonucleotide on the product target, so that therefore a separation of the amplification occurs and a reduction of the amplification rate by blo precluding false annealing is excluded.

In further embodiments, in particular of variant 2, 2 or more nucleic acids having 2 or more differences (polymorphic sites, X 1 , X 2... XN) can be detected in the nucleotide sequence, wherein the nucleic acids described in (Nucl. Acids Res. Vol. 16, 23, 11141-11155) described method can be used (multiplex method).

There are several possibilities according to the invention. In a first embodiment, the polymorphic sites are separately made the subject of the method of the invention. As many oligonucleotides of the invention are then used as polymorphic sites to be detected on different nucleic acids. For 2 polymorphic sites on 2 nucleic acids, therefore, at least one set of 4 oligonucleotides P 1 , P 2 , P 4 , P 5 according to the invention is used. Such a case is shown in FIG . The amplified regions, which are also bounded by the two counterstrand primers, do not overlap here, but are preferably of different lengths. This makes separate detection easy. Alternatively, it is possible to dispense with primer P 6 . Then in the amplification for both polymorphic sites the opposite strand primer P 3 is used.

A second embodiment is based on the fact that the sets of oligonucleotides according to the invention belonging to different sites hybridize with different strands of the nucleic acids to be detected ( FIG. 9). In this case, the amplified regions may also overlap, it being possible by analogous use of Y positions (in the overlapping region of the oligonucleotides P 1 , P 2 , P 4 and P 5 ) to ensure that the amplification of the two polymorphic sites remains separated. Here too, 4 oligonucleotides (P 1 , P 2 , P 4 and P 5 ) and two counterstrand primers are required by way of example.

A third, preferred embodiment ( Figure 10), in which the detection of polymorphisms is coupled to each other, does not require any counterstrand primers, since the oligonucleotides according to the invention act as such. In a simple manner, four different amplification products can be formed and detected here, depending on whether corresponding alleles are present. Thus, a direct determination of the cis / trans location of mutations is possible.

The above embodiments are analogous to more than two polymorphic places usable.

A particularly preferred embodiment is based on Use of the PCR (EP-A-02 01 184), hereinafter referred to content is referred to. To prove similar Nucleic acids require only a primer a set of oligonucleotides of the invention  replace. The remaining reaction conditions for the Amplification, in particular the use of the Gegenstrangprimers, can be applied analogously.

Also, the so-called ligase chain reaction (LCR, EP-A-03 20 308) can be improved according to the invention. Again, a set of oligonucleotides of the invention is used instead of at least one oligonucleotide. In Fig. 13 it is shown that this increases the number of mismatches in cross products. Depending on the position of Y, the extension or / and the annealing of the wrong oligonucleotides is thereby largely suppressed. Position X in this embodiment may be at the 3 'end of one or the 5' end of the other oligonucleotide. Incidentally, in principle, analogous to the COP, an LCR can be carried out in which X is close to the middle of the oligonucleotide. Preferred counterstrand oligonucleotides used are those which, in positions corresponding to y, have nucleotides which are not complementary to any of the nucleotides of positions Y of the oligonucleotides according to the invention.

However, the improvement according to the invention can be just as in promoter-primed, cyclical Amplification reactions are used, as in Method of EP-A-03 10 229, EP-A-03 29 822 or EP-A-03 73 960. The contents of these documents are hereby incorporated by reference Referenced. These amplifications will help with a promoter primer a variety of product targets (RNA) formed again using the promoter primer be amplified.

The specific proof of the extension products, which is a measure of the presence or amount of is to be detected nucleic acid in the sample is For example, possible because the used Differentiate oligonucleotides in a further feature.  

The difference may be, for example, that the Oligonucleotides of a set a different length respectively. This is caused by the extension different oligonucleotides of different lengths Extension products. The oligonucleotides can become but also differ in that they are different detectable groups D included. Such detectable Groups D are for example color or Fluorescent molecules or chemical residues in one subsequent reaction with a detectable residue can be determined. Such chemical groups are for example haptens, such as digoxin or digoxigenin. Haptens can be detected by using a labeled antibodies are reacted against the hapten and the mark is measured. Another hapten could be, for example, biotin, which is different labeled antibody or detectable with streptavidin is done.

