WO2001075154A2 - Novel method for the parallel sequencing of a nucleic acid mixture on a surface - Google Patents

Abstract

The invention relates to a method for the parallel sequencing of at least two nucleic acids that are contained in a nucleic acid mixture. According to said method, a surface is provided which has islands of nucleic acids, each of the same type, i.e. tertiary nucleic acids; - the tertiary nucleic acids are treated in such a way that the products are only connected to the surface by the 5' end of one strand of a double-strand; - the tertiary nucleic acids are cleaved by restriction endonucleases of the IIS type, in such a way that 3' overhanging ends or 5' overhanging ends are created; - one or more bases of the tertiary nucleic acids are determined; - linker molecules are connected by ligation to the free ends of the tertiary nucleic acid molecules, whereby the linker molecules have one or more recognition sites for restriction endonucleases of the IIS type; - the tertiary nucleic acids are cleaved once again by one or more restriction endonucleases of the IIS type, which recognize the sequence parts that have been introduced into the tertiary nucleic acids by the linker molecules in step (e); and - steps (d), (e) and (f) are repeated until the desired sequence information is obtained.

Description

 New methods for parallel sequencing of a nucleic acid mixture on a surface

The invention relates to a method for solid-phase-supported parallel sequencing of at least two different nucleic acids contained in a nucleic acid mixture.

An important method of biological analysis is the sequence analysis of nucleic acids. The exact base sequence of the DNA or RNA molecules of interest is determined here. Knowing this sequence of bases allows, for example, the identification of certain genes or transcripts, i.e. the messenger RNA molecules belonging to these genes, the detection of mutations or polymorphisms, or the identification of organisms or viruses that can be clearly identified by means of certain nucleic acid molecules , The sequencing of nucleic acids is usually carried out using the chain termination method (Sanger et al. (1977) PNAS 74, 5463-5467). For this purpose, an enzymatic addition of a single strand to the double strand is carried out by extending a "primer" hybridized to said single strand, usually a synthetic oligonucleotide, by adding DNA polymerase and nucleotide building blocks. A small addition of terminating nucleotide building blocks, which after their incorporation into the growing strand no longer allow any further extension, leads to the accumulation of partial strands with a known end determined by the respective terminating nucleotide. The mixture of strands of different lengths thus obtained is separated by size by means of gel electrophoresis. The nucleotide sequence of the unknown strand can be derived from the resulting band patterns. A major disadvantage of the method mentioned is the required expenditure on instruments, which limits the achievable throughput of reactions. For each sequencing reaction, assuming the use of termination nucleotides labeled with four different fluorophores, at least one track on a flat gel or, when using capillary electrophoresis, at least one capillary is required. The resulting effort limits the number of sequencing processes that can be processed in parallel to a maximum of 96 on the currently most modern commercially available sequencing machines. Another disadvantage is the limitation of the reading length ", ie the number of correctly identifiable bases per sequencing, by the resolution of the gel system alternative method for sequencing, the sequence determination via mass spectrometry, is faster and therefore allows the processing of more samples in the same time, but on the other hand is limited to relatively small DNA molecules (for example 40-50 bases)

BESTATIGUNGSKOPIE other sequencing technique. Sequencing by hybridization (SBH. Sequencing By Hybridization; cf. Drmanac et al., Science 260 (1993), 1649-1652), base sequences are identified by the specific hybridization of unknown samples with known oligonucleotides. For this purpose, said known oligonucleotides are fixed in a complex arrangement on a support, hybridization with the labeled nucleic acid to be sequenced is carried out, and the hybridizing oligonucleotides are determined. The sequence of the unknown nucleic acid can then be determined from the information about which oligonucleotides have hybridized with the unknown nucleic acid and from their sequence. A disadvantage of the SBH method is the fact that the optimal hybridization conditions for oligonucleotides cannot be predicted exactly and accordingly a large set of oligonucleotides cannot be designed which on the one hand contain all possible sequence variations with their given length and on the other hand require exactly the same hybridization conditions. Thus unspecific hybridization leads to errors in the sequence determination. In addition, the SBH method cannot be used for repetitive regions of nucleic acids to be sequenced.

In addition to the analysis of the expression strength of known genes, as is possible by dot blot hybridization, Northern hybridization and quantitative PCR, methods are also known which enable the de ovo identification of unknown genes differentially expressed between different biological samples.

Such a strategy for expression analysis consists in the quantification of discrete sequence units. These sequence units can consist of so-called ESTs (Expressed Sequence Tags). Sufficient numbers of clones from cDNA banks. which are derived from samples to be compared with one another, identical sequences can be recognized and counted and the relative frequencies of these sequences obtained in the different samples can be compared with one another (cf. Lee et al. Proc. Natl. Acad. Sei. USA 92: 8303-8307 (1995)). Different relative frequencies of a certain sequence indicate differential expression of the corresponding transcript. However, the method described is very complex since the sequencing of many thousands of clones is already necessary for the quantification of the more common transcripts. On the other hand, a short sequence section of approximately 13-20 base pairs in length is generally required for the unambiguous identification of a transcript. This fact is exploited by the method of "Serial Analysis of Gene Expression" (SAGE) (Velculescu et al, Science 270 (1995), 484-487). Here short sequence sections ("tags") are concatenated, cloned, and the resulting clones are sequenced. In this way, about 20 tags can be determined with a single sequence reaction. Nevertheless, this technique is not yet very efficient because it is already used to quantify the more common transcripts, many conventional sequence reactions have to be carried out and analyzed. Due to the high effort, reliable quantification of rare transcripts using SAGE is very difficult.

Another method for sequencing tags according to US Pat. No. 5,695,934 is to coat small spheres with nucleic acid to be sequenced in such a way that each sphere receives numerous molecules of only one nucleic acid species. The method of "stepwise ligation and cleavage" is then used for sequencing, in which the nucleic acid to be sequenced is degraded base by base from an artificial linker by using a type IIS restriction enzyme and its sequence is determined in the process. So that the sequencing process can be observed and recorded, the balls used are placed in a flat cuvette which is only slightly higher than the ball diameter in order to allow the formation of a single layer. Furthermore, the balls must be in the densest packing in the cuvette, so that there is no change in the ball arrangement during the sequencing process due to the necessary exchange of the reaction solutions or due to vibrations of the device. Although many sequencing reactions can be carried out in a small space in this way, the arrangement in a very narrow cuvette (a few micrometers in height) has considerable disadvantages, since uniform filling of the cuvette is difficult to achieve. Another disadvantage is the high level of equipment required for the method. For example, high pressures must be used so that the necessary reaction solutions can be exchanged efficiently despite the small cuvette size. Another disadvantage is the slight clogging of the cuvette, which is also favored by the necessarily small dimensions of the cuvette.

The known methods for nucleic acid analysis have one or more of the following disadvantages:

They only allow the parallelized execution of individual sequencing reactions to a very limited extent. - You need relatively large amounts of the nucleic acid whose sequence is to be determined.

They are only suitable for determining the sequence of short sequence sections and are expensive in terms of equipment.

The object of the invention is to provide a method which overcomes the disadvantages of the prior art. The object is achieved according to the invention by a method for parallel sequencing of at least two different nucleic acids contained in a nucleic acid mixture, wherein

(a) a surface is provided, comprising islands of nucleic acids of the same type, tertiary nucleic acids;

(b) the tertiary nucleic acids are treated so that the products are only connected to the surface with the 5 'end of a strand of a double strand;

(c) the tertiary nucleic acids are cut by type IIS restriction endonucleases; (d) one or more bases of the tertiary nucleic acids are determined;

(e) Linker molecules are connected by ligation to the free ends of the tertiary nucleic acid molecules, the linker molecules having one or more recognition sites for type IIS restriction endonucleases;

(f) the tertiary nucleic acids are cut again by one or more type IIS restriction endonucleases which recognize sequence segments which are shown in

Step (e) through which linker molecules have been introduced into the tertiary nucleic acids: and

(g) Repeat steps (d), (e) and (f) until the desired sequence information has been obtained.

Steps (b) and (c) or (d) and (e) can be implemented by a single process measure. The sequence of steps (d), (e) and (f) depends on the cleavage characteristics of the restriction endonucleases used and can be changed in this respect (see the text below). The following is a special embodiment of the method according to the invention, in which in step (a)

(a1) a surface is provided on which at least primer molecules of a first primer and a second primer and optionally a nucleic acid mixture comprising the nucleic acid molecules with which both primers can hybridize have been irreversibly immobilized, both primers forming a pair of primers; (a2) nucleic acid molecules of the nucleic acid mixture are hybridized with one or with both primers of the same primer pair; (a3) the irreversibly immobilized primer molecules are extended in a complementary manner to the opposite strand with the formation of secondary nucleic acids; (a4) the surface in one of nucleic acid molecules. that are provided in a liberated form that are not bound to the surface by irreversible immobilization;

(a5) the secondary nucleic acids are amplified to form tertiary nucleic acids.

Tertiary nucleic acids in accordance with step (a) can be provided by starting from a surface to which at least a first primer and a second primer and optionally a nucleic acid mixture comprising the nucleic acid molecules. with which both primers can hybridize, have been irreversibly immobilized. Both primers form a pair of primers, so they can bind to the strand or counter strand of the nucleic acid molecules. If the nucleic acid molecules of the nucleic acid mixture are already bound to the surface, the hybridization in step (a2) can be brought about by merely heating and cooling. Otherwise, the nucleic acid molecules of the nucleic acid mixture must be brought into contact with the surface in step (a2). In this context, reference is also made to WO 00/18957. The following is a special embodiment of the method according to the invention, in which in step (a) a surface is provided on which at least one primer molecule forming at least one primer pair has been irreversibly immobilized.

