DE19812103A1 - Iteratively synthesizing nucleic acid from oligonucleotides which contain cleavable sites, useful for preparation of genes, ribozymes etc. - Google Patents

Abstract

Synthesis of a nucleic acid (NA) comprises: (1) preparing a first NA (1) with at least one end that permits annealing and/or linkage of another NA (2); (2) annealing/linking (2) to the end of (1), with the other end of (2) masked; (3) masking the end of NA where (2) has annealed; (4) cleaving (2) at a predetermined site to remove the mask and to generate an end that can react (as above) with further NA; and (5) (v) repeating the cycle (ii)-(iv) at least once, using any suitable NA in step (ii). An Independent claim is also included for a kit comprising at least a ligase. a polymerase, a type II S-restriction enzyme and/or uracil-DNA-glycosylase, a pyrimidase and/or endonuclease III and a formamidinopyrimidine DNA glycosylase and/or a mismatch repair enzyme. The kit may also comprise: (1) a phosphatase, (2) a terminal transferase, and/or exonuclease; and (3) a synthetic matrix, optionally with starter NA attached to it.

Description

The invention relates to a process for the synthesis of Nucleic acid molecules. In particular, the invention relates such a procedure that is performed recursively. The nucleic acid components are preferably synthetic or semi-synthetic origin. The principle of invent The inventive method is based on the fact that on or with a provided nucleic acid molecule another nuclein acid molecule is attached and / or linked, the end masking the nucleic acid molecule provided, if there is no further nucleic acid on or with this added and / or linked, the further Nucleic acid molecule cleaved at a predetermined location is, whereby preferably again an end arises to which or with which another nucleic acid molecule is attached and / or can be linked, and the aforementioned procedure Repeat steps as often as necessary until the desired product is synthesized. The invention be  also meets a kit for performing the fiction procedure.

Recombinant techniques for manipulating nucleic acids have had many scientific in the past twenty years disciplines, but also the pharmaceutical industry and an enormous boost for medical research awarded. It is desirable in many areas of application worth, a nucleic acid molecule with a precisely defined sequence in the simplest possible way with only little time and provide cost. The currently most far most common methods of providing such Nucleic acid molecules involve the cloning of DNA for example from cDNA libraries, optionally coupled with subsequent sequencing of the isolated cDNA. Other On the other hand, DNA with the desired sequence can be synthesized for example via the conventional phosphoamidite process, getting produced.

Usual procedures for providing the desired double stranded nucleic acid molecules are subsequently on the case game of the provision of DNA molecules explained. Inte DNA molecules that need to be treated, for example by a cDNA or position cloning, isolated and in appropriate Vectors are cloned. The multiplication of the resulting Vectors and thus the DNA molecules of interest takes place in vivo. To do this, the vectors must be placed in suitable host cells, for example bacteria or yeast. For further manipulation of the DNA, for example for the Be provision of modified constructs, the new phenotypes mediate properties, the DNA must again from the Host organisms are isolated. Only then does it stand again available for manipulation purposes. For further propagation tion must in turn be introduced into suitable host organisms be brought. So there are often many procedural steps and / or laborious manipulations necessary to a ge wanted to generate DNA. It is also easy to imagine and  well known to those skilled in the art that this effort is still ver multiple, provided a large number of different types DNAs are to be produced.

Another method known in the art for the in vitro synthesis of double-stranded DNA is the PCR Technology. A prerequisite for such a production is the availability of suitable template DNA. The subcloning suitable DNA fragments and the sometimes lengthy Setting the correct reaction conditions for the PCR can significantly delay the experimental work.

Those described above, known in the prior art Procedures are still relatively time-consuming and therefore also cost-effective very expensive. They are also, as in the case of the cDNA clony tion, not always successful. The syntheti generation of longer nucleic acid fragments Often there are significant difficulties in practice. Also the Generation of DNA by PCR, although it is the recombinant DNA tion technology has advanced widely in individual cases not be successful or difficult encounter as described above.

The object of the present invention was therefore a method to provide the synthesis of nucleic acid molecules desired sequence and length on simple and time-saving Way. This task is carried out by the in the An solved marked embodiments.

The invention thus relates to a method for the synthesis of nucleic acid molecules, which comprises the following steps:

• a) Provision of a nucleic acid molecule, the min at least one end that has an attachment and / or Linking of or with a further nucleic acid mo reading allowed;  
• b) attachment and / or linking of at least one wide one Ren nucleic acid molecule to or with the nucleic acid remolecule, wherein one end of the at least one white tere nucleic acid molecule to or with the mind attached at least one end of the nucleic acid molecule and / or is linked and the other end of the minde at least one further nucleic acid molecule in the case of a Link is masked;
• c) masking the at least one end of the nucleic acid molecule to which or with which no further nucleic acid remolecule was attached and / or linked;
• d) cleavage of the at least one other attached and / or linked nucleic acid molecule on one specific location, removing the mask, and an end is generated which is an attachment and / or Linking of or with a further nucleic acid mo reading allowed; and
• e) at least one, possibly repeated, repetition of steps (b) to (d), wherein in step (b) in each case suitable nucleic acid molecules are used.

In a preferred embodiment of the invention The other nucleic acid molecule is a nuclein acid single strand molecule.

In a particularly preferred embodiment, the method according to the invention comprises the following step after step (b):

• a) Filling the second to the single strand in its Se complementary nucleic acid strand through a Po lymerase activity, the mas is removed.

In a further particularly preferred embodiment, the method according to the invention comprises the following step after step (d) or (e):

• 1. (d / ea) filling the second, to the single strand in its Sequence of complementary nucleic acid strands through a Polymerase activity.

As mentioned above, the method according to the invention is suitable for the synthesis of single-stranded (ssDNA), double-stranded (dsDNA) or partially double-stranded DNA. The principle of the method according to the invention is shown in FIG. 1. Further embodiments are shown in FIGS. 2 to 7.