The different extension products which a measure for the amount and presence of the detected Nucleic acid can, either after division of the Separate approach, sequential and upon receipt of the approach when using suitable markings also simultaneously be detected by known methods. For the use of steroid hormones as markers has in EP-A-03 24 474 described method proved to be preferred. A Another method is to use different ones Fluorescent dyes. The oligonucleotides can either be marked directly, or z. B. antibodies against chemical groups could be appropriately fluorescently labeled his. The proof of the different allelic products is then by simultaneous fluorescence measurement in several Channels possible. A marking of a part of the products with fluorescent markers and other products from the same  Approach with enzymes is also possible. All other conceivable marking methods and detection methods are in principle also suitable.

For sequential or simultaneous detection, for example, two different chemical groups, preferably haptens, which are recognized by different antibodies, can be used to label the two oligonucleotides. Examples of such haptens are digoxigenin, biotin and fluorocein. The antibody against fluorescein and the antibody against digoxigenin then preferably have different enzymes as a label. By sequential or simultaneous contacting with enzymes that are specifically detectable for the enzymes, both nucleic acids can be detected. Alternatives on how the extension products can be detected are given in Figs. 5-7.

The mixture of the common extension reaction is subjected to a common denaturation reaction in the embodiment shown in FIG. 5 (left side). The single-stranded extension products are bound to a solid phase F with a so-called capture probe which is immobilized or can be immobilized via a group I (eg biotin). The extension products of the reaction are immobilized with all nucleic acids to be detected. The different detectable groups enable a sequential detection of nucleic acids to be detected (separated by a washing step). If the signals finally to be detected can also be generated and detected side by side in a vessel, the washing step can be dispensed with.

On the right-hand side of FIG. 5, a variant is shown in which, after the common extension reaction, a sufficient amount of fluid is withdrawn from the reaction mixture at least twice (or correspondingly more frequently upon detection of more than 2 alleles and using more than 2 labeling enzymes) individually tested for the extension products. From a multiplex reaction aliquots can be removed and depending on the number of different labels used on the oligonucleotides correspondingly many amplification products per aliquot can be detected.

FIG. 6 shows a method in which a mononucleotide or oligonucleotide modified with an immobilisable group was also incorporated in the extension reaction. Thereby, the extension products can be bound directly (ie without separate capture probe and without denaturation) to a solid phase (analogous to DE-A-40 01 145). Detection is also possible as shown in FIG. 5, simultaneously, sequentially or side by side.

FIG. 7 shows an embodiment based on the fact that the extension products for different nucleic acids to be detected differ preferably by at least three nucleotides from each other. This can be exploited for selective immobilization via a capture probe. Detectable groups D are introduced by incorporation of a modified mono- or oligonucleotide. This group may be identical for the extension products of both alleles. If the amplification of the nucleic acids according to variant 2 has been carried out, the variations Y are in the vicinity of X and the capture probe can be selected to be identical to a part of the oligonucleotide sequence and to a part of the extension sequence; at the same time, selection for the correct extension product (artifacts lacking the extension sequence) and for the various alleles takes place at the same time.

Further embodiments are conceivable, for example via selective immobilization via built-in Oligonucleotides that are different for each allele have immobilizable group. The proof can then via built-in detectable mononucleotides, a Built-in detectable Gegenstrangprimer or means done a detectable detection probe.

The proof when using different length Oligonucleotides are preferably based on gel chromatographic separation of the extension products, which are as long as the amplified area (inclusive the oligonucleotides). The detection can be right after Separation in the gel, (eg ethidium bromide staining), after Incorporation of labeled oligonucleotides, mononucleotides or Hybridization with labeled probes done. On Difference in electrophoretic mobility can also be achieved in other ways, for. B. by 5'-end labeling of an oligonucleotide with biotin; after Amplification will be an aliquot of the product Streptavidin incubated and electrophoresed subjected: by binding of streptavidin to the biotin labeled product becomes the electrophoretic Mobility of this product significantly reduced while the other product behaves normally.

Unless one of the similar nucleic acids in the sample fails is preferably not for this nucleic acid Signal measured by extension of your corresponding oligonucleotide would be caused. With Therefore, the method of the invention can only one  the similar nucleic acids are detected, or also the absence of all similar nucleic acids detected become.