In this embodiment, for example, the individual work steps to be carried out can also be configured as follows:

• Primer molecules that form at least one pair of primers are irreversibly immobilized on a surface; • Nucleic acid molecules are hybridized with one or with both primers of the same primer pair by bringing the nucleic acid mixture into contact with the surface;

• The irreversibly immobilized primer molecules are extended to complement the opposite strand with the formation of secondary nucleic acids; • The nucleic acid molecules that are not bound to the surface by irreversible immobilization are removed from the surface;

The secondary nucleic acids are amplified to form tertiary nucleic acids:

• The tertiary nucleic acids are treated so that the products are only connected to the surface with the 5 'end of a strand of a double strand;

• The tertiary nucleic acids are cut by type IIS restriction endonucleases so that 3 ^' overhanging ends or 5 ^' overhanging ends are produced; one or more bases of the overhanging ends are determined;

Linker molecules are connected by ligation to the free ends of the tertiary nucleic acid molecules, the linker molecules having one or more recognition sites for type IIS restriction endonucleases;

The tertiary nucleic acids are again cut by one or more type IIS restriction endonucleases which recognize sequence segments which have been introduced into the tertiary nucleic acids by the linker molecules; and the penultimate step and the subsequent steps are repeated until the desired sequence information has been obtained.

The nucleic acid mixture of step (a2) can be, for example, a library, that is to say nucleic acid molecules which have an identical sequence over long distances, but differ greatly in a partial area in the middle of the identical areas. The libraries often consist of optionally linearized plasmids, into which various nucleic acid fragments have been cloned, which are to be sequenced later. Furthermore, the nucleic acid mixture can be restriction fragments, on the sections of which linker molecules of the same sequence have been ligated. As a rule, the linkers which are bound to the 5 'end of the fragments differ from the linkers which are bound to the 3' end of the fragments. In any case, the sequence section of interest in the nucleic acid molecules of the nucleic acid mixture is generally surrounded by two flanking sequence sections which are essentially identical for all nucleic acid molecules, at least one of the two sequence sections preferably having a self-complementary sequence. The sequence section in question has a pronounced tendency in single-strand form to form a so-called hairpin structure.

The primers or the primer molecules in step (a1 to a3) are single-stranded nucleic acid molecules with a length of about 12 to about 60 nucleotide building blocks and more, which are broadly suitable for use in the context of PCR. They are DNA molecules. RNA molecules or their analogs which are intended for hybridization with a nucleic acid which is complementary at least over a partial region and which, as a hybrid with the nucleic acid, represent a substrate for a Doppler strand-specific polymerase. The polymerase is preferably DNA polymerase I, T7 DNA polymerase, the Klenow fragment of DNA polymerase I, polymerases. which are used in PCR or a reverse transcriptase.

The pair of primers in step (a2) represents a set of two primers that bind to regions of a nucleic acid that flank the target sequence of the nucleic acid to be amplified and have a "polarity" with respect to the orientation of their binding to the nucleic acid, that an amplification is possible (the 3 'terms point towards one another). These regions are preferably sequence segments which are associated with the nucleic acid molecules The nucleic acid mixture can be a plasmid library, for example, and the primers would then preferentially bind in the region of the so-called multiple cloning site (MCS), one above and one below the cloning site bind the primers to the sequence segments which correspond to the linkers which, as described above, were ligated to restriction fragments on both sides. The method according to the invention is preferably carried out with only one pair of primers, such as that in US Pat. No. 5,641,658 (WO 96 / 04404) described method, in which only one pair of primers is used, preferably binding according to the invention n the primers of the primer pair or of the primer pairs at sequence regions which are essentially identical for all or almost all nucleic acids of the nucleic acid mixture (so-called conserved regions). The primers of a primer pair can also have the same sequence. This can be advantageous if the conserved areas flank the sequence to be amplified. Have sequences that are complementary to each other.

One of the primers of a primer pair can have a sequence which enables the formation of an intramolecular nucleic acid double hex (a so-called hairpin structure), although a region of the 3 'terminus consisting of at least 13 nucleotide units remains unpaired.

The surface in step (a, al and a2. A4) is the accessible surface of a plastic body. Metal, glass, silicon or similar suitable materials. This is preferably flat, in particular flat. The surface can have a swellable layer, for example made of polysaccharides, poly sugar alcohols or swellable silicates.

Irreversible immobilization means the formation of interactions with the surface described above, which are stable on an hourly scale at 95 ° C. and the usual ionic strength in the PCR amplifications of step (a5). These are preferably covalent bonds, which can also be cleavable. The primer molecules in step (a) are preferably irreversibly immobilized on the surface via the 5 'termini. Alternatively, immobilization can also take place via one or more nucleotide building blocks. which lie between the terms of the primer molecule in question are immobilized, although a sequence segment of at least 13 nucleotide building blocks calculated from the 3 'terminus must remain unbound. The immobilization is preferably carried out by forming covalent bonds. Here, of course, a to ensure that there is sufficient occupancy that enables the primers and nucleic acids involved in the polymerase chain reaction to be contacted. If two primers are immobilized, the primers should have an average distance on the surface which at least corresponds to the maximum length with full extension of the nucleic acid molecules to be amplified or is smaller. The procedure is essentially as described in US Pat. No. 5,641,658 or WO 96/04404.

Methods are known from the prior art for binding chemically suitably derivatized oligonucleotides to specific surfaces. Carbodiimide-mediated binding of 5'-phosphorylated oligonucleotides to activated polystyrene supports is particularly suitable (Rasmussen et al., Anal. Biochem 198 (1991), 138-142). Another known method takes advantage of the high affinity of gold for thiol groups to bind thiol-modified oligonucleotides to gold surfaces (Hegner et al, FEBS Lett 336 (1993), 452-456). Another possibility is to couple derivatized oligonucleotides to glass surfaces. For example, terminal primary amino groups (“aminolink”), which are bonded to the 5 ^′ end of the oligonucleotide via a polyatomic spacer and which can easily be incorporated in the course of oligonucleotide synthesis, are bound to isothiocyanate-modified glass surfaces. For example, Guo et al. (Nucleic Acids Res. 22 (1994). 5456-5465) described a method of activating glass surfaces with aminosilane and phenylene diisothiocyanate and then binding 5'-amino-modified oligonucleotides to them. Preferred here is the use of commercially available glass supports which are activated on the one hand for binding aminolink-modified oligonucleotides and on the other hand have a structured surface which has a greater binding capacity than a flat surface (Surmodics. Eden Prairie, Minnesota, USA).

The term secondary nucleic acid in step (a3) denotes those nucleic acid molecules which are formed by complementary extension of primer molecules, the extension being complementary to the nucleic acid molecules of step (a2) which have been hybridized with the primers.

The surface is made up of one of nucleic acid molecules. which are not bound to the surface by irreversible immobilization are provided in the released form [step (a4)]. If the nucleic acid molecules from step (al) have already been irreversibly immobilized on the surface in step (al), no nucleic acid molecules are generally brought into contact with the surface in step (a2). As a result, they do not need to be removed in the subsequent steps. If, in step (a2), nucleic acid molecules are brought into contact with the surface for the purpose of hybridization with the primers, for example because the nucleic acid molecules are not already on the surface in step (a1) have been irreversibly immobilized, then these can be removed in step (a4) by denaturation and washing. It is possible to remove the aforementioned nucleic acid molecules only after one or more amplification cycles of step (a5) have been completed.

The term tertiary nucleic acids denotes secondary nucleic acids and those nucleic acid molecules which are formed from the secondary nucleic acids by the polymerase chain reaction method in step (a5). It is important here that the surface and the liquid reaction space surrounding the surface are free of nucleic acids to be amplified, which are not irreversibly immobilized on the surface. As a rule, the amplification results in real islands, that is to say discrete regions on the surface which carry tertiary nucleic acids of the same type, that is to say identical or complementary nucleic acid molecules.

In step (b) the tertiary nucleic acids are treated in such a way that the products are only connected to the surface at the 5 ′ end of a strand of a double strand (“half-sided dissolving”). This can be done by the tertiary nucleic acids being cleaved, particularly by restriction with a restriction endonuclease of nucleic acid double strands on one side of the surface, while the other side remains connected to the surface, which means that the double-stranded nucleic acid is only connected to the surface via a nucleic acid strand This is done by incubation with a restriction endonuclease, the recognition site of which has been integrated into a primer of the primer pair used in step (a1) or which is statistically sufficiently high at least at another location within the nucleic acid molecules, which is preferably a restriction ction endonuclease of type IIS, the interface of which lies outside the primer sequence and which produces a 3 ^' overhang, a 5' overhang or a so-called blunt end. The creation of a 5 'overhang is preferred. In this respect, step (b) and step (c) can be implemented by a single restriction cleavage, which is particularly preferred. The restriction cut effecting the “half-sided release” can therefore take place in the area of a primer sequence, in the area adjacent to it or in the interior of the tertiary nucleic acids flanked by the primer sequences. You can first cut in the vicinity of the primer sequence of a primer of a primer pair and the method according to the invention in this way carry out and another experiment with a different surface, but which has the same tertiary nucleic acids, carry out the method according to the invention as well, but with the modification that cutting is now carried out in the vicinity of the primer sequence of the other primer of a primer pair, the counterprimer Such a measure has the advantage that the The process of sequencing described can only be used at one end and then at the other end of a tertiary nucleic acid, which approximately doubles the read range that can be achieved if the flanking primer sequences are arranged differently and with respect to a tertiary nucleic acid in the same orientation. It is of course advantageous to determine overlapping areas and to assign the sequence information to a tertiary nucleic acid in this way.

However, it is also possible to achieve the "half-sided detachment" in another way, for example by chemical cleavage of the bond between one of the primers of the primer pair used in step (a) and the surface. Furthermore, a measure similar to the result can be taken, in which the tertiary nucleic acid molecules are denatured and supplemented to form the double strand by providing counter-strands of tertiary nucleic acids.