In one embodiment of the method according to the invention becomes a single-stranded nucleic acid molecule, a partial double-stranded nucleic acid molecule with an overhanging 5 'or 3' end or a double-stranded nucleic acid mole provided with a smooth end. At this end of the nucleic acid molecule provided is next Step with the aid of a ligase activity, for example egg ner T4RNA ligase, a single-stranded nucleic acid molecule covalently bound. The single-stranded nucleic acid molecule can end with its 5'-phosphate or 3'-hydroxy the nucleic acid molecule provided is bound. Accordingly, the method according to the invention comprises a Nucleic acid synthesis in the 3'-5 'or in the 5'-3' direction. In In these embodiments, it is essential that the end of the single-stranded nucleic acid molecule that is not with the be linked nucleic acid molecule is masked is. For the purposes of the present invention, masked means that this end in this ligation approach does not end with a other single-stranded nucleic acid molecule of the same Kind can be linked, and thereby single-stranded moles cooler arise from multiple copies of the same Nucleic acid molecule exist and also with the ready provided nucleic acid molecule can be linked. in the For the purposes of the invention, masking is a chemical cymatic or other modification of the end that the  o. g. Link prevented. Masking in the sense of this Invention are described in more detail below. After The ligation becomes the ends of the nuclein provided masked acid molecules that with no single-stranded Nuc linseed acid molecule have been linked. In the next step becomes the single-stranded nucleic acid molecule attached to the be provided nucleic acid molecule was ligated to one predetermined location split, the mask ent is removed, and an end is created that is a link with a next single-stranded nucleic acid molecule leaves. This ensures through the last two steps The inventive method in an advantageous manner that in further ligation steps only the nucleic acid molecules to be extended further to those in the previous step a single-stranded nucleic acid molecule was ligated. Of the Ligation, masking and cleavage step can be done in this Order with each new molecule to be added can be repeated as often as required, with a suitable one cell-stranded nucleic acid molecules are used.

In another preferred embodiment of the Invention according to the method is after the synthesis of the complete ge desired single strand of the complementary in its sequence Counter strand synthesized with a polymerase activity. The single strand was synthesized in the 3'-5 'direction and was a double-stranded nucleic acid molecule with a smooth end or a partially double-stranded nucleic acid Provided remolecule, the complementary nucleic acid restricted directly from the free 3 'end of the provided Nucleic acid molecule can be synthesized. However, was a provided single-stranded nucleic acid molecule and he followed the synthesis of the single strand in the 3'-5 'direction, must via hybridization of a suitable single-stranded Nucleic acid oligomers to the provided nucleic acid mo a 3 'end is available before the polymerase reaction be put. The nucleic acid was synthesized cell strand in 5'-3 'direction, the last single strand  nige nucleic acid molecule, which on the synthesized Single-stranded nucleic acid is advantageously ligated chosen that the 3 'end forms a hairpin structure, so that a 3 'end for the synthesis of the complementary Nucleic acid strand by polymerase activity for ver is provided.

In a further preferred embodiment of the invented The process according to the invention is carried out immediately after (each) cleavage of the ligated single-stranded nucleic acid molecule before Ligation of the next single-stranded nucleic acid molecule the corresponding complementary nucleic acid strand using synthesized a polymerase activity. Essentially otherwise the procedure is as described above for Syn thesis of the complete complementary nucleic acid strand be wrote. In the case of a 5'-3 'synthesis direction each single-stranded nucleic acid molecule is selected so that it is on 3'-end advantageously forms a hairpin structure. In addition, before the polymerase reaction that occurs before the Mas Labeling of the ends of the nucleic acid molecules provided masking at the 3 'end of the ligated nucleic acid remolecule removed.

In a further embodiment of the Ver driving are the other nucleic acid molecules attached to the provided nucleic acid molecule attached and / or there be linked with, double-stranded. In this execution form is the nucleic acid molecule provided single stranded or partially double-stranded with an overhang the 3 'or 5' end. Has the other nucleic acid moles cool a corresponding one in its sequence complementary overhanging 3 'or 5' end, attaches through Hybridization of the single-stranded overhanging ends instead of. Preferably the other end is further Nucleic acid molecule smooth. Described by the above This masking ensures that there is none at this end  Attachment by hybridizing cohesive ends of others Nucleic acid molecules of the same kind take place.

In yet another embodiment of the fiction According to the method, the further nucleic acid molecule is single stringy and it attaches to the ready put nucleic acid molecule on hybridization of comple mental terminal nucleotides instead. In this version tion form is the nucleic acid molecule provided single-stranded or partially double-stranded with an overhang 3 'or 5' end. If necessary, the individual stranded further nucleic acid molecule additionally covalent with the nucleic acid molecule provided by means of a Ligase activity can be linked. Find the hybridization over 3'-terminal nucleotides can take place in the next Step by means of a polymerase activity of the complementary Strand to be synthesized. Finds hybridization via 5'-terminal nucleotides instead, the 3 'end becomes further single stranded Nucleic acid molecule chosen so that it is a hairpin structure train forms, so that a 3 'end for the subsequent Po Polymerization reaction for the synthesis of the complementary Nucleic acid strand is provided. In the next step the synthesized nucleic acid duplex on a predetermined site split, being the one for the split necessary recognition sequence and the smooth end or the Hairpin structure is removed and a preferably cohesive ves end arises that is an attachment via hybridization and optionally a covalent linkage of the nuclein acid molecule with another single-stranded nuclein acid molecule allowed.

The present invention also encompasses methods thereof Addition, masking and / or splitting steps combi nations of the corresponding steps of the aforementioned Aus represent management forms. For example, in one first synthesis cycle a single-stranded nucleic acid mole  cool covalently with a provided nucleic acid molecule linked, then the complementary nucleic acid be synthesized tightly, the double strand as before described cleaved, and in the next synthesis cycle another single-stranded nucleic acid molecule can be added via hybridization.

Are single-stranded nucleic acid molecules using a Ligase activity with a provided nucleic acid mole kül linked, the synthesis of the complementary nuclein acid strands after each addition, masking and / or cleavage step or at the end of the synthesis of the com complete nucleic acid single strand. Point of time filling the complementary strand can be any ge be chosen in the sense that, for example, after a any permuting attachment, masking and / or cleavage steps is selected.