In that no cross reactions of the Extension product from an allele with Oligonucleotides used to detect the other allele be held, it is possible the Amplification and, if desired, the detection of similar nucleic acids in a batch simultaneously and in to make a single reaction vessel. this is not to an optimum extent possible if the positions of the Incompleteness Y (Mismatches) with the detected Nucleic acids identical for different oligonucleotides are.

The inventive method can be applied in many ways become. For example, the occurrence of alleles in a sample drawn by a single subject was (individual diagnostics), in correlation to certain Diseases (eg metabolic disorders) are.

In addition, the procedure is because of the good Specificity is also suitable for procedures on the Principle of pool screening of samples based. To be a large number of individual samples mixed by different subjects. For the diagnosis For example, the defect in the apo B 3500 gene has it has proved expedient to add 64 samples in one pool combine. It is with the method according to the invention possible to determine whether a sample of the pool Defective had. Through several times in succession Implementation of the method according to the invention Subpools of a pool with a positive result can be in, compared with the prior art, few provisions the sample to be identified, which is the gene with the defect having.  

In the method according to the invention, it turns out to be particularly advantageous that at the same time always the gene without the defect can be detected if it exists is. In pool screening, alternatively, the mutant Product can be detected or, if no mutant allele is present, the normal product can be detected. The ability to detect both alleles poses in addition to a control of the reaction as such also a means of quantifying the to be detected Nucleic acids.

Pool screening procedures can be guaranteed because of Sample anonymity especially in epidemiological interesting diseases, such as the rate of infection with the AIDS virus or the enforcement of Population groups with z. Hypercholesterolemia, Hypertension or diabetes (mass screening) used become. The frequency of occurrence of alleles can be be determined with it.

The same applies to the species diagnostics of bacteria or optionally for virus diagnostics. Here is especially the simultaneous detection of closely related pathogenic / non-pathogenic species or strains Interesting. Also for the safe detection of E. coli K12 (Assessment of the Genetic Engineering Act) on the known AT sequence variants with distinction from E. coli B strains is the method of the invention suitable. It can thus be used in environmental analysis become.

Main advantage of the invention over the prior art is the possibility to add allelic compounds in mixtures practically negligible background reaction to be able to detect simultaneously in a vessel. On Restriction digestion is not required. The goal is  achieved by a quasi-separation of the amplifications (Reduction of cross-reactions) of the individual alleles, although an amplification in a common vessel is carried out.

Likewise provided by the invention is a set of Oligonucleotides containing at least two oligonucleotides contains, which hybridize with similar nucleic acids where the oligonucleotides are present in at least 2, preferably at least in 3 defined positions as well preferably distinguish another structural feature. Preferred are those oligonucleotides which are in their 3'-terminal nucleotides (corresponding to position X) and in each case two further nucleotides as well as preferred differentiate one another from another structural feature. The detectable structural feature may be the different length of the oligonucleotides or a be detectable or immobilizable group. The Oligonucleotides may be present as a mixture. also this mixture may contain further oligonucleotides included in the amplification of nucleic acids with the help of the sentence of Oligonucleotides are required, in particular Gegenstrangoligonukleotide or primers.

Also show an effect according to the invention Oligonucleotides that differ in position Y Have nucleotides, but these are each not too the nucleotides of Templatnukleinsäuren complementary are when this position in the oligonucleotides this Sentence has the same distance from X has. a attenuated effect shows such a set of Oligonucleotides, of which only one in position Y has a nucleotide that is not the corresponding  Position of the associated nucleic acid to be detected is complementary. The inventive set of Oligonucleotides will solve the problem in the used according to the invention.

Another object of the invention is a reagent kit for the detection of similar nucleic acids containing a Inventive set of oligonucleotides and a Enzyme for the extension of nucleic acids and possibly further required to extend the oligonucleotides Contains reagents and excipients. In addition, that can Reagent kit further oligonucleotides, for example Counterstrand primer, included. Lie in the reagent kit prefers the enzyme and the oligonucleotides in of remaining reagents separate containers before.

Another object of the invention are the use of the Inventive method for simultaneous determination of alleles in a sample (eg also for the typing of DNA-based transplantation antigens), for simultaneous determination of topologically closely related Infectious agents (eg also in the course and Therapy Control) and for the screening of sample pools the presence of closely related nucleic acids or their frequency. In addition, the use of the Method claimed for the detection of mutations.