Counterstrands of the tertiary nucleic acids (GTN) can be provided, for example, by one of three measures, which are listed below: - On the one hand, in step (a) primer molecules or optionally in

Step (al or a2) nucleic acid molecules (of the nucleic acid mixture) with flanking sequence sections are used, which have self-complementary regions and are therefore capable of intramolecular base pairing, which is expressed in a so-called hairpin structure (see also FIG. 3: ligation of “masked hairpins "in the form of double-stranded linker molecules).

In this case, preferably only one primer of a pair of primers or only one flanking sequence section of two is able to form a shark instruction to ensure that the incorporation of nucleotides takes place only on one of two complementary nucleic acid molecules, so that interference of the sequence signals of both nucleic acid molecules is excluded is.

The tertiary nucleic acids formed in step (a5) then, in the single-stranded state, which is brought about by removing one of the two strands under denaturing conditions, have a refolding in the form of a hairpin near their 3 'terminus. The double-stranded portion of the hairpin preferably extends up to and including the last base of the 3'-

In the end, so that said hairpin can serve directly as a substrate for a polymerase used for sequencing. This can be ensured by a suitable choice of the sequence of the primer molecules or of the sequence sections flanking the nucleic acid molecules.

Secondly, GTNs can be provided in the form of hairpins by ligation of oligonucleotides which are capable of hairpin formation and, if appropriate (but not necessarily), already in the form of hairpins for ligating gation are used (see also Fig. 2). This can be done in such a way that the tertiary nucleic acids are cut in the double-stranded (ie not denatured) state and are thus separated from the surface on one side. This is preferably done by incubation with a restriction endonuclease which has a recognition site in exactly one of the sequences originating from one of the two primers (primer sequences) or in a sequence adjacent to these primer sequences. After the restriction cut has taken place, a free end of the tertiary nucleic acids then protrudes into the solution space, which depending on the restriction endonuclease used has an overhanging end of a predictable sequence and to which the

Oligonucleotide can be hybridized and ligated. An oligonucleotide which has already formed a hairpin structure, that is to say is therefore partially double-stranded, and which has an overhang complementary to the free end of the tertiary nucleic acids would be particularly suitable for this. To ensure. that a ligation takes place exclusively on the irreversibly immobilized strand of the double strand of the tertiary nucleic acids, the 5 'end of the oligonucleotide can carry a phosphate group, while the 3' end of the irreversibly immobilized strand and the 5 'end of the counter strand hybridized with it Have OH group (see Fig. 2, steps 1 and 2). After ligation, the non-irreversibly immobilized strand becomes the tertiary

Nucleic acids removed under denaturing conditions. As an alternative to this, as proposed in US Pat. No. 5,798,210 (see there in particular FIG. 7), an oligonucleotide folded back to the hairpin could be ligated to the single-stranded immobilized strand of the tertiary nucleic acids. The problem with this second measure is that an often observed inadequate efficiency of the ligation step before sequencing can no longer be compensated for by amplification steps as in the first measure. This can result in an insufficient signal strength in the subsequent sequencing.

Third, it is also possible. Oligonucleotides. which are unable to form a hairpin structure to hybridize with the tertiary nucleic acids to form GTN (cf. US Pat. No. 5,798,210, FIG. 8).

The length of the oligonucleotides plays a minor role in the measures described. As a rule, the oligonucleotides will have a length of less than 100 or less than 50 nucleotide building blocks, so that one can also generally speak of nucleic acids (here: polymeric nucleotides which comprise more than three nucleotide building blocks). Single-stranded oligonucleotides longer than 45 Nucleotide building blocks are difficult to handle due to unspecific interactions if they do not have a sequence that enables hairpins. By ability. Training hairpins reduces non-specific interactions through competition. If double-stranded oligonucleotides are used, then the length of the oligonucleotides hardly plays a role (see also FIG. 3).

All of the measures described have the consequence that the tertiary nucleic acids have a double-stranded partial region which enables a strand extension on the opposite strands of the tertiary nucleic acids (GTN) by means of a DNA polymerase or reverse transcriptase.

At the end of such a process step, each of the islands of tertiary nucleic acid molecules provided in step (a) by the polymerase chain reaction process has double strands, half over the 5 'end of one and half over the 5' end of the other Strands (namely the counter strand) are bound to the surface.

These measures described with the provision of gene strands of tertiary nucleic acids have the advantage that the sequencing process described below can only be used at one end and then at the other end of a tertiary nucleic acid, which approximately doubles the read range that can be achieved without having to do so with two different ones Restriction enzyme-treated surfaces which have the same tertiary nucleic acids must be subjected to the process according to the invention.

The cut in step (c) with a restriction endonuclease of type IIS is preferably carried out in such a way that at least part of the sequence to be determined is exposed in the form of a single-stranded overhang. This overhang can be a 3 'or a 5' overhang. The latter is preferred. However, it is also possible first to produce a smooth end in step (c), to dephosphorylate the tertiary nucleic acid, then to carry out a ligation via blunt ends in step (e), to make up step (d), whereby the base sequence of the tertiary nucleic acid in question takes place with displacement of a strand of the tertiary nucleic acid (strand displacement) and in step (g) steps (f). Dephosporilation of the tertiary nucleic acid, repeat steps (e) and (d) in this order.

It is particularly preferred that the part of the nucleic acid molecules to be exposed by the restriction cut is in the immediate vicinity of a part of the nucleic acid molecules which is already known. This already known part can, for example, if the nucleic acid mixture of step (a1 or a2) is a library of restriction fragments, be one of the restriction sites of known sequence used to produce the library. One or more bases of the overhanging ends in step (d) can be determined, for example, by means of one of the following measures: The sequence is determined in I) and II) via a replenishment reaction with the aid of a nucleic acid polymerase.

(I) In one embodiment, a (single-stranded) 5 'overhang at the end of a double strand is used as a template for the 3' extension of the opposite strand by a polymerase. If the length of the single-stranded overhang is only one base, labeled nucleotides or labeled termination nucleotides such as dideoxynucleotides can be used for the primer extension. In any case, it is necessary to determine the identity of the incorporated nucleotides, which is preferably done via a fluorescent labeling group attached to the nucleotide. If the length of the single-stranded overhang is more than one base, an unambiguous sequence determination requires the use of termination nucleotides, the identification of which in turn is done by a group of molecules attached to the nucleotide. The sequencing of the resulting transition preferably takes place by entschützbare Abbruchnukleotide by the following steps ^• Extension of the 3 'end of the 5' generated -Überhangs by one nucleotide, wherein the nucleotide at the 2'-OH position or at the 3'-OH Position bears a protective group that prevents (or greatly slows down) further elongation, - the nucleotide bears a group of molecules that enables the identification of the nucleotide:

• identification of the incorporated nucleotide;

• the removal of the protective group and removal or change of the molecular group used for identification and • if necessary repeating the previous steps until the desired sequence information has been obtained.

In this connection reference should also be made to US-A 5 714 330 and US-A 5 798 210. Canard and Sarfati (Gene 148 (1994) 1-6) describe 3 ^' -esterified nucleotides which contain a fluorophore which can be split off together with the protective group. These nucleotide building blocks can, however, be incorporated into a growing strand by various polymerases with low efficiency and then as termination nucleotides. therefore do not allow any further strand extension. The esters described may be split off enzymatically or alkaline, so that free, more Nukleotidinkorporation permitting 3 ^'-OH groups occur. However, the ester cleavage takes place very slowly (within 2 hours), so that the compounds described for sequencing longer DNA sections

(e.g. more than 20 bases) are unsuitable. As long as the protective group is bound in the 3'-OH or, if appropriate, the 2'-OH position, the quaternary nucleic acid extended by this nucleotide no longer represents a substrate for a nucleic acid polymerase. Only the removal of the protective group makes it possible for the polymerase to extend the nucleic acid further , The protective group generally also carries a group of molecules which enables identification of the incorporated nucleotide and thus the sequencing of the growing nucleic acid strand and which leaves the nucleotide when the protective group is split off. However, the identifying group of molecules can also be bound at another location on the nucleotide, for example at the base. In this case it is necessary to delete the signal of the identifying group of molecules. This can usually be done in two ways. For example, in the case of a fluorophore, the molecular group can be changed by bleaching. In addition, the identifying group of molecules can also be removed, for example by photochemical cleavage of a photolabile bond. Preferably everyone wears the four suitable for installation

Nucleotide building blocks (G, A, T, C) another identifying group of molecules. In this case, the four types of nucleotides can be offered simultaneously. If different or even all nucleotides carry the same identifying group, the identification step is usually to be broken down into four sub-steps in which the nucleotides of one variety (G, A.T. C) are offered separately.

The molecular group is, for example, a fluorophore or a chromophore. The latter could have its absorption maximum in the visible or in the infrared frequency range. The detection is both spatially and time-resolved, so that the islands on the surface can be sequenced by nucleic acids in parallel. Other examples of the aforementioned group of molecules are biotin, digoxygenin (DIG) or dinitophenol (DNP). In this case, the built-in nucleotide is not identified directly, but rather is identified indirectly by treating with fluorescence-labeled streptavidin to detect incorporated nucleotides. Anti-DIG-Ig or Anto-DNP-Ig is treated. Termination nucleotides allow the complete sequencing of almost any length single-stranded 5 ^' overhangs by the process consisting of incorporation. Identification, deprotection (ie release of the protective group and removal of the labeling group) of the termination nucleotides is repeated until the desired one Reading range is reached or until an asynchronicity between the processes associated with sequencing occurs on identical nucleic acid molecules, which prevents the generation of uniform and clearly interpretable signals.

An alternative to deprotection of demolition nucleotides is to replace incorporated dideoxynucleotides by incubating the insert with a polymerase with exonuclease properties (roofreading activity) and regular nucleotide building blocks, so that a smooth strand end is formed by final filling. In the case of a previous link ligation, one of the two linker strands is replaced by the newly synthesized strand via beach displacement.