The term "masking" means in the sense of the present Invention that a covalent linkage of two nucleic acid remolecules is not possible by means of a ligase activity. Masked single-stranded 3 'ends can, for example by incorporating an amino block, a dideoxynucleo tids, a 3-phosphate or by an artificially installed 5 'end are generated. Masked single-stranded 5 'ends stand out in the sense of the present invention for example by a missing phosphate group or by the Ein construction of a 5'-modified nucleotide (e.g. biotin-dNTP, Digoxygenin-dNTP). Finds an extension of a be provided nucleic acid molecule via hybridization com complementary terminal nucleotides instead, a dop pelstring nucleic acid molecule with a smooth end that is no further nucleic acid molecule with its terminal can hybridize gene nucleotides, in the sense of the present the invention also referred to as masked. A partial double-stranded nucleic acid molecule with overhanging one cell-stranded ends can thus be masked by a  overhanging 3 'end by means of exonuclease activity is removed, or the complementary strand becomes one over hanging 5'-end by means of a polymerase activity synthe is tized, so that in both cases a double-stranded Nucleic acid molecule with smooth ends is formed.

The term "providing a nucleic acid molecule" summarizes any form of provision, e.g. B. cloning of a gene with subsequent restriction cleavage and iso lation of a fragment with z. B. an overhanging or smooth end, which as a starting material for the fiction, ge appropriate procedure. In another embodiment the nucleic acid molecule by joining two at least partially complementary synthetic oligo nucleotides provided by the juxtaposition overhang can arise. In another version Formation are single-stranded oligonucleotides poses.

The term "at at least one end", as in the invention used means that the synthesis is unidirectional or bidirectional nal can run.

The "attachment" of the single-stranded nucleic acid molecules follows preferably by hybridization. The necessary Hybridization conditions can, if necessary, be from Specialist for every step of the addition of a new single strand based on his specialist knowledge be modified.

The nuclein used in the method according to the invention single-stranded acid molecules have a maximum length of approx. 150 nucleotides. A length between 15 and is preferred 130 nucleotides. Generally, when choosing the length of the Single-stranded molecules note that the yield is more intact Oligonucleotide in Chemical Synthesis of Single-stranded Precursor molecules decrease with increasing length, namely we against incorrect incorporation of nucleotides. So it's a Compromise between the length of the oligonucleotides and their yield. An impact on the yield of desired  ter nucleic acid with the inventive method also the quality of the individual used for the synthesis strand molecules. By oligonucleotide purification with the help HPLC is the single strand of nucleic acid cooler for further synthesis intact. Eventually it will the length of those used for further syntheses Oligonucleotides according to the amount required for a synthesis step and the yield in chemical synthesis orien animals.

The term "predetermined position" as ver according to the invention means either that this sequence by its Pri märsequenz or by their relative positioning to the egg generic cleavage agency is defined.

A predetermined site for cleaving a nucleic acid cell strand can be, for example, by incorporating a artificial or modified nuleotide, a Basenana logons or a chemical group are generated by means of a physical, chemical or enzymatic Process can be split, so that a 3'-OH and / or a 5'-phosphate end is formed. Nucleotides that are for the Processes according to the invention are, for example, 5-Hy droxy-2-deoxycytidine, 5-hydroxy-2-deoxyuridine or 5-Hy droxy-2'-deoxyuridine. While the first two nucleotides E. coli endonuclease III and formamidopyrimi substrates the DNA glycosylase may be nucleotide on the latter using uracil DNA glycosylase and apyrimidase or alkali Treatment to be split.

Another way to cleave the nucleic acid zelstranges at a predetermined location consists in the Introducing a "miss match" in an artificial hair needle structure. Using a "miss match repair" enzyme this structure split efficiently and precisely.

Which (molecular) agent as restriction activity for Cleavage of a predetermined site in a nucleic acid  Ultimately, use double strand in the method according to the invention finding is not essential to the invention. Essential is against for embodiments that split the dop pelstring nucleic acid include that, as before mentioned the recognition sequence on the nucleic acid and the actually split sequence from each other locally are separated. According to the invention, the Recognition sequence by splitting from the growing Removed nucleic acid double-stranded molecule. The restriction Class II S endonucleases have properties that meet the requirements for such an agent. Here are depending on the embodiment of the inventive method rens representative of this class, which a free, cohesive 3'- End or create a protruding, cohesive 5 'end, ge is suitable.

The properties of the restriction activities which can be used in the process according to the invention can be summarized as follows:

• - the restricting agent can be of various types: this includes all nucleic acids specifically cleaving syn thetic agents such as synthetic peptides, PNA (peptides nucleic acid), triple-helical DNA-forming oligonucleo tide used for the specific processing of the Nucleic acid terminus / i suitable for the purposes of this invention are, like naturally occurring DNA-cleaving enzymes. The person skilled in the art is able to for his particular purposes use appropriate restriction activities;
• - These can, for example, restriction endonucleases of the Be Type II S;
• - Asymmetric recognition sequences (restriction endonuclea Class II S), as well as symmetrical recognition sequences can be used;
• - As already mentioned above, the gaps, generated by the restriction activity, not lie within the specific recognition sequence, son they must be located 5 'or 3' distal to it;  
• - the removal of the interface from the recognition sequence must be precisely and clearly defined;
• - the specificity of the attachment of the nucleic acid single stranges in one embodiment of the invention Ensure procedural and efficient ligations guarantee reaction, if desired, he the restricting agent is preferably cohesive End up. This also eliminates the need discussed above maneuverability of masking the single strands, which, for. B. on the smooth ends are attached.

A suitable selection of restricting agents can be obtained from the Consult a specialist from the attached list of literature.

The execution of step (e) or the frequency is The implementation ultimately depends on the length of the ge desired end product as well as the strand length of the Available raw material.

The advantages that the present invention has over the State of the art includes:

1. Availability:
Sequences of nucleic acids, for example of genes, are available anytime, anywhere and quickly (within days), provided the nucleotide sequences are known, for example, from databases. Knowing these sequences and the teaching according to the invention, the person skilled in the art can synthesize any desired nucleotide molecule.

2. Storage and transport:
Nucleic acid molecules the size of genes no longer have to be physically stored in refrigerators with high energy consumption. Your sequences can be managed in EDP systems and, if necessary, easily synthesized in a biological system. This also eliminates the need for transport. DNA sequences can be sent by email.

3. Manipulation:
Any nucleotide, e.g. B. DNA sequences and gene sequences are mutated to achieve certain properties, such as stability to heat, pH changes or solubility in certain solvents, as well as the optimization or change in the biological behavior of their gene products in vitro. Despite many different methods, in vitro mutagenesis is a complex undertaking. The synthesis option enables the insertion of any number of mutations, even at widely spaced sequence positions, since the sequence can be freely defined and predefined. Any number of variants can be created.