Fig. 1 shows in theoretical form a method according to the invention, which is based on competitive priming.

Fig. 2 shows the arrangement of nucleotide sequences, as underlying Fig. 3 and the examples.

Fig. 3 shows the method according to the invention for the use of 3'-mismatch oligonucleotides.

Fig. 4 shows a method in which the mismatch nucleotides are both located in the same position near the end of the oligonucleotides.

FIGS. 5 to 7 show embodiments for detecting the extension products of the method according to the invention.

Figs. 8 to 10 show the application of the method according to the invention for the simultaneous detection of several polymorphic sites.

Fig. 11 shows an analysis of the samples amplified in Example 1.

Fig. 12 shows the analysis of the samples amplified in Example 2 and a negative control.

Fig. 13 shows the method according to the invention using an LCR.

LIST OF REFERENCE NUMBERS I Group for immobilization D Groups for detection D 1 , D 2 different groups for detection F solid phase P 1 , P 4 Primers 1, 4 according to the invention P 2 , P 5 Primer 2, 5 according to the invention P 2 ' comparative primer P 3 , P 6 , P 7 Strand primer X, X 1 , X 2 allelic positions Y 1 , Y 2 Positions of Mismatches

The following examples serve to clarify the Invention.  

example 1

Detection of the two alleles for the CGG → CAG mutation in codon 3500 of the apolipoprotein B gene in one Reaction.

sample preparation

Human genomic DNA was prepared from EDTA blood by the method by Higuchi (in PCR Technology, Principles and Application for DNA Amplification, ed. Ehrlich, H.A. (Stockton press, New York), 31-38 (1989)), wherein the final volume of DNA solution equal to that Volume of EDTA blood is used. On average For example, 7500 leukocytes / μl will be a 2 μl sample contain up to 15,000 leukocytes, so with up to about 100 ng DNA can be calculated in 2 ul solution. The DNA was at the end of the method of Higuchi at 100 ° C. heated, allowing further denaturation before implementation the PCR is no longer necessary.

Amplification according to the invention with the aid of PCR

The PCR according to EP-A-02 00 362 was in 0.5 ml Tubes (Sarstedt No. 72 699) and a GENE ATAQ controller (Pharmacia) using the following reagent mixture carried out:

10 μM each dATP, dGTP, dTTP, dCTP
1 mM MgCl₂
6 μl of 10 × PCR buffer without Mg²⁺
0.6 μl Asa short (primer P 1 , 95.7 ng / μl), SEQ ID no. 1
1 μl of Asa lang (primer P 2 , 81 ng / μl), SEQ ID no. 2
0.6 μl of counterstrand primer P 3 (92.4 ng / μl), SEQ ID no. 4
0.3 μl of Taq polymerase (5 units / μl, Beckmann)
60 μl total volume.

The 10X PCR buffer is: 100mM Tris-HCl pH 8.3, 500mM KCl, 0.1% gelatin

Temperature profile:
45 times (1 min. 95 ° C, 1 min. 65 ° C, 1 min. 72 ° C)
4 times (2 min. 65 ° C, 1 min. 72 ° C)
1 time (5 min. 72 ° C)
Cool to room temperature

Detection of extension products

10 μl of the PCR products were then mixed with 2 μl Application buffer (40% sucrose, 0.25% bromophenol blue, 0.25% Xylenecyanol, 0.1 M EDTA pH 8.0) and mixed in one Agarose gel examined electrophoretically. The Composition of the gel was 3% NuSieve GTG (FMC / Biozym), 1% agarose NA (Pharmacia), 1 x TAE, 0.5 μg / ml ethidium Bromide; (50 x TAE = 242 g Tris base, 57.1 ml glacial acetic acid, 100 ml of 0.5 M EDTA pH 8.0). The duration of the gels is 0.5-1 h at about 5 V / cm. After the photograph of the Fluorescence at 300 nm with Polaroid 667 black & white instant film using a Camag Reprostar Setup, the evaluation is based on the photo.