The procedure can also be as under I), but a 3 'overhang is generated instead of a 5' overhang. This can be done at the 3 'overhang

Linker molecules with an at least partially complementary but shorter overhang are hybridized and optionally ligated so that the double strand formed from the nucleic acid molecule and linker molecule has an internal single-stranded region, a “gap” (see FIGS. 10 and 11) Primer extension then takes the form of an extension of the corresponding linker strand

The resulting transition can be sequenced by deprotectable termination nucleotides (see FIG. 11) and then comprises the following steps

• Extension of the 3 'end of the linker connected to the 5' overhang by one nucleotide, the nucleotide at the 2'-OH position or at the 3'-OH position being one

Bears protective group that prevents (or greatly slows down) further elongation, the nucleotide bears a group of molecules that enables the identification of the nucleotide; ^• identification of the incorporated nucleotide;

• Removal of the protective group and removal or change of the molecular group used for identification and

• if necessary, repeating the previous steps until the desired sequence information has been obtained. In order to generate a double strand with an internal single-stranded region in the manner described, the backward linker strand of a linker with an overhang must be phosphorylated at its 5 ^' end before the ligation. Even in the case of a 3'- Overhang, there is an alternative to incorporating deprotectable termination nucleotides by incorporating labeled dideoxynucleotides, which are then replaced by means of a /? Roo / reα <img polymerase with strand extension and strand displacement of the opposite strand (see under I)). Then cut with a type IIS enzyme to restore an overhang for the next round of sequencing. Subsequently, the tertiary nucleic acids are dephosphorylated, so that when the relevant tertiary nucleic acid with the linker is subsequently ligated via blunt ends, a “nick”, that is to say a breakage of the sugar phosphate backbone within a nucleic acid double strand results, as is shown, for example, in FIG. 11, numbers 4 and 5.

III) Alternatively, sequencing can be done by ligation. The sequence of the overhanging ends in step (d) will be determined by ligation of the tertiary nucleic acid with a linker which in turn has an overhang which is completely or partially complementary to the overhangs of the nucleic acid molecules to be sequenced and which identifies one or more bases of the sequencing overhangs allowed. In this case, steps (d) and (e) are performed together (see Fig. 12). For example, as described in US Pat. No. 5,714,330, ligation by a ligase, which preferably ligates hybrid mutually complementary ends, for example DNA

Ligase from E. coli, to a given overhang, preferably one from a selection of different linker molecules, which differ from each other in their overhang. In this case, it can be concluded from the nature of the ligation products formed that one or more bases of the overhang to be sequenced. Here, the linker molecule connected to the nucleic acid molecule by ligation can be identified, for example, by one or more marker groups. As an alternative to the ligation of a double-stranded linker molecule, a first single-stranded DNA molecule can first be hybridized with the overhang of the nucleic acid molecule to be sequenced and attached by ligation, then in a next step a further second DNA that is wholly or partly complementary to the first DNA molecule -Molecule hybridized to the overhanging part of the first DNA molecule and exposed again to ligation conditions, wherein ligation only takes place in the case of a completely complementary to the opposite strand 3 ^' end of said second DNA molecule. The nature of the ligated second DNA molecule, such as an included labeling group, allows the identification of one or more bases of the overhang to be sequenced. In order to ensure that both strands are linked in the course of the ligation, care must be taken to ensure that the linker strand to be ligated to the nucleic acid molecule with its 5 ^′ end is attached to its side. big end is in phosphory cheerful form. In any case, it is also preferred here that the double-stranded region formed from the first and the second DNA molecule contains a recognition site for a type IIS restriction endonuclease.

The linker molecule in step (e) represents an at least partially double-stranded piece of nucleic acid. The simplest way to produce a linker is by hybridizing two oligonucleotides that are complementary to one another in sections. It is also possible to use a linker that is capable of intramolecular refolding (formation of a hairpin structure). The linkers preferably have an end which is capable of ligation, for example a smooth end, and an end which is not capable of ligation to the end of the fragment provided, which end can be characterized, for example, by a non-palindromic overhang or a multi-base mismatch. It is possible to provide the linker with a phosphate group at its end capable of ligation (see II). In any case, the linker should carry the recognition site for a type IIS restriction endonuclease.

In step (f), the tertiary nucleic acids are cut again with one or more type IIS restriction endonucleases, the recognition sites of which lie in the linker molecules attached in step (e) and which produce 3 'overhanging ends or 5' overhanging ends. It is preferred here that the overhang of the tertiary nucleic acids exposed by step (f) borders directly on the region of the tertiary nucleic acids previously sequenced in step (d).

In step (g), steps (d), (e) and (f) are repeated in any order until the desired sequence information has been obtained. The order of the steps is determined by the cleavage characteristics of the type IIS restriction endonucleases used in steps (c) and (f). Furthermore, the sequence information obtained for each of the islands provided in step (a) in successive cycles, each consisting of steps (d) to (f), is combined to form sequence information related to each island, the sequence information of different islands preferably being determined in parallel becomes. If the area sequenced in a given cycle borders on the area sequenced in the previous cycle, the total reading distance results from the product of the number of cycles and the number of bases determined per cycle.

The method according to the invention can be designed in more detail by the following embodiments.

In one embodiment of the method according to the invention, step (b) and / or step (c) are carried out by cleaving the tertiary nucleic acids with the aid of a restriction sendonuclease that cleaves a 5 'overhang - or in another embodiment - creates a 3' overhang.

In one embodiment of the method according to the invention, primer or nucleic acid molecules with flanking sequence sections are used in step (a1), of which at least one primer of a primer pair has at least one restriction site and one of these restriction sites is used for the above-mentioned restriction cleavage.

If a 5 ′ overhang is generated by the restriction cleavage, step (d) of the method according to the invention can be configured by the following steps:

(dl) Extension of the 3 'end of the 5' overhang generated by one nucleotide, the nucleotide having a protective group at the 2'-OH position or at the 3'-OH position which prevents further extension (or greatly slowed down). the nucleotide carries a group of molecules which enables the identification of the nucleotide;

(d2) identification of the incorporated nucleotide;

(d3) removing the protecting group and removing or changing the molecular group used for identification;

(d4) optionally repeating steps (dl) to (d3).

As a rule, steps (dl) to (d3) are repeated until the overhang is filled. In this case, in the subsequent step (e), a linker is ligated over smooth ends, as is described in FIG. 9. In a further embodiment of the method according to the invention, in which a 3 ′ overhang is generated by the restriction cleavage, the sequence of steps (d) and (e) is interchanged and step (d) is carried out by the following steps:

(dl) Extension of the 3 'end of the linker connected to the 5' overhang by one nucleotide, the nucleotide at the 2'-OH position or at the 3'-OH position carrying a protective group which carries another Extension prevented (or greatly slowed),

- The nucleotide carries a group of molecules that enables the identification of the nucleotide;

(d2) identification of the incorporated nucleotide; (d3) removing the protecting group and removing or changing the molecular group used for identification;

(d4) optionally repeating steps (dl) to (d3).

This embodiment is described in FIG. 10. The repetition of steps (dl) to (d4) results in the displacement of one strand of the double strand of the tertiary nucleic acid (so-called beach displacement). In step (f), one or more restriction cleavages are carried out using a type IIS restriction endonuclease, which recognize sequence sections which were introduced into the tertiary nucleic acids by the linker molecules in step (e). This is followed by step (g). that is, steps (e), (d) and (f) are repeated in this order until the desired sequence information has been obtained.

In a further embodiment of the method according to the invention, the primer molecules are irreversibly immobilized in step (a1) by forming a covalent bond to a surface.

In a further embodiment of the method according to the invention, in step (d2) the base carries the molecular group which enables the nucleotide to be identified.

In a further embodiment of the method according to the invention, the nucleotide at the 3′-OH position bears the protective group in step (dl).

In a further embodiment of the method according to the invention, in step (dl) the nucleotide at the 2'-OH position bears the protective group.

In a further embodiment of the method according to the invention, the protective group in step (dl) has a cleavable silyl ether, ester. Ether. Anhydride or peroxide group or an oxygen-metal group.

In a further embodiment of the method according to the invention, the protective group is removed in step (d3) by a complex-forming ion, preferably by cyanide. Thiocyanate, fluoride or ethylenediaminetetraacetate.

In a further embodiment of the method according to the invention, the protective group is split off photochemically in step (d3). In a further embodiment of the method according to the invention, the protective group has a fluorophore in step (dl) and the nucleotide is identified fluorometrically in step (d2).

In a further embodiment of the method according to the invention, steps (d) and (e) are implemented by the ligation of identifiable linkers to the tertiary nucleic acids. This embodiment is described under III). The base sequence is determined using the specificity of the ligase. The base sequence is inferred from the nature of the ligation products.

To carry out the method according to the invention, a device is further provided with the following components:

A reaction chamber in the form of a flow cell,

At least one light source to excite fluorophores used for marking,

A device for scanning the surface in the XY direction,

At least one photodetector for measuring the emitted fluorescence radiation,

Optionally at least one vessel for reaction solutions and waste,

• if necessary, an electronic computer, which stores the measured fluorescence intensity as a function of the position on the surface and, if necessary, processes the data records thus obtained.

The reaction chamber contains the surface on which the described processes for sequencing and possibly previously for amplification take place. For example, said surface can simultaneously serve as a wall, preferably as the upper or lower wall, of the reaction chamber. The chamber for observing the processes taking place on the surface must be at least partially transparent. In order to be able to produce suitable reaction conditions, the reaction chamber is preferably designed to be temperature-controllable, for example by means of a Peltier element. It is particularly preferred here to provide the reaction chamber with a temperature control by methods known per se which, in terms of tolerance and response behavior and other criteria, meets the high requirements of the solid-phase polymerase chain reaction (see step e) in order to include all process steps of the process according to the invention to be able to carry out only one apparatus.