The free synthesizability of the DNA sequences becomes many the current methods of time-consuming DNA manipulation relocate them from the laboratory to the synthesis machine, whereby a great cost, and associated with it a great time savings result. The Experi ment then consists of designing a nucleic acid sequence on a computer editor and checking whether the properties desired by the sequence manipulations on set biological model or in vitro. That invented The method according to the invention is therefore also a contribution to Wei Development of techniques of reverse genetics.

In a preferred embodiment of the invention Procedures are not provided in the nucleic acid Molecularly Incorporated Additional Nucleic Acid Molecules, Frag elements thereof and / or nucleotides after step (b), (ba), (c), (d), (da), (e) and / or (ea) separated.

The separation of the unincorporated nucleic acid single strand molecules are preferred, but not absolutely necessary maneuverable and can be carried out by a person skilled in the art using standard methods, e.g. B. by column chromatographic procedures the. The concentration of free nucleotide triphosphates could be desired in particular for the overall yield Nucleic acid becoming limiting, nucleotides consumed the  Disrupt synthesis and ligation reaction, which, for. B. in the case of Generation of smooth ends or the addition of Single-stranded nucleic acid strands at blunt ends and follow the generation of the complementary strand during execution tion of the method according to the invention is necessary, such as has been described above. A high concentration ver Different single-stranded DNAs increase the risk of undesirable By-products. It is therefore a practical advantage if every single synthesis step under optimal conditions can expire. It is therefore advisable to separate the single strands not required before the next syn thesis step, for example in a matrix coupled Re action, after the expiration of used nucleotides and over single stranded nucleic acids are eluted. The separation can of course also after or while performing step (e).

The optional ligation described above embodiment of the method according to the invention, the Anla hybridization of complementary terminal Nucleotides may include, for example, before, simultaneously with or after step (d). In another version It can take the form after or during step (s) consequences. An example of the ligation after step (e) lie fert the case that bacteria, e.g. B. E. coli with which not ligated synthesis product to be transformed and the league tion is made by endogenous ligases. So be knows that with increasing size of the complementary overlap pung cohesive ends a transformation of e.g. E. coli with suitable DNA using the endogenous Ligase activity for circularization is possible. Here who the gaps and protruding single-stranded DNAs are absolutely great repaired because the integrity of the circular Restore the double strand. Single strand areas who the replenished and repaired if at least one Phosphodi backbone of the ester is intact. A ligation is preferred made when the overhangs are only a few nucleotides  are long. In the case of longer overhangs it is conceivable that between the terminal nucleotides of single and dop Pelstranges gaps occur before a ligation freak tion closed, for example, by a polymerase activity will be. Because the previously known restriction enzymes too usually only produce relatively short cohesive ends, too a C and G or A and T "tailing" with terminal trans ferase conceivable that creates long overlap areas that can be transformed directly without "in vitro" ligation. Finally, the synthesis product can also after the step Isolation of the finished synthesis product to a ligation be performed.

As already mentioned above, one is preferred Embodiment of the method according to the vorbe correct location of the nucleic acid molecule by incorporation an artificial or modified nucleotide, a Ba analogues, a chemical group, or a "miss match "in an artificial hairpin structure will be carried out by means of a physical, chemical or enzymatic process can be split.

In a particularly preferred embodiment, this is artificial or modified nucleotide 5-hydroxy-2-deoxy cytidine, 5-hydroxy-2-deoxyuridine or 5-hydroxy-2'-des is oxyuridine.

As also mentioned above, this concerns lying invention in a further preferred embodiment form of a process, the linkage being via a 3'- Hydroxy and a 5'-phosphate end of two terminals Nucleotides using a ligase activity and the attachment hybridization of complementary sequences follows.

In another preferred embodiment of the Invention According to the method, the nucleic acid is DNA.  

In a further preferred embodiment of the invented The method according to the invention is the nucleic acid RNA. The Ge also includes the method generation of DNA / RNA hybrids.

In a further preferred embodiment of the invented The method according to the invention is carried out in step (c) additive and subtractive by adding or removing a chemical group or a chemical molecule. In a preferred embodiment of the invention A 5 'end is masked by the method Removing the phosphate group (s) or installing a 5'- modified nucleotides (e.g. biotin-dNTP, digoxygenin-dNTP Etc.). As mentioned above, by masking the 5'-end of a single-stranded nucleic acid mole to be attached cool in the corresponding embodiment of the invention according to an undesired ligase side reaction 5 'and 3' ends of the single-stranded nucleic acid molecules with prevented each other and thus the possible formation of Kon katemeren, whereby an optimal yield of the process of teaching according to the invention is ensured.

In a particularly preferred embodiment of the invent The method according to the invention is carried out by the masking Incorporation of at least one 5'-modified nucleotide.

In a further preferred embodiment of the invented process according to the invention is, as already mentioned, a masked 3 'end by the presence of an ami noblocks, a dideoxynucleotide, a 3'-phosphate or an artificial 5 'end.

In a further preferred embodiment, this forms additional nucleic acid molecule on the provided Nucleic acid molecule after attachment and / or linkage  removed a hairpin loop that acts as a primer serves for the polymerase activity.

In a further preferred embodiment, the invention relates to rungform a method, wherein the cleavage on a prep agreed place in step (d) by a sequence-specific cleaving triple helical DNA occurs. A triple helical DNA is e.g. B. formed when a single-stranded DNA, at the end of which is a heavy metal (SM) is coupled to a DNA double strand and, so if the sequence conditions are suitable, a trippleheli kale structure with a DNA double strand. Of the Nucleic acid duplex is attached to the heavy metal on one defined position split.

In addition, as already mentioned, according to the invention any specific physical, chemical and enzymatic Nucleic acid cleavage are used, which for the Anla succession of a single-stranded nucleic acid molecule ligation with the nucleic acid double-stranded molecule is beneficial. Other examples of this are methodological Approaches based on designed peptides or PNA (peptides nucleic acid). An overview of the aforementioned molecules and examples of their possible uses can be found in the subject refer to the list of literature below.