To demonstrate that the two allelic selective primers are necessary for the reliable identification of the alleles, the following samples were analyzed (see Fig. 11):

1: length standard
2: heterozygous subject
3: homozygous normal subject and
4: cloned mutant allele, amount (molar) according to sample 3

The samples were amplified using the two allelic selective primers P 1 , P 2 and the counterstrand primer P 3 . The normal sample yields, as expected, only one 134 bp product, the mutant sample only one 114 bp product. The heterozygous subject (lane 4 in Figure 11), on the other hand, delivers both products as expected. If, on the other hand, only one of the allelic selective primers is used, the PCR product corresponding to the primer is obtained, irrespective of which of the templates was amplified. In particular, the normal subject also provides the band corresponding to the mutant allele (lane 6) and the cloned mutant DNA the product of the normal selective primer (lane 10). In this case, amplification with the original ARMS concept (CR Newton et al., Nucleic Acids Research 17, 2503-2516 (1989)) is therefore not suitable for distinguishing the alleles under the given reaction conditions. Only the use of the invention described here allows in the present case, the correct and unambiguous (lane 2, 3, 4) determination of the genotype and also allows the determination of both alleles in one approach.

The cloned DNA is a fragment 10573-1070 of the Apo B cDNA sequence includes in the vector Gem3zf (-) (Atlanta / Promega). Before the Used in the PCR reaction, the DNA was linearized with BamHl and diluted to 0.06 pg / μl. This dilution contains about 15 000 DNA molecules / μl, ie in 2 μl approximately as often the Apo B sequence as 2 ul DNA solution from a DNA isolation from EDTA blood.

These experiments show that the formation of nonspecific Products, despite additional measures to Increase of specificity in reaction in separate Vessels occur by using the invention Oligonucleotides are suppressed in a reaction vessel can.  

Example 2 Screening for the CGG → CAG mutation in codon 3500 of the Apolipoproteins B gene (pool screening)

This example demonstrates that the described methodology is suitable for detecting a carrier gel in a pool of other subjects' DNA. First, the sensitivity was checked. For this purpose, DNA obtained by DNA isolation according to Higuchi (see Example 1) was mixed as follows:
heterozygous DNA: normal homozygous DNA
1:50, 1: 100, 1: 200, 1: 400 and 1: 800. 2 μl of the obtained DNA solutions were amplified in three reaction steps:

1st reaction step

In this pre-reaction, only the normal-selective primer was used used around the DNA that denatures on preparation was, so single-stranded, again in duplex to convict. This will cause wrong priming by the mutant-selective primer at large excess normal Alleles avoided, what at low temperature, at the PCR is set, otherwise easily occurred. By This pre-reaction ensures that the mutant selective primers only at the desired Annealing temperature in the following Reaction steps to single-stranded normal template meets. In this pre-reaction is the dNTP concentration due to the relatively small total volume of only 12 μl relatively high, since in this reaction already the entire dNTP amount was used for the  2nd reaction step is needed. This facilitates the complete transfer of single-stranded normal Alleles in duplex.

2 μl of DNA solution
10 μl aliquot of the reagent mix:
1.4 μl dNTP mix (20 μM stock solution)
0.9 μl Mg²⁺ (20 mM stock solution)
1.2 μl of 10 × PCR buffer without Mg 2 O (see Example 1)
0.28 μl primer Asa lang (81 ng / μl stock solution) SEQ ID no. 2
5.99 .mu.l H₂O
0.23 μl of Taq polymerase (5 U / μl, Beckmann)
12 μl total volume

Temperature profile:
4 × (2 min 60 ° C, 2 min 72 ° C), 5 min 72 ° C, without oil overlay.

2nd reaction step

PCR with the primers P 1 , P 2 and the counterstrand primer P 7 (5 'to counterstrand primer P 3 from FIG. 2, SEQ ID No. 5).

The additional counterstrand primer was used to eliminate primer timer problems. Both primer P 7 and primer P 3 provide primer dimers for a very large number of cycles, with the result that the desired PCR product could possibly no longer be obtained. By amplification with primer P 7 (this second reaction step) and subsequent further amplification with primer P 3 (reaction step 3), the formation of the primer dimers is suppressed.

The concentration of the normal-selective primer in this reaction is significantly lower than that of the mutant-selective primer, since only the amount of normal-selective primer present in reaction 1 has been taken over. As a result, a preferred amplification of the mutant allele is possible. However, the amount of normal selective primer present is sufficient in the event that no mutant allele is present in the batch to obtain a visible amplification product after the 3rd reaction step and thus also to allow in the pool screening an internal control of the PCR reaction.