Furthermore, the reaction chamber has at least one inlet and one outlet through which used or no longer required reaction solution can be exchanged for fresh or another solution. Inlet and outlet are usually through suitable hose or pipe lines with one or more storage vessels or waste containers. ß connected, which serve to hold fresh or used solutions and which in turn can be temperature controlled. Furthermore, the apparatus described preferably has at least one device for conveying liquids, which can be designed, for example, as a peristaltic pump and is used to exchange solutions. Finally, the lumen of the reaction chamber is given a shape which favors the most efficient solution exchange possible.

The light source preferably emits monochromatic light of a wavelength which is as close as possible to the absorption maximum of the fluorophore or fluorophores used for sequencing. Lasers of a suitable wavelength are preferred for this, but other light sources, for example mercury vapor lamps, can also be used.

The device for scanning the surface located in the reaction chamber can be designed as an XY table which carries the reaction chamber and moves perpendicular to the direction of irradiation, so that the entire area of said surface can be scanned. Alternatively, it is possible to keep the reaction chamber stationary and instead to deflect the light beam used for excitation so that it can scan the entire surface. It is also conceivable to design the reaction chamber to be movable in only one direction and to deflect the excitation light beam in a direction perpendicular thereto.

The photodetector should have a sensitivity suitable for detecting the emitted fluorescent radiation, and on the other hand should allow a reliable differentiation when different fluorophores are used at the same time. For example, photomultipliers or avalanche diodes are suitable. which may be preceded by suitable optical filters or beam splitters for masking out undesired radiation.

For example, commercially available Windows, Macintosh or Unix-based systems are suitable as electronic computers. It is preferred that said computer first generates a "horizontal" data set for each individual sequencing step, consisting of the identification of one base per "island" of identical tertiary nucleic acid molecules, which correlates fluorescence intensity and, if appropriate, detected wavelength and or fluorescence lifetime and position , After recording the desired number of horizontal data records, corresponding to the desired reading distance, the generation of a "vertical" data record is further preferred, in that each position of the horizontal data records, sequencing step by sequencing step, the fluorescence intensity measured at this position as well as any detected wavelength and or fluorescence life. duration is assigned. Finally, it is preferred to translate vertical data sets obtained in this way using the sequencing protocol and the fluorophores used for labeling into sequence information of the individual islands.

The invention further relates to the solid-phase-coupled libraries of nucleic acid molecules produced by the process according to the invention.

The invention is described in more detail by the drawing. 1 shows the amplification of individual nucleic acid molecules by means of surface-bound primers to form islands of identical amplified nucleic acid molecules; 2 shows the sequencing of surface-bound amplification products; 3 shows the provision of counter-strands of tertiary nucleic acids by forming a hairpin structure in sequence sections which originate from linkers;

4 shows the parallel sequencing on a surface;

5 shows the assembly of the detection and identification results into connected sequences; 6 shows the provision of primary nucleic acids for use in expression analysis;

7 shows the provision of primary nucleic acids for sequencing genomic clones; FIG. 8 shows the result of the amplification of individual nucleic acid molecules according to FIG. 1; FIG. 9: sequencing of surface-bound amplification products; 10: Another alternative sequencing of surface-bound amplification products;

Fig. 11: Another alternative sequencing of surface-bound

amplification; 12: Another alternative sequencing of surface-bound amplification products; Fig. 13: A device for performing parallel sequencing

1 shows the amplification of individual nucleic acid molecules by means of surface-bound primers to form islands of identical amplified nucleic acid molecules, in particular 1 the irreversible immobilization of oligonucleotides forming a primer pair,

2 the hybridization of the primary nucleic acids with the surface-bound primers,

3 the formation of secondary nucleic acids by extending the length of the primers, 4 the removal of the non-irreversibly bound primary nucleic acid molecules and amplification of the secondary nucleic acids,

5 islands each with identical tertiary nucleic acid molecules.

Figure 2 illustrates the sequencing of surface-bound amplification products. in which

1 the dissolution of the amplification products by restriction duration of the amplification products (underlined: the recognition site for the restriction endonuclease Sphl); 2 dephosphorylation;

3 the ligation of a hairpin oligonucleotide (bold)

4 the removal of the non-irreversibly immobilized strand of nucleic acid;

5 the installation and identification of a first protected nucleotide;

6 removal of protective group and labeling group with restoration of a free 3'OH group;

7 the incorporation and identification of a second protected nucleotide;

8 shows the repetition of steps 5 and 6.

3 shows the provision of a GTN by forming a hairpin structure in sequence sections from which the linkers originate. The nucleic acid to be sequenced (restriction fragment with two different ends, one of which is generated by the restriction endonuclease Nlalϊl) is grayed out. CATG, overhang generated by restriction endonuclease αlll; GCATGC, recognition site for restriction endonuclease Sphl (contains the recognition site for αlll, CATG); NNNNNN NNN and MMMMMMMMMM, "inverted repeats" (mutually complementary sequences that allow intramolecular refolding of a single strand); XXXXX and YYYYY, spacer region to the surface. Described in detail

1 ligation of a linker with "inverted repeat" and Sp interface to the fragment to be sequenced; 2 denaturation and hybridization with primer immobilized on a surface;

3 the amplification with two primers immobilized on the surface (counter primer not shown);

4 the "half-side detachment" of the amplification products from the surface by restriction endonuclease Sphl (arrows):

5 denaturation and removal of the strand not immobilized on the surface;

6 renaturation with the formation of a hairpin. Beginning of the sequencing by incorporation of deprotectable nucleotides. 4 describes parallel sequencing on a surface. In this figure, “islands” of identical nucleic acid molecules are symbolized simply by a single strand. In detail, FIG. 1 shows the attachment of a sequencing primer, installation of the first termination nucleotide and parallel detection and identification of the first nucleotide building block in each case,

2 the removal of the protective group and the marking group of the first nucleotide, incorporation of the second termination nucleotide and parallel detection and identification of the second nucleotide building block in each case;

3 the detection and identification result of the first base;

4 the detection and identification result of the second base.

5 describes the assembly of the detection and identification results into coherent sequences, wherein

1 the detection and identification result of the first base,

2 the detection and identification result of the second base,

3 the detection and identification result of the nth base,

4 denotes the assembled sequences of the nucleic acid molecules in individual islands.

6 shows the provision of primary nucleic acids for use in expression analysis, in particular

1 cDNA synthesis with biotinylated primer, binding of the double-stranded cDNA to streptavidin-coated surface; 2 the restriction cut with the first enzyme (RE1). Washing away the released fragments, and the second restriction cut with the second enzyme (RE2);

3 the ligation of two different linkers;

4 mRNA; 5 double-stranded cDNA immobilized on solid phase;

6 shows a cDNA fragment flanked by two different "overhanging" ends.

7 shows a cDNA fragment flanked by two different linkers (L1, L2). 7 shows the provision of primary nucleic acids for sequencing genomic clones, in detail

1 shows a restriction section of a genomic clone in parallel with two different restriction endonucleases (RE1-4), ligation of different linkers (L 1-4);

2 a genomic clone:

3 denotes two overlapping sets with linkers of ligated fragments. Fragments (crossed out) flanked symmetrically by identical linkers cannot be sequenced.

8 shows the result of the amplification of individual nucleic acid molecules using surface-bound primers to form islands of identical amplified nucleic acid molecules, visualized by staining with SYBR Green I.

9 shows an alternative sequencing of surface-bound amplification products by means of deprotectable termination nucleotides and restriction endonucleases of type IIS, in detail

1 incorporation of a first labeled deprotectable termination nucleotide by strand extension on a 5 'overhang;

2 the identification of the incorporated nucleotide, removal of the protective group and incorporation of a second labeled deprotectable termination nucleotide;

3 the repetition of incorporation and identification of a protectable termination nucleotide and removal of the protective group, up to 5 '-

Overhang is filled to smooth end, then again deprotection;

4 the ligation of a linker with a smooth and a non-smooth end (overhang or mismatch) and a recognition site (shaded) for a type IIS restriction endonuclease;

5 the incubation with said IIS restriction endonuclease;

6 the repetition of step 1;

7 shows the repetition of steps 2 to 5.

FIG. 10 shows a further alternative sequencing of surface-bound amplification products by means of termination nucleotides and restriction endonucleases of type IIS, in detail 1 the ligation of a linker molecule to a 3 ^" overhang which contains a recognition site (shaded) for a type IIS restriction endonuclease;

2 incorporation and identification of a termination nucleotide; 3 the removal of the termination nucleotide by incubation with a polymerase with proofreading activity and all four nucleotides, the replacement of the counter strand to the strand to be sequenced beginning with beach displacement;

4 the complete removal of the original counter strand by strand displacement, restoring an unbranched double strand;

5 the incubation with a type IIS restriction endonuclease, the recognition site of which is present in the linker, followed by the dephosphorylation of the relevant tertiary nucleic acids and by the blunt end lambda ion of a new linker on the tertiary nucleic acids, the re-incorporation and identification of a termination nucleotide under beach displacement;

6 shows the repetition of steps 3 to 6.

11 shows a further alternative sequencing of surface-bound amplification products by means of deprotectable termination nucleotides and restriction endonucleases of type IIS, in detail

1 the ligation of a linker molecule to a 3 "overhang which contains a recognition site (shaded) for a type IIS restriction endonuclease, a single-stranded region flanked by double-stranded regions being formed;

2 the incorporation, identification and deprotection of a first labeled deprotectable termination nucleotide by strand extension on the linker molecule;

3 the repeated repetition of incorporation, identification and deprotection of labeled, deprotectable termination nucleotides;

4 incubation with a type IIS restriction endonuclease. the recognition site of which is present in the linker, followed by the dephosphorylation of the tertiary nucleic acids concerned and by the blunt ligation of a new linker to the tertiary nucleic acids and the reincorporation, identification and deprotection of a labeled deprotectable termination nucleotide;

5 the repeated repetition of incorporation, identification and deprotection of labeled deprotectable termination nucleotides: whereby strand displacement takes place: 6 shows the complete removal of the original counter-strand of the strand to be sequenced by strand displacement, with the restoration of an unbranched double strand;

7 shows the repetition of steps 4 to 6.