Another preferred embodiment of the invention meets a procedure whereby the cleavage passes agreed place in step (d) by a type II S restriction endonuclease occurs. Type or class II S enzymes sit an asymmetrical or non-palindromic bay sequence. The cleavage sites are either 5 'or 3' distal to the recognition sequence. Either 5 '(e.g. BspMI) or 3'- (e.g. RleAI) protruding ends or smooth Ends (e.g. BsmFI).  

In a particularly preferred embodiment of the invent The method according to the invention is the Type II S restriction end nuclease the Rle AI enzyme from Rhizobium leguminosarum (Vesely Z., Müller A, Schmitz G, Kaluza K, Jarsch M, Kessler C (1990) RleAI: a novel class-IIS restriction endonuclease from Rhizobium leguminosarum recognizing 5'-CCCACA (N12 / 9) - 3 ', Gene 95: 129-131).

In another preferred embodiment of the Invention according to the method is / are the nucleic acid double strand mo lekül and / or the nucleic acid single-stranded molecules synthe table or semi-synthetic origin. The use of synthetic is particularly preferred for synthesis single stranded molecules. Semisynthetic molecules can be produced in that Nucleic acid fragments from "in vivo" (bacteria, yeast) ampli infected DNA (dsDNA, ssDNA) or RNA on one or more intermediate steps of the synthesis according to the invention at de Finished places can be installed by ligation. This strategy can significantly reduce costs in individual cases help decorate. For example, this can be used as a starter molecule Nucleic acid molecule also an "in vivo" generated DNA Be a molecule to which any by recursive DNA synthesis DNA sequences are attached.

In a further preferred embodiment of the invented According to the inventive method, the synthesis is at least partially automated. For example, in a nucleic acid (gene) synthesis automatic machines for nucleic acid double strands of nucleic acid Single rods a battery of automated chemical Oligonucleotide syntheses (one already on a large scale practiced technology) the raw material for the synthesis of biologically active, double-stranded DNA molecules (e.g. whole genes). These are derived from the chemically syn thetized oligonucleotides in a likewise automati based process.  

The dop to be extended are in a synthesis chamber pelstrangnucleic acids bound to the synthesis matrix. In this synthesis chamber run in a cyclic reaction always follow the same steps described above from. The reaction byproducts of the previous reaction are synthesized before a new reaction begins always washed out. That extended by one nucleic acid molecule Starter molecule remains bound to the synthesis matrix. At each synthesis step is a nucleic acid with another Ren sequence sequence built in, so that ultimately a counter also double-stranded nucleic acid with the desired one Nucleotide sequence arises.

In a particularly preferred embodiment, the invention relates to embodiment a method, the synthesis being matrix-bound which is carried out. All carrier materials to which a nucleic acid binds lets and their properties with the desired recursive Nucleic acid synthesis is compatible come as a synthesis scheme trix in question, e.g. B. streptavidin-coated surfaces where in the nucleic acid double used as the starter molecule strand molecule via a built-in biotinylated nucleotide is coupled to the synthesis matrix. Other examples are Nylon surfaces to which polydT-containing sequences by UV Radiation can be coupled and tosy-activated surface Chen, where the binding takes place via an "amino link", glass Silicate, latex, polystyrene, epoxy or silicon.

In another preferred embodiment of the Invention according to the method, the synthesized nucleic acid mole cool isolated after synthesis.

This is done on the one hand by incorporating an affinity ver averaging agent in the last synthesis step, such as. B. Bio tin, digoxiginine, a histidine tag or a malto serestes. The synthesis end products labeled in this way can thus simple and inexpensive by means of appropriate columns iso  be lated. Alternatively, in the last synthesis step the inventive method a plasmid with the Synthe end product and the resulting nuclein acid molecule, if necessary after its recircularization, introduced and reproduced in bacteria. Alternatively, will in the provided nucleic acid molecule as well as in the last one Synthesis step used nucleic acid molecule defined Incorporated sequences to which primers can specifically bind nen. With the help of these primers, the fully synthesized Nucleic acid molecule amplified in a PCR reaction the, whereby the final synthesis product from the affinity matrix be isolated.

In another preferred embodiment of the Invention According to the method, single-stranded nucleic acid molecules by denaturing the double-stranded nucleic acid molecule isolated.

This embodiment of the method according to the invention is suitable for arbitrary nucleic acid single-stranded molecules Manufacture composition. In this context Another point to mention is the possibility of such RNA molecules to provide.

Finally, the invention relates to a kit, at least containing

• a) a ligase;
• b) a polymerase; and
• c) a type II S restriction enzyme; and or
• d) a uracil DNA glycosylase and an apyrimidase and / or an endonuclease III and a formamidopyrimi din DNA glycosylase and / or a "miss match repair" En zym;
• e) optionally a phosphatase, a terminal trans ferase and / or an exonuclease;  
• f) optionally a washing buffer for the elution of reactants by-products and not in the product of the inventor inventive synthesis of built-in material;
• g) optionally a synthesis matrix with a given if already attached nucleic acid molecule as Starter molecule; and
• h) optionally suitable reaction buffers for the in (a) to (e) listed enzymes.

Based on the teaching of the present invention as well as general knowledge in this technical field known to the manufacturer of the kit according to the invention as he the individual components of the kit, e.g. B. the buffers represents and formulates. If necessary, the fiction kit also includes a starter motor that is not bound to a matrix lekül and / or a set of suitable single-stranded molecules contain.  

The figures show:

Fig. 1. "In vitro" SSDNA synthesis in the 3'-5 'direction (1). A starter molecule (s) coupled to a matrix is linked by means of a ligase activity around an n + 1-th single-strand molecule by means of a 3'-5'-phosphodiester bond. The n + 1-th ssDNA has a uracil deoxynucleotide at the terminal. The glycosidic bond of the base uracil is cleaved by the uracil glycosylase, which creates an apyrimidine position. This in turn is cleaved by an apyrimidine endonuclease activity (exonuclease III) so that a 5'-phosphate and a 3'-OH end are formed. (3) The n + 2-th ssDNA molecule is linked to the released 5'-phosphate end in the n + 2-th ligation reaction. A subsequent phosphate reaction (not shown, see FIG. 4) inactivates all DNA chains for the n + 3 th ligation step for all subsequent ligation steps, provided that no n + 2 th ssDNA molecule is incorporated in the n + 2 th step has been. Through DNA-uracil glycosylase and the apyrimidine endonuclease activity, a 5'-phosphate can be made available for the next reaction sequence (n + 3) through processing. All steps are repeated k times until the last ssDNA molecule has been inserted in the mth step.