Approach:
12 μl = batch of the 1st reaction step
58 μl aliquot of the reagent mix:
1.4 μl of primer P 7 (51.1 ng / μl stock solution) SEQ ID no. 5
0.7 μl Asa short (95.7 ng / μl stock solution) SEQ ID no. 1
0.85 .mu.l Mg²⁺ of 20 mM MgCl₂ solution
5.8 μl 10 × PCR Buffer without Mg²⁺
0.12 μl Taq polymerase
49.13 μl H₂O
70 μl total volume

The samples were washed with approx. 5 μl heavy white mineral oil (Sigma Cat No. 400-5).

Temperature profile:
20 × (1 min 95 ° C, 1 min 60 ° C, 1 min 72 ° C),
4 × (2 min 60 ° C, 2 min 72 ° C) 5 min 72 ° C

The last four cycles (only annealing and extension, without denaturation) should ensure that before setting of the third reaction step no single-stranded DNA more present.

3rd reaction step

PCR with counterstrand primer P 3

7 μl of the product of the 2nd reaction step
43 μl aliquot of the reagent mixture:
0.5 μl of primer P 3 (92.4 ng / μl) SEQ ID no. 4
0.65 μl Asa short, SEQ ID no. 1
0.2 μl Asa lang, SEQ ID no. 2
5 μl 10 × buffer without Mg²⁺
2.14 .mu.l Mg²⁺ of 10 mM MgCl₂ solution
2.5 μl of dNTP mix containing the four dNTPs per 100 μm
31.76 μl H₂O
0.25 μl of Taq polymerase (5 U / μl, Beckmann)
50 μl total volume

Temperature profile:
40 × (1 min 95 ° C, 1 min 65 ° C, 1 min 72 ° C),
4 × (2 min 65 ° C, 2 min 72 ° C), 5 min 72 ° C

In the second reaction, the selectivity is essentially determined by the low dNTP concentration (2 μM), while the annealing temperature at 60 ° C is relatively low is to ensure efficient annealing. In the 3. Reaction, on the other hand, becomes a higher amount of dNTP used, so that a larger amount of product can be formed. Since the additional mutations in the Primers with the second reaction in the PCR products  were installed, the annealing temperature in the 3. Reaction be increased to 65 ° C.

For the screening application for the B3500 mutation, the scheme for pooling the samples of frequency of mutation in the general population was adjusted. The B3500 mutation occurs in about one in 500 to 700 people. The analysis of the samples should be done in three stages:
1. analysis of relatively large pools,
2. Analysis of smaller pools, from which the larger pools were formed,
3. Analysis of the individual samples.

For this purpose, 8 EDTA blood samples each were mixed into primary pools and of each 8 primary pools an aliquot was used to generate a 64 pool. This scheme is optimal for a three-step analysis with a frequency of mutation of 1 in 8 ^3, ie 1 in 512, but can also be used over a wide range of other frequencies.

When mixing the EDTA samples, the different concentration of leukocytes the individual samples considered by different Volumes, so that each subject an equal number of leukocytes went into the pools. For this purpose were initially 100 μl of EDTA blood per subject on microtiter plates pipetted so that the subsequent pipetting of a pipetting robot could be taken over (Tecan RSP 5052, Zinsser Analytic, Frankfurt). Through the machine Each sample was first diluted with 100 μl of 0.9% NaCl and aliquots with approximately 80,000 leukocytes (volumes per Computer calculated from leukocyte concentrations) united to the primary 8 pools. From the 8 pools were (after mixing by machine) about 160 000 each Leukocytes pipetted to 64 pools. The isolation of  DNA was initially performed only for the 64 pools. Approximately 4100 samples were analyzed in this way. there 6 64 pools were found, the mutant sequence contained. The DNA of the corresponding 8 pools was isolated and analyzed in the same way as the 64er Pools. Eventually the DNA became the most positive Found 8 pools belonging to individual samples isolated and examined according to Example 1. In this way were Identified 6 heterozygous carriers.

For the negative control, only the normal ande receive. With slightly different mixture of the oligonucleotides (a little less ASA long) can be You can always do that for negative controls as well a slightly weaker band at the mutant's position Product receives. This is the highest level of Sensitivity of this system is reached, since now already by Fehlpriming or error of the tag polymerase in the Counterstrand synthesis just emerged background becomes visible. This ensures that even small Amounts of actual mutant original be detected (then the normal band disappears), if they are above the background.