FIG. 12 shows a further alternative sequencing of surface-bound amplification products by means of fluorescence-coded linkers with an overhanging end and restriction endonucleases of type IIS, in detail

1 the sequence-specific ligation of a linker molecule to a fragment overhang, whereby a combination of all possible linker overhangs (for example 16 in the case of a double-base overhang) is made available and the sequence of the overhang of each linker species is unambiguous via its labeling group or its combination of labeling groups (* 1, * 2 ) is encoded;

2 the identification of the ligated linker species on the basis of their labeling group or labeling groups;

3 incubation with a type IIS restriction endonuclease, the recognition site of which is present in the linker; 4 shows the repetition of steps 1 to 3.

13 shows an apparatus for carrying out the method according to the invention, wherein

1 a laser; 2 a mirror;

3 a temperature-controlled reaction chamber on an XY table;

4 a semi-transparent mirror;

5 shows a detector.

The invention is explained in more detail below by the examples.

Example 1:

Preparation of nucleic acid molecules

4 μg of total RNA from rat liver was precipitated with ethanol and dissolved in 15.5 μl of water. 0.5 μl 10 μM cDNA primer CP28V (5 '-

ACCTACGTGCAGATTTTTTTTTTTTTTTTTTN-3 ^' ) added. Denatured for 5 minutes at 65 ° C and placed on ice. The mixture was mixed with 3 ul 100 mM dithiothreitol (Life Technologies GmbH, Karlsruhe), 6 ul 5x Superscript buffer (Life Technologies GmbH. Karlsruhe). 1.5 μl 10 mM dΝTPs, 0.6 μl RΝase inhibitor (40 U / μl; Röche Molecular Bio- chemicals) and 1 μl Superscript II (200 U / μl. Life Technologies) and incubated for 1 hour at 42 ° C. for cDNA first strand synthesis. For the second strand synthesis, 48 μl of second strand buffer (cf. Ausubel et al. Current Protocols in Molecular Biology (1999), John Wiley & Sons), 3.6 μl of 10 mM dNTPs, 148.8 μl of H ₂ O, 1.2 μl RNaseH (1.5 U / μl, Premega) and 6 μl DNA Polymerase I (New England Biolabs GmbH Schwalbach, 10 U / μl) were added and the reactions were incubated at 22 ° C. for 2 hours. It was extracted with 100 μl phenol, then with 100 μl chloroform and precipitated with 0.1 vol. Sodium acetate pH 5.2 and 2.5 vol. Ethanol. After centrifugation for 20 minutes at 15,000 g and washing with 70% ethanol, the pellet was dissolved in a restriction mixture consisting of 15 μl 10X universal buffer, 1 μl Mbol and 84 μl H ₂ O and the reaction was incubated at 37 ° C. for 1 hour. It was extracted with phenol, then with chloroform and precipitated with ethanol. The pellet was prepared in a ligation mixture from 0.6 μl lOx ligation buffer (Röche Molecular Biochemicals), 1 μl 10 mM ATP (Röche Molecular Biochemicals), 1 μl linker ML2025 (produced by hybridization of oligonucleotides ML20 (5 ^' -TCACATGCTAAGTCTCGCGA-3') and LM25 (5 -GATCTCGCGAGACTTAGC ATGTGAC-3 ^' ), ARK), 6.9 μl H ₂ O and 0.5 μl T4 DNA ligase (Röche Molecular Biochemicals) dissolved and the ligation carried out overnight at 16 ° C. The ligation reaction was made up to 50 μl with water, extracted with phenol, then with chloroform and, after adding 1 μl glycogen (20 mg / ml, Röche Molecular Biochemicals), with 50 μl 28% polyethylene glycol 8000 (PromegaVlO mM MgCl ₂₎ . The pellet was precipitated was washed with 70% ethanol and taken up in 100 ul water.

Example 2:

Coating with oligonucleotides

Lyophilized oligonucleotides Amino-M13rev (5'-amino-CAGGAAACAGCGATGAC-3 ^' ) and Amino-T7 (5'-amino-TAATACGACTCACTATAGG-3') (ARK Scientific GmbH, Darmstadt) were lyophilized at their 5 'end carrying amino link groups added to a final concentration of 1 mM in 100 mM sodium carbonate buffer pH 9. Microscope slides made of glass ("Slides"; neoLab Migge Laborbedarf-Vertriebs GmbH, Heidelberg) were cleaned in chromosulfuric acid for 1 hour and then washed 4x with distilled water. After air drying, the slides were 5 minutes in a 1% solution of 3-aminopropyltrimethoxysilane ("Fluka": Sigma Aldrich Chemie GmbH. Seelze) treated in 95% acetone / 5% water. It was then washed ten times for 5 minutes each in acetone and heated to 110 ° C. for 1 hour. The slides were then placed in 0.2% 1,4-phenylene diisothiocyanate (“Fluka”: Sigma Aldrich Chemie GmbH, Seelze) in a solution of 10% pyridine in dry dimethylformamide (Merck KGaA, Darmstadt) for 2 hours. After 5 washing steps in methanol and 3 washing steps in acetone, the slides were air-dried and processed directly for coating. Self-adhesive .. Frame Seal "frames for 65 μl reaction chambers (MJ Research Inc., Water- town. Minnesota, USA) were applied, 65 μl of oligonucleotide solution were pipetted into the reaction chambers thus formed, and the chambers were sealed by adhering a polyester cover sheet (MJ Research Inc.) to the exclusion of air bubbles. The exact position of the reaction chamber was marked with a waterproof felt tip pen on the underside of the slides. The binding of the oligonucleotides via aminolink to the surface of the activated slides took place over 4 hours at 37 ° C. The adhesive frame was then removed and the slides rinsed with deionized water. In order to inactivate remaining reactive groups, the slides were for 15 minutes in a blocking solution heated to 50 ° C. (50 mM ethanolamine (“Fluka”: Sigma Aldrich Chemie GmbH, Seelze), 0.1 M Tris pH 9 (“Fluka”: Sigma Aldrich Chemie GmbH. Seelze), 0.1% SDS ("Fluka": Sigma Aldrich Chemie GmbH, Seelze). To remove non-covalently bound oligonucleotides, the slides were placed in 800 ml 0.1X SSC / 0.1% SDS ( See Ausubel et al., Current Protocols in Molecular Biology (1999), John Wiley & Sons) The slides were washed with deionized water and air dried.

Example 3:

Coating with oligonucleotides

Lyophilized, at its 5 '-end bearing Aminolink groups oligonucleotides amino M13rev ^(5' -amino-CAGGAAACAGCGATGAC-3 ') and amino-T7 ^(5' TAATACGACTCACTATAGG-amino-3 ') (ARK Scientific GmbH, Darmstadt) were taken up to a final concentration of 100 pmol / μl in deionized water. 1.4 μl each of these primer solutions were mixed with 32.2 μl water and 35 μl 2x binding buffer (300 mM sodium phosphate pH 8.5). Self-adhesive “frame seal” frames for 65 μl reaction chambers (MJ Research Inc. Watertown, Minnesota, USA) were attached to “SD-Link activated slides” (glass slides activated for binding amino-modified nucleic acids; (Surmodics, Eden. Prairie, Minnesota, USA), 65 μl oligonucleotide solution were pipetted into the reaction chambers thus formed, and the chambers were sealed by adhering a polyester cover sheet (MJ Research Inc. Watertown. Minnesota, USA) to the exclusion of air bubbles The exact position of the reaction chamber was marked with a waterproof felt-tip pen on the underside of the slides. The binding of the oligonucleotides via Aminolink to the surface of the activated slides took place overnight at room temperature. The adhesive frame was then removed and the slides were rinsed with deionized water. In order to inactivate remaining reactive groups, the slides were heated at 50 ° C. for 15 minutes blocked blocking solution (50 mM ethanolamine ("Fluka": Sigma Aldrich Chemie GmbH, Seelze). 0.1 M Tris pH 9 ("Fluka": Sigma Aldrich Chemie GmbH, Seelze). 0.1% SDS ("Fluka": Sigma Aldrich Chemie GmbH. Seelze) treated. To remove non-covalently bound oligonucleotides, the slides were placed in 800 ml 0.1 x SSC / 0.1% SDS for 5 minutes (cf. Ausubel et al., Current Protocols in Molecular Biology (1999), John Wiley & Sons). The slides were washed with deionized water and air dried.