Fig. 2. "In vitro" dSDNA synthesis in the 3'-5 'direction. All steps take place as in FIG. 1, but then in the last step, starting from the 3 'end of the starter molecule, a polymerization reaction is started, which fills the newly synthesized single strand into a double strand. Alternatively, a primer of a pair of primers can be installed in the first and last step and a double-stranded molecule can thus be generated by amplification by means of the PCR reaction.

Fig. 3. "In vitro" DSDNA synthesis in the 3'-5 'direction. All steps are carried out as in FIG. 1. Within each synthesis cycle, a polymerization step is initiated after the phosphatase treatment, which converts the ligated ssDNA into a dsDNA. The processing can also be carried out using a restriction endunuclease of type IIS, provided that a recognition sequence is built into each of the ssDNA fragments.

Fig. 4. "In vitro" DSDNA synthesis in the 3'-5 'direction. All steps take place as in FIG. 1. A phosphate sereaction carried out after a ligation step inactivates all DNA molecules for the next ligation step and for all subsequent ligation steps, provided that no ssDNA molecule has been incorporated in the n-1st synthesis cycle. Due to the DNA uracil glycosylase and the apyrimidine endonuclease activity in each synthesis cycle, a 5'-phosphate can again be made available for the next reaction sequence by processing. All steps are repeated k times until the last ssDNA molecule has been incorporated in the mth step.

Fig. 5. "In vitro" SSDNA synthesis in the 5'-3 'direction (1). A starter molecule (s) coupled to a matrix is linked by means of a ligase activity around an n + 1-th single-strand molecule by means of a 3'-5'-phosphodiester bond. The n + 1-th ssDNA has a uracil deoxynucleotide, is 5'-phosphorylated and 3'-blocked (-X). The glycosidic bond of the base uracil is cleaved by the uracil glycosylase, which creates an apyrimidine position. This in turn is cleaved by an apyrimidine endonuclease activity (exonuclease III) so that a 5'-phosphate and a 3'-OH end are formed. (3) the n + 2-th ssDNA molecule is linked to the released 5'-phosphate end in the n + 2-th ligation reaction. A subsequent terminal transferase reaction with a dideoxytrinucleotide (not shown, see FIG. 7) inactivates all DNA chains for the n + 3 th ligation step for all subsequent ligation steps, provided there is no n + 2 th ssDNA molecule in the n + 2 -th step was installed. The 3-OH for the next reaction sequence (n + 3) can be made available again by processing due to the DNA uraclyglycosylase and the apyrimidine endonuclease activity. All steps are repeated k times until the last ssDNA molecule has been inserted in the mth step.

Fig. 6. "In vitro" SSDNA synthesis in the 5'-3 'direction (1). A starter molecule (s) coupled to a matrix is linked by means of a ligase activity around an n + 1-th single-strand molecule by means of a 3'-5'-phosphodiester bond. All further steps take place as shown in FIG. 5. In the last step, initiated by, for example, a 3'-terminal hairpin structure, a 3 'end can be made available for a DNA polymerization reaction or dsDNA polymerization can be carried out as described in FIG. 2.

Fig. 7. "In vitro" SSDNA synthesis in the 5'-3 'direction (1), representation of the reaction to inactivate non-ligated ends. A terminal transferase reaction with a dideoxytrinucleotide inactivates all DNA chains for the next ligation step for all subsequent ligation steps, provided that no ssDNA molecule has been incorporated in the n-1 th synthesis step. Due to the DNA uraclyglycosylase and the apyrimidine endonuclease activity, a 3'-OH can again be made available for the next reaction sequence by processing. All steps are repeated until the last ssDNA molecule was inserted in the mth step.

The examples illustrate the invention:

example 1

Recursive DNA synthesis "in vitro" can be used for manipulation of DNA sequences are used "in vitro". On the one hand can mutations, such as deletion mutagenesis, several Deletions in one gene at the same time, gem fusions under Er generation of new properties, insertion mutagenesis, substi tution mutagenesis and sequence inversions performed become. Furthermore, you can have one to any number Introduce point mutations into a sequence. All DNA sequences zen can be done in parallel without intermediate cloning steps Syntheses can be generated directly.

The functio resulting from the sequence manipulations changes in biological activity "in vivo" can affect the protein level, so far the coding sequences into functional proteins can be translated. The method can thus be used to Implementation of considerations in enzyme or protein design be set.

On the other hand, it is possible to regulate the DNA sequences to manipulate the cis elements in order to change the activity of transactivators and suppressors, de to investigate their behavior or even a completely new combination to create cis elements. Furthermore, the Activity of RNA molecules can be manipulated (e.g. Ribo zyme), provided the manipulated DNA is transcribed.

The following example of the application of recursive DNA "In vitro" synthesis method is the manipulation of DNA-Se sequences for analyzing the binding activity of a transactive Regulatory protein on a bacterial promoter region. Through the "in vitro" mutagenesis of the binding sites investigated the effects on the DNA-binding protein.  

The DNA wild-type sequence and the mutants of the cis-active element are shown in FIG. 7, the sequence manipulations are explained in the text. The aim of the experiments was to be able to investigate the functionality of the binding of the regulator in a different sequence context “in vitro” and possibly also “in vivo”.

On a SauIIIA DNA fragment in the area of a bacterial promoter there are palindromic sequence sections whose structure is very similar to sequences which are also involved in the transcriptional regulation in other systems. The transcriptional activity of the S ⁷⁰ -like promoter in the 5 'sequence region of the 6-HDNO gene, on which cis-active elements lie, was described by Mauch et al., (1990) "in vivo" in the heterologous E. coli system examined in detail. The cloning of this DNA fragment, which was used in most of the DNA binding studies of this work, is referred to below as the WT-6-HDNO promoter fragment ( FIG. 7A (1-3)).

Incubation of raw extracts from Arthrobacter ricatinovorans cells (10-40 mg total protein) with a ra dioactively labeled 6-HDNO binding fragment of the 6-HDNO gene Presence of a competitor (usually pdloC) from the promo gate area, shows this after the separation of the components Incubation approach in the electric field of a native PAA Gels compared to a clear retention of the DNA fragment a control batch without the addition of crude extract protein.