Fig. 12 shows the analysis of the amplified samples and a negative control. While only the long PCR product occurs in the negative control (134 bp, lane 2), only the mutant PCR product (114 bp) is found at 3 and 4. At dilution 5 to 7, one finds dominant the mutant PCR product, and also a very small proportion of the normal product. Thus, the method described makes it possible to detect DNA of a heterozygous subject even in a mixture with DNA of 800 normal subjects. Even greater dilutions have not been tested.

1: length standard
2: negative control
3: 1: 50
4: 1: 100
5: 1: 200
6: 1: 400
7: 1: 800

Example 3 Procedure using a non-renewable Oligonucleotide for one of the alleles

If necessary, especially for multiplexing also blocks oligonucleotides against extension be z. B. by incorporation of a Dideoxynukleotids on 3 'end (Cozzarelli et al., Journal of Molecular Biology 45, 513 (1969)). In a multiplex system that serves to determine whether of several known (rare) mutations one (or more) is present, so can the normal Alleles are excluded from the amplification, so that the number of amplification products for each subject in usually limited to 1-3 bands (1 normal Gang is mitamplifiziert to control the Amplification, 1-2 bands of mutant alleles). A wrong one Annealing on the original template in cycles that after the primary annealing step, may additionally by subsequent addition of blocked oligonucleotides which are perfectly complementary to the original target are.

In the event that roughly the concentration of an allele or an isolate of an infectious agent or a somatic mutant (oncogenes)) with variant 2 is supposed to be a third artificial allele than Standard and for this and the allele, which is to be estimated becomes an inventive Primers with 3'-OH end used while the  Amplification of the other alleles by Dideoxyoligonucleotides is prevented. The artificial 3. Allele can be the Y-deviations of the corresponding primer already included and insofar as the primer then matched directly to the template relatively many Y-positions contribute to a safe separation of the amplifications achieve.

In the polymerization of nucleotides to polynucleotides there is a link between the 5'-OH group and the 3'-OH group via a phosphodiester bond. On Dideoxynucleotide has no 3'-OH group. A Extension of a nucleic acid with dideoxynucleotide on End is not possible. This effect is yes z. B. used in sequencing. When multiplexing occur now but numerous gangs on, the first confuse rather. z. B. was already in the laboratory with us a multiplexing for 5 mutations in one approach performed with the variant 2 (allele-specific primers). In doing so, at least occur 5 gangs on (normal person without mutations). If one Person heterozygous for two mutations is even 7 gangs. But if for a particular gene (in this case, LCAT, z. But also LDL receptor) and a specific person only It should be checked if any of the known mutations may be present on the amplification of normal alleles (except one band as PCR control) can be omitted. This reduces the number of bands in one Normal person on a gang. Each existing mutation comes only a gang to it. The overall yield of the PCR is distributed So on significantly fewer bands and the band pattern is simplified. Do you have such z. B. of 40 different possible defects one proved (in several Multiplex reactions), so can subsequently on Heterozygosity or homozygosity are tested (this time instead of dideoxy- with normal primer). To be in one such multiplexing the normal allele by an oligo cover, and so a hybridization with z. B. the  to exclude false COP primer can (depending Mutation site ever) used a Dideoxyoligonukleotid become. By otherwise according to the invention fits primer then the mutant primer is perfect for a possible formed PCR product from the mutant allele. Also at Variant 2 (allele-specific primers) could be one Dideoxyoligonucleotide can be used to form a incorrect annealing and priming of the other primer to reduce.

In LCR reactions analogous 5'-OH oligonucleotides be used.