Example 4: Plasmids pRNODCAB (contains bases 982 to 1491 of the transcript of rat ornithine decarboxylase, AC number J04791, cloned into vector pCR II (Invitrogen BV, Groningen, the Netherlands) and pRNHPRT (contains bases 238 to 720 of the transcript Hypo Rat xanthine phosphoribosyl transferase, AC number M63983, cloned in vector pCR II (Invitrogen)) were linearized by adding 1 μg plasmid in a volume of 20 μl lx restriction buffer H (“Röche Molecular Biochemicals”: Röche Diagnostics GmbH, Mannheim) with each 5 U of the restriction enzymes BgHl and Seal (Röche Molecular Biochemicals) were incubated for 1.5 hours at 37 ° C. The vector inserts were then amplified by adding 1 μl of the restriction mixtures in a volume of 100 μl PCR buffer II (Perkin-Elmer, Foster City, California, USA) with 4 μl 10 mM primer T7 (5'-TAATACGACTCACTATAGG-3 ^' ), 4 μl 10 mM primer M13 (5 ^' - CAGGAAACAGCGATGAC-3 ') (ARK), 4 μl 50 mM MgCl ₂ ("Fluka": S igma Aldrich Chemie GmbH, Seelze), 5 μl dimethyl sulfoxide (“Fluka”: Sigma Aldrich Chemie GmbH, Seelze), 1 μl 10 mM dNTPs (Röche Molecular Biochemicals), and 1 μl AmpliTaq DNA Polymerase (5u μl; Perkin-Elmer) was transferred. The reactions were then subjected in a Gene Amp 9700 thermal cycler (Perkin-Elmer) to a temperature program consisting of 20 cycles of denaturation for 20 seconds at 95 ° C, primer annealing for 20 seconds at 55 ° C and primer extension for 2 minutes at 72 ° C. The amplification products were examined electrophoretically on a 1.5% agarose gel for their correct size. To remove unincorporated primers, the reactions were purified using QiaQuick columns (Qiagen AG, Hilden) according to the manufacturer's instructions and eluted in 50 μl deionized water.

Example 5:

Amplification In order to attach the nucleic acids prepared in Example 2 to glass supports, annealing mixtures of 1 .mu.l undiluted or in parallel batches 1:10, 1: 100 or 1: 1000 amplification product solutions diluted with water, 4 .mu.l 50 mM each MgCL solution, 1 μl bovine serum albumin (20 mg / ml: Röche Molecular Biochemicals), 5 μl dimethyl sulfoxide, 1 μl 10 mM dNTPs and 1 μl AmpliTaq each in a total volume of 100 μl lx PCR buffer II. Taking into account the felt-tip pen markings on the underside of the slide, frame-seal chambers were applied to the slides prepared in Example 1 in the positions used for the oligonucleotide coating. Then 65 μl each of the annealing mixtures were pipetted into the reaction chambers and the chambers were sealed as above. The slides were placed on the heating block nes UNO Il-in-situ thermal cycler (Biometra biomedizinische Analytik GmbH, Göttingen), covered with a cushion of paper towels and pressed against the heating block using the height-adjustable heated lid. The following temperature program was used for the annealing and the subsequent primer extension: denaturation for 30 seconds at 94 ° C, annealing for 10 minutes at 55 ° C, primer extension for 1 minute at 72 ° C. After the reaction, the reaction chambers were removed and the slides were rinsed with deionized water. To remove the non-covalently bound strands, the mixture was boiled in 800 ml of 0.1 × SSC / 0.1% SDS for 1 minute, the slides were rinsed with water and air-dried. In order to carry out a compartmentalized amplification of the nucleic acid molecules bound to the support, reaction chambers were again applied at the previously selected positions and 65 μl of an amplification mixture were applied, composed as follows: 4 μl 50 mM MgCl ₂ , 1 μl bovine serum albumin (20 mg / ml) , 5 ul dimethyl sulfoxide. 1 μl AmpliTaq (5 U / μl), 1 μl 10 mM dNTPs, in 100 μl lx PCR buffer II. After sealing the chambers, the following temperature program was applied to the in situ thermocycler: denaturation at 93 ° C for 20 seconds, Primerannealing 20 seconds at 55 ° C, extension 1 minute at 72 ° C, for 50 cycles. After amplification was complete, the chambers were removed and the slides rinsed with water and air dried. To detect the clonal islands created by compartmentalized amplification, 40 μl of SYBR Green I solution (Molecular Probes: solution 1: 10,000 in water) were pipetted onto the slides and covered with cover glasses # 2 (MJ). Detection was carried out on a DMRBE confocal microscope (Leica Microsystems Heidelberg GmbH, Heidelberg) with an excitation wavelength of 488 nm and a detection wavelength of 530 nm. Clonal islands of compartmentally amplified nucleic acid molecules could be detected, which are arranged in a random arrangement over the slide surface in the loading area. distributed across the reaction chambers (see Fig. 8). In contrast, in the area of reaction chambers in which no oligonucleotides had been bound to the support as a negative control or in which the amplification reaction had been carried out without prior hybridization of template molecules, no signals originating from clonal islands were detected. Furthermore, the comparison of the slide surfaces in the area of reaction chambers in which different concentrations of matrices were used showed an approximately linear dependence of the number of clonal islands formed on the amount of molecules used.

Example 6: To identify the nucleic acid molecules in the detected clonal islands, the slides were decolorized in water for 10 minutes after detection of the double-stranded DNA stained with SYBR Green. Reaction chambers were then stuck on again at the same positions as before and a restriction mixture consisting of 12 μl lOx universal buffer (Stratagene GmbH, Heidelberg), 1 μl bovine serum albumin, 3 μl restriction tion endonuclease Mbol (1 U / μl; Stratagene) and 64 μl water, pipetted in. To restrict the nucleic acid molecules by means of the internal bol interface, incubation was carried out at 37 ° C. for 1.5 h, then the reaction chambers were removed and the slides were washed with water. The strand fragments which were not covalently bound to the glass support were removed by denaturation for 2 minutes in 800 ml of boiling 0.1 × SSC / 0.1% SDS. After washing again in water and air drying, new reaction chambers were applied. For each hybridization experiment, a hybridization solution consisting of 8 μl 10 x PCR buffer II, 3.2 μl 50 mM MgCl ₂ , 2 μl 100 pmol / μl oligonucleotide probe Cy5-HPRT (5'-Cy5-TCTACAGTCATAGGAATGGACCTATCACTA-3 ^' ; ARK), 2 μl 100 was used pmol / ul oligonucleotide probe Cy3-ODC (5 ^' -Cy3-ACATGTTGGTCCCCAGATGCTGGA-TGAGTA-3') and 65 ul water. For each hybridization experiment, 65 μl of this was added to the respective reaction chamber and hybridized at 50 ° C. for 3 hours. After the hybridization had ended, the reaction chambers were removed and the slides were washed for 5 minutes at room temperature in 30 ml of 0.1 × SSC / 0.1% SDS. The slides were rinsed briefly with distilled water, air dried and used for detection. The detection was carried out as above using a confocal laser microscope. 568 nm and 647 nm were used as excitation wavelengths; signals at 600 nm and at 665 nm were detected. It could be shown that the clonal islands previously detected with SYBR Green were partly caused by the Cy3-ODC probe and partly by the Cy5 probe -HPRT were detected.

Example 7:

Expression Analysis by Highly Parallel Sequencing of Nucleic Acid Molecules The ligation products obtained in Example 1 were diluted 1: 1000 with water and 1 μl of this dilution was amplified in a compartment as described in Example 5 for 50 cycles. Glass slides coated with the amplification primers amino-CP28V (5 ^' - amino-ACCTACGTGCAGATTTTTTTTTTTTTTTV-3') and amino-ML20 (5'-amino-TCACATGCTAAGTCTCGCGA-3 ^' ) were used for this as described. In order to detach the amplification products from the support on one side, the amplification mixture was replaced by a restriction mixture consisting of 12 μl 10 × Universal buffer (Stratagene), 1 μl bovine serum albumin, 4 μl restriction endonuclease Mbol. in a final volume of 65 μl. After incubation at 37 ° C. for 2 h, the restriction mixture was replaced by a dephosphorylation mixture of 1 U alkaline phosphatase from arctic crabs (Amersham) in 65 μl of the reaction buffer supplied. After incubation for 1 hour at 37 ° C. and inactivation for 15 minutes at 65 ° C., reaction chambers and the dephosphorylation mixture were removed, the slides were washed thoroughly with distilled water, reaction chambers were applied again and filled with 65 μl of a ligation mixture consisting of 3 U T4 DNA ligase (Röche Diagnostics) and 500 ng am 5 "end of phosphorylated Haiφin sequencer SLP33 (5 ^' -TCTTCGAATGCACTG-AGCGCATTCGAAGAGATC-3') in 65 μl of the supplied ligation buffer. It was ligated for 14 hours at 16 ° C., then the ligation mixture and reaction chambers were removed. To remove the non-covalently strand fragments bound to the glass slide were treated for 2 minutes in 800 ml boiling 0.1x SSC / 0.1% SDS and washed with distilled water.To prepare suitable deprotectable nucleotides, dATP, dCTP, dGTP and dTTP (Röche Molecular Biochemicals) were used on their 3rd '-OH group esterified with 4-aminobutyric acid These derivatives were labeled with the fluorescent groups FAM (dATP, dCTP) and ROX (dGTP, dTTP) (Molecular Probes Inc., Eugene, Oregon, USA) for the parallel determination of the first base Reaction chambers were again applied to the slides and a primer extension mixture of 1 mM FAM-dATP, 1 mM ROX-dGTP and 2 U Sequenase (United States Biochemical Coφ., Cleveland, Ohi o, USA) in 65μl reaction buffer (40 mM Tris-HCl pH 7.5, 20 mM MgCl ₂ , and 25 mM NaCl). After incubation at 37 ° C. for 5 minutes, the mixture was washed with reaction buffer and detected on the laser scanning microscope. Excitation wavelengths were 488 nm and 568 nm. Detection was carried out at 530 nm and at 600 nm. After the detection, primer extension mixture was again added, which now contained the other two labeled nucleotides, FAM-dCTP and ROX-dTTP. After incooration was carried out, washing and detection were carried out again. and the protecting groups were removed by enzymatic cleavage. For this purpose, treatment was carried out with 5 mg / ml Chirazyme L lipase (Röche Diagnostics) in 100 mM potassium phosphate buffer pH 9 for 1 h at 35 ° C. The sequencing was then carried out for 15 further cycles as described above.