Used to identify NicR1 binding activity The experimental approach is called gel retention analysis draws. With the help of this method the kinetic and functional behavior of DNA binding proteins in dependent quality and various parameters "in vitro" be examined quantitatively. You can also be did the conditions for statements on the structure of the Make DNA / protein complex.  

Using raw extracts you can usually in Gel retention experiment a dominant, retarded band detect. A second gang is sometimes also recognized bar. This DNA binding activity could be caused by large amounts of unlabeled, non-specific competitor DNA not sup be primed, but probably by unmarked binding fragment in very small quantities. It was therefore assumed that this DNA binding activity is specific and with which transcriptional regulation of nicotine regulation together menhang stands. It was abbreviated to NcR1 (nicotine regulator 1) (Mauch et al., 1990, Bernauer et al., 1992).

The behavior of NicR1 binding activity in gel retention expe riment was analyzed in this work to obtain statements about the location, specificity, kinetics and stoichiometry of the bin reaction and the reaction of the DNA Binding function on manipulations on the WT-6 HDNO promo gate fragment and on potential effector substances search.

These experiments should provide information about which moles specific mechanisms for the regulation of the 6-HDNO gene ver could be answerable. The breeding conditions were also increased conditions and the induction status of Arthrobacter ricatinovorans cells, from which eventually Rohex tract produced for analysis in the gel retention experiment has been. These experiments should shed light on whether the binding behavior of NicR1 changes or the An presence of additional factors depending on one the test parameters are to be demonstrated. The pro reaction standard puf used in tein / DNA binding experiments fer is based on that used by Garner and Revzin (1981) reaction buffer. The NicR1 binding activity is through Ammonium sulfate fractionation can be enriched. The enrichment NicR1 binding activity was the prerequisite for Versu  for the analysis of the binding behavior of NicR1 at the same time early binding of both palindromic sequences that can be found on the WT-6 HDNO promoter fragment.

The WT-6-HDNO promoter fragment from the 5 'control area of the 6-HDNO gene has some very interesting sequence features (see FIG. 7). It is characterized by extensive inverted sequence repetitions (IR) and other striking sequence motifs. Characteristic sequence arrangements within the 6-HDNO gene promoter region are shown in FIG. 7. This shows two inverted repeats, IR1 and IR2, which have extensive homologies with each other ( Fig. 7). The right palindromic half of IR2 is repeated in the 5 'area. Such palindromes are structural features found in many bacterial cis-active regulator regions.

IR1 and IR2 are separated by a 50 bp interpalindromic sequence. The palindromic half-sides of IR1 are over 17 bp homologous, those of IR2 over 9 bp. The palindrome of IR1 reaches an extension of twelve base pairs, but shows two insertions of two and one base pair (AT, A) in this area. Ten of twelve base pairs of IR1 in the 5 'half and 9 of 12 base pairs in the 3' half of the sequence are homologous to IR2 ( Figure 7A, sequences of IR1 and IR2). These sequence-specific features could have structural and functional significance in the binding of the "in trans" binding-active protein NicR1 and an S ^70- like RNA polymerase. IR1 represents an almost perfect S ⁷⁰ -like promoter sequence, with a remarkable modification. The -10 region differs from the consensus sequence TAT AAT by the insertion of a C, which results in the sequence TAT-CAAT. A -30 region is found in the sequence of IR2, but is not similar to the known -10 region of an S ⁷⁰ -like promoter sequence. The distance between the -10 and -30 region corresponds to the S ⁷⁰ ideal of 17 at 16 bp. Integrated into the sequence of IR2 is the 35 region of an S ⁷⁰ -like promoter, a consensus-like -10 region is missing. Some other sequence features could also reflect the activity of other regulatory elements transactive at the 5 'sequence region of the 6-HDNO gene. Within the palindromes IR1 and IR2 there are three Nlal (CATG) recognition palin dromes in a homologous position. It is interesting that behind the left palindromic half of IR2, in a non-homologous position, there is also such an interface. The question arises of coincidence or the need for such a structure. Outside the palindromic sequences of IR1 and IR2 there are also striking sequence motifs. Sequences rich in GC and AT are arranged alternately. An interesting sequence feature of this domain is the presence of GC-rich sequence segments, which are interrupted by an AT-rich sequence segment in the 6-HDNO-5 'sequence. The GC sequence blocks are located above the 5 'region of the S ⁷⁰ -like promoter. A detailed base neighborhood analysis according to the algorithm described in Ebbole and Zalkin (1989) showed that this sequence is largely non-statistical. To show this, a computer program in "Pascal" was specifically written. While the sequences within the palindromic regions consist of fairly regularly alternating short GC and AT regions with a very balanced GC content, there are larger sequence sections 5 'to the palindromes with a very unbalanced GC content ( FIG. 7A). First, the GC content increases from 5 'outside the palindrome IR2, first a GC maximum, then a GU minimum is run through ( FIG. 7A). The situation repeats itself in front of the Palin drom IR1. Alternating GC and AT-rich sequence sections are associated with structural properties of protein binding. The AT-rich positions turn into the protein with their small DNA groove, the GC-rich sequence blocks point outwards. GC-rich promoters, which no longer have sequence similarity with the known S ⁷⁰ -like promoters, can be found in Streptomyces species.