In promoter-controlled variants can analogously one of above oligonucleotides are used or a primer, the one non-promoter variant of the Promoter sequence contains.

sequence Listings

SEQ ID no. 1 Sequence length: 29 bases Type of sequence: deoxyribonucleotide Stranded form: single strand Topology: Linear Position: 10658-10686 [J. Biol. Chem. 261, 122918-12921 (1986)]

3'-TGAGAAGTCA CTTCGACGTC CCGTGAAGG-5 '

SEQ ID no. 2 Sequence length: 49 bases Type of sequence: deoxyribonucleotide Stranded form: single strand Topology: Linear Position: 10658-10706

3'-CCATAAGTCA CTTCGACGTC CCGTGAACCT TTTAACTACT ATAGACCTT-5 '

SEQ ID no. 3 Sequence length: 49 bases Type of sequence: deoxyribonucleotide Stranded form: single strand Topology: Linear Position: 10658-10706

3'-CGAGAAGTCA CTTCGACGTC CCGTGAACCT TTTAACTACT ATAGACCTT-5 '

SEQ ID no. 4 Sequence length: 28 bases Type of sequence: deoxyribonucleotide Stranded form: single strand Topology: Linear Position: 10573-10600

5'-GATGTCAAGG GTTCGGTTCT TTCTCGGG-3 '

SEQ ID no. 5 Sequence length: 31 bases Type of sequence: deoxyribonucleotide Stranded form: single strand Topology: Linear Position: 10,532 to 10,562

5'-GCCTCACCTC TTACTTTTCC ATTGAGTCAT C-3 '

Claims (23)

1. A method for detecting similar nucleic acids whose nucleotide sequences differ in at least one position (X), which contains at least the following substeps:

• - Implementation of a set of oligonucleotides whose nucleotide sequences differ in the position corresponding to position X, with the nucleic acids to be detected under hybridization conditions;
• Extension of the hybridized oligonucleotides using the nucleic acids to be detected as template; and
• - proof of extension products,

characterized in that the oligonucleotides of the set also differ from one another in at least one further position (Y). 2. The method according to claim 1, characterized in that it contains the following further substeps:

• Formation of nucleic acids complementary to the extension products or a substantial part thereof;
• Extension of a new set of oligonucleotides to polynucleotides using the complementary nucleic acids as target nucleic acids.

3. The method according to claim 1, characterized each X corresponding nucleotide of a Oligonucleotide to the nucleotide X a to be detected nucleic acid, but not the other nucleic acids to be detected, complementary is. 4. The method according to claim 3, characterized that the oligonucleotides with the position X corresponding nucleotide end. 5. The method according to claim 1 or 2, characterized characterized in that the positions Y from the range the oligonucleotides are selected, the 1 to 8 Nucleotides of the terminal nucleotide of Oligonucleotides is removed. 6. The method according to any one of the preceding claims, characterized in that the Extension reactions of the oligonucleotides simultaneously he follows. 7. The method according to any one of the preceding claims, characterized in that the extension reaction for both oligonucleotides in a single Reaction vessel is made. 8. The method according to any one of the preceding claims, characterized in that the oligonucleotides distinguish detectable. 9. The method according to any one of the preceding claims, characterized in that the oligonucleotides are different lengths.   10. The method according to any one of the preceding claims, characterized in that the oligonucleotides have different detectable groups. 11. set of oligonucleotides, which at least 2 Contains oligonucleotides that are similar Nucleic acids can hybridize, thereby characterized in that the oligonucleotides from each other at least in 2 defined positions X and Y differ. 12. Set according to claim 11, characterized in that the oligonucleotides are in a position X and distinguish at least 2 positions Y. 13. sentence according to claim 11 or 12, characterized characterized in that the oligonucleotides in a different structure feature. 14. Set according to claim 13, characterized in that the detectable structural feature a different length or relative position of Oligonucleotides is. 15. Set according to claim 13, characterized in that the detectable structural feature is a detectable one Marker group is. 16. sentence according to any one of claims 11-15, characterized characterized in that the oligonucleotides as a mixture available.   17. Reagent kit for the detection of similar nucleic acids containing a set of oligonucleotides according to any one of claims 11-16, an enzyme for extension of nucleic acids and optionally further to Extension of the oligonucleotides required Reagents and excipients. 18. Use of the method according to claim 1 for simultaneous determination of alleles in one Sample. 19. Use of the method according to claim 1 for simultaneous determination of topologically close related infectious agents. 20. Use of the method according to claim 1 for Screening of sample pools for the presence of closely related nucleic acids. 21. Use of the method according to claim 1 for Detection of one or more nucleic acids, wherein one to the or the nucleic acids to be detected similar nucleic acid as a control nucleic acid is detected. 22. Use of the method according to claim 1 for Determination of mutations.