Example 8:

Coating polyacrylamide supports with oligonucleotides

Microscope slides (Merck Eurolab GmbH, Darmstadt) were cleaned with ethanol and treated with binding silane (Amersham Pharmacia Biotech GmbH. Freiburg; 50 μl silane and 50 μl acetic acid in 10 ml ethanol). After removing excess silane by means of ethanol and wipes, an acrylamide polymerization mixture was prepared, consisting of 10 μl 50% “long range” acrylamide solution (Biozym Diagnostik GmbH, Hessisch Oldendorf), each 20 μl 200 μM acrydite-modified primer Acryl-Tι ₅ - T7 (5 ^' -Acrydite-TTT TTT TTT TTT TTT AAT ACG ACT CAC TAT AGG-3'; Eurogen- tec, Seraing / Belgium) and Acryl-TA, ₅ -M13 (5 '-Acrydite- TTA TTA TTA TTA TTA CAG GAA ACA GCG ATG AC-3 '; Eurogentec). 0.5 μl 10% ammonium persulfate (Fluka) and 0.5 μl 10% TEMED (Fluka). 5 μl each of this mixture was placed on a slide. ben and covered with a cover slip (Merck) for polymerization. After the polymerization, the coverslips were removed and frame seal reaction chambers applied.

Example 9:

Coating polystyrene supports with oligonucleotides

A primer binding solution was prepared, consisting of 19 mg l-ethyl-3- (3-dimethylaminopropyl) carbodiimide (Sigma-Aldrich Chemie GmbH, Steinheim), 100 μl 1 M 1 -methylimidazole (Sigma-Aldrich), each 20 μl 100 μM amino-modified primer Amino-Tι ₅ -T7 (5'-Amino-TTT TTT TTT TTT TTT AAT ACG ACT CAC TAT AGG-3 '; ARK) and Amino-TAι ₅ -M13 (5 ^' -Amino- TTA TTA TTA TTA TTA CAG GAA ACA GCG ATG AC-3 ': ARK). 100 μl each of this solution were placed in NucleoLink tubes (Nunc GmbH & CO.KG, Wiesbaden) and incubated at 50 ° C. overnight. The primer binding solution was then removed and the NucleoLink tubes were washed according to the manufacturer's instructions.

Example 10:

Expression analysis by highly parallel sequencing of nucleic acid molecules

Nucleic acid molecules were prepared analogously to Example 1, except that BL2123 (produced by hybridization of the oligonucleotides BL23 [5 '- GCTCAGATCGCAGCTTAGCGAT-3'] and LB 21 [5'-

ATCGCTAAGCTGCGATCTGA-3 '] in ligase buffer) was used instead of ML2025. After coating slides with amplification primers Acryl-TAι ₅ -CP28V (5 ^' - Acrydite-TTA TTA TTA TTA TTA ACC TAC GTG CAG ATT TTT TTT TTT TTT TV- 3'; Eurogentec) and Acryl-T, ₅ -BL23 (5 ' -Acrydite-TTT TTT TTT TTT TTT GCT CAG ATC GCA GCT TAG CGA T-3 ') as described in Example 8 as in Example 5 after hybridization of the nucleic acid molecules to the surface-bound oligonucleotides, primer extension and removal of the non-surface-bound strands for 50 cycles amplified. The amplification products thus obtained were incubated with a restriction mixture consisting of 10 U restriction endonuclease Bbvl (New England Biolabs) and 1 μl bovine serum albumin in 65 μl lx NEBuffer 2 (New England Biolabs) for 1 h at 37 ° C. to detach the amplification products from the support. After removing the Frame Seal reaction chambers, the slides were distilled Washed water and provided with new glass reaction chambers, which had a lumen of 150 μl and had two closable openings for exchanging solutions. As described in Example 7, the first base of the 5 'overhang produced was determined by inco-fluorescence-labeled, deprotectable termination nucleotides and the protective group was then removed. This was repeated three more times so that the overhang was completely filled to a blunt end bearing a free 3 'OH group. Then, after thoroughly rinsing the reaction chamber with distilled water, a ligation mixture consisting of 10 U T4 DNA ligase (Röche) and 3 μg of the Rbvl linker BL2123P was applied in 150 μl to 1 mM hexamine cobalt (III) chloride (Fluka). 0.3 mM ATP and 0.5 mM spermidine trihydrochloride (Sigma-Aldrich) supplemented ligase buffer. The Rbvl linker BL2123P was previously prepared by hybridizing the oligonucleotides BL23 (5'-GCTCAGATCGCAGCTTAGCGAT-3 '; ARK) and LB21P (5'-ATCGCTAAGCTGCGATCTGA-3'; 5 ^' -phosphorylated; ARK) in ligase buffer. After ligation for 8 hours at 16 ° C., Bbvl was cut again as described above, the chambers were rinsed thoroughly and the overhangs were sequenced. This process of sequencing, linker ligation and renewed generation of an overhang shifted towards the inside of the fragment via Rbvl restriction was repeated three more times, so that a reading range of 20 bases in total was achieved.

Claims

claims

1. A method for parallel sequencing of at least two different nucleic acids contained in a nucleic acid mixture, wherein

(a) a surface is provided, comprising islands of nucleic acids of the same type, tertiary nucleic acids;

(b) the tertiary nucleic acids are treated such that the products are only connected to the surface at the 5 'end of a strand of a double strand;

(c) the tertiary nucleic acids are cut by type IIS restriction endonucleases;

(d) one or more bases of the tertiary nucleic acids are determined;

(e) Linker molecules are connected by ligation to the free ends of the tertiary nucleic acid molecules, the linker molecules being one or more

Have recognition sites for type IIS restriction endonucleases;

(f) the tertiary nucleic acids are cut again by one or more restriction endonucleases of type IIS, which recognize sequence sections which were introduced into the tertiary nucleic acids by the linker molecules in step (e); and

(g) Repeat steps (d), (e) and (f) until the desired sequence information has been obtained.

2. The method according to claim 1, wherein in step (a) (a1) a surface is provided on which at least primer molecules of a first primer and a second primer and optionally a nucleic acid mixture comprising the nucleic acid molecules with which both primers can hybridize have been irreversibly immobilized are, where both primers form a Primeφaar; (a2) nucleic acid molecules of the nucleic acid mixture are hybridized with one or with both primers of the same prime pair; (a3) the irreversibly immobilized primer molecules are extended to complement the opposite strand to form secondary nucleic acids; (a4) the surface is provided in a form freed from nucleic acid molecules which are not bound to the surface by irreversible immobilization; (a5) the secondary nucleic acids are amplified to form tertiary nucleic acids:

3. The method according to Anspmch 2. wherein in step (a) a surface is provided on which at least one prime molecule forming primer molecules has been irreversibly immobilized.

4. The method according to Anspmch 1 to 3, characterized in that step (b) and / or step (c) are carried out by cleavage of the tertiary nucleic acids with the aid of a restriction endonuclease.

5. The method according to Anspmch 4. characterized in that the cleavage produces a 5 'overhang.

6. The method according to Anspmch 4, characterized in that the cleavage produces a 3 'overhang.

7. The method according to any one of claims 4 to 6, characterized in that in step (al) primers or nucleic acid molecules are used with flanking sequence sections, of which at least one primer of a prime pair has at least one restriction interface and one of these restriction interfaces for the restriction cleavage according to Anspmch 4th is used.

8. The method according to Anspmch 5, characterized in that step (d) comprises the following steps:

(dl) Extension of the 3 'end of the generated 5' overhang by one nucleotide, where the nucleotide at the 2'-OH position or at the 3'-OH position is one

Protective group, which prevents (or greatly slows down) further elongation, the nucleotide carries a group of molecules which enables the identification of the nucleotide; (d2) identification of the incorporated nucleotide;

(d3) removing the protective group and removing or changing the molecular group used for identification and (d4) if necessary repeating steps (dl) to (d3).

9. The method according to Anspmch 1 to 8, characterized in that the order of steps (d) and (e) is interchanged.

10. The method according to Anspmch 6, characterized in that the order of steps (d) and (e) is interchanged and step (d) comprises the following steps: (dl) Extension of the 3 'end of the linker connected to the 5' overhang by one nucleotide. wherein the nucleotide at the 2'-OH position or at the 3'-OH position carries a protective group which prevents (or greatly slows down) further elongation, - the nucleotide carries a group of molecules which enables the nucleotide to be identified; (d2) identification of the incorporated nucleotide and

(d3) removing the protective group and removing or changing the group of molecules used for identification; (d4) optionally repeating steps (dl) to (d3).

11. The method according to any one of claims 2 to 10, characterized in that in step (al) the primer molecules are irreversibly immobilized by forming a covalent bond to a surface.

12. The method according to any one of claims 8 to 10, characterized in that in step (d2) the base carries the molecular group which enables the identification of the nucleotide.

13. The method according to any one of claims 8 to 10, characterized in that in step (d2) the protective group carries the molecular group which enables the identification of the nucleotide.

14. The method according to any one of claims 8 to 10, characterized in that in step (dl) the nucleotide at the 3'-OH position carries the protective group.

15. The method according to any one of claims 8 to 10, characterized in that in step (dl) the nucleotide at the 2'-OH position carries the protective group.

16. The method according to any one of claims 8 to 10, characterized in that in step (dl) the protective group has a cleavable silyl ether, ester, ether, anhydride or peroxide group or an oxygen-metal group.

17. The method according to Anspmch 16, characterized in that in step (d3) the removal of the protective group by a complex-forming ion. preferably by cyanide.

Thiocyanate. Fluoride or ethylenediaminetetraacetate takes place.

18. The method according to any one of claims 8 to 10, characterized in that the protective group is removed photochemically in step (d3).

19. The method according to any one of claims 8 to 10, characterized in that in step (dl) the protective group has a fluorophore and in step (d2) the nucleotide is identified fluorometrically.

20. The method according to any one of claims 1 to 6, characterized in that steps (d) and (e) are carried out by the ligation of identifiable linkers to the tertiary nucleic acids.