As a starter molecule (see FIG. 7 AA (0)) for the recursive DNA synthesis, the plasmid pUC19 (Yanish-Perron et al., 1985) was double-digested with BamHI and KpnI and purified using an agarose gel. KpnI has the recognition sequence 5'-GGTAC'C-3 '. An oligonucleotide of complementary sequence in the presence of a T4 ligase, T4 DNA polymerase and 0.2 mM dNTP was attached to the 3 'protruding KpnI end under standard conditions (Sambrook et al., (1989)), ligated and double-stranded The oligonucleotide has at the 5 'end the recognition sequence of the restriction endonuclease RleAI plus a few additional bases (see FIG. 7A (1)). The now double-stranded DNA molecule (filled in, supernatant synthetic oligonucleotide) was enriched with a restriction endonuclease RleAI from Rhizobium leguminosarum restricted (respectively Fig. 7 (2) and (3 ')) The reaction conditions were taken from Veseley et al., (1990). This enzyme produces 3' protruding ends outside of its asymmetric binding site Specificity has so far been unique and allows the repeated attachment of an oligonucleotide and the priming for a DNA polymerisation.The short DNA fragment with the RleAI - Detection sequence was purified from the plasmid using an agarose gel. At the 3 'protruding end resulting from the restriction reaction, a complementary oligonucleotide ( FIG. 7A (2)) was added to the end and filled in as mentioned above (see also FIGS. 1 and 2). The same reaction was carried out with the oligonucleotide ( Fig. 7A (3)) and the variants Fig. 7B-1 to B-7. By using synthetic oligonucleotides, seven sequence variants and the wild-type sequence could be generated in parallel. After the reaction sequence FIG. 7A (3) the newly DNAs formed (Fig s. Sequence. 7A (3 ")) were recut with BamHI, the vector (pUCl9 + binding fragment) circularized and transformed according to standard methods in E. coli. Found the RleAI recognition sequence terminally at the end of the synthetic oligonucleotides as well, the DNA synthesis reaction can be extended by one step as in this example In order to characterize the binding properties of NicR1, sequence changes were carried out in the IR1- bearing the S ⁷⁰ -like promoter. Binding site introduced and the length of the interpalindromic sequence (IS, Fig. 7B-5, -6, -7) was varied, the latter attempts to investigate the steric requirements for the palindromic binding sequence IR1. 6- HDNO promoter fragment introduced are shown in FIG. 7.

The changes introduced by "recursive DNA synthesis in vitro" in the sequence of the promoter-containing IR1 palindrome and in the interpalindromic sequence are shown in Fig. 7B. The reduction of IR1 to an octamer ( FIG. 7B-3) as well as the deletion of the central G position ( FIG. 7B-4) destroy the ability of NicR1 to bind to IR1.

If the cleavage products are used in the gel retention experiment, only IR2 is retarded, but not the mutant IR1-containing fragment. The construct shown in Fig. 7B-4 shows retention only by the binding of NicR1 to IR2. Since the size of the complex at IR2 is the same size as the complex at IR1, this is an indication of the binding of the same protein to both palindromes.

Contrary to the pronounced effect created by the changes in both the length and the symmetry of the palindron IR1 on the NicR1 bond, changes in the number of helical turns in the interpalindromic sequence did not result in a difference in the NicR1 bond to both palindromes. The length of the interpalindromic sequence was changed by deletions as well as inserts of 5 bp each ( FIGS. 7B-6 and -7). These changes correspond to half a helix turn. The consequence of this is that in these DNA mutants the IR2 binding site is rotated by 180 ° relative to the IR1 binding site. In addition, the 50 bp interpalindromic sequence was reduced by 20 bp ( Fig. 7B-5). The pattern of the gel retention experiment bearing these changes ( Fig. 7B-5, -6, -7) was identical to the control pattern seen with the unchanged 242 bp 6 HDNO promoter fragment ( Fig. 7B-1) was.

The right half of IR1 contains the -10 region of the promoter of the 6-HDNO gene which differs from the consensus sequence of the promoter of the S ⁷⁰ RNA polymerases by the insertion of an additional base position in the TATAAT sequence containing cytosine ( FIG . 7B-1). The question arose whether this unusual S ⁷⁰ -10 region contributed to the specificity of the NicR1 binding to IR1. In the gel retention experiment ( Fig. 7B-2) with NicR1, the deletion of the cytosine residue at the corresponding position ( Fig. 7B-2) showed no change in the protein binding pattern compared to the pattern obtained with the unchanged DNA fragment, but did in binding the S ⁷⁰ -like RNA polymerase from E. coli.

The statement that the two mutations FIGS. 7B-3 and -4 greatly reduce, if not completely prevent, the NicR1-binding ability to the palindrome IR1 is supported by further experiments not described here.

Example 2

Example of the synthesis of:
PLASMIDS π-AN7 885 bp: Huang, Little, Seed (1985) in vectors: A survey of molecular cloning and their applications ", Rodriguez, R., ed., Butterworth Publishers, Stoneham, MA, USA.

STARTER MOLECULE:

AAUGCGGCCGCTCACGAGCCGCGCGGTTAATTAACTCGAGAABTCCGCGGTGCAATTAATT-X

Restriction enzymes: EacI, Bst2BI, AccBSI, NotI, PacI, XhoI, EcoRI, SacI
B = biotin
X = AminoBlock

Brief protocol:
A µg biotinylated starter molecule was bound to streptavidingecoatete DynalBeads. Then 0.2 mM biotin deoxyuracil was set (0-5 h incubation at RT each) to block all biotin binding sites.

In 16 synthesis cycles (ligation, T4-RNA ligase; uracil-DNA- Glycosyllase, exonuclease III; Phosphatase) the 17 DNA Molecules to a single strand, the whole plasmid sequence comprehensive DNA linked.

From the 3 'end of the starter molecule, the T4 DNA Polymerase replenished the ssDNA to the dsDNA. With NotI Released dsDNA from your attachment to DynalBeads. The same amount of fresh DynalBeads were added. Only the molecules with biotin were bound to the column. Molecules without biotin from the last ligation reaction were washed away.

The restriction endonuclease PacI was subsequently cleaved. The Dynabeads were pelletized with a magnet. The molecules were precipitated with ethanol from the supernatant and circularized the molecule with the T4 DNA ligase. The circularized synthetic plasmid molecules were then transformed into E. coli DH10a / P3 according to standard protocol and selected against Tet / Amp on LB plates.

Bibliography

Bernauer, H., Mauch, L. and Brandisch, R. (1992) Interaction of the regularory protein NicR1 with the promotor region of the pA01-encoded 6-hydroxy-D-nicotine oxidase gene of Arthrobacter oxidans, Mol Microbiol 6: 1809 -1820.
Bigey, P., G. Pratviel, et al. (1995). "Cleavage of double stranded DNA by 'metalloporphyrin-linker-oligonucleotide' molecules: influence of the linker. Nucleic Acids Res 23 (19): 3894-900.
Francois, JC and C. Helene (1995). "Recognition and clea vague of single-stranded DNA containing hairpin structures by oligonucleotides forming both Watson-Crick and Hoogsteen hy drogen bonds. Biochemistry 34 (1): 65-72.
Gao, X., A. Stassinopoulos, et al. (1995). "Structural basis for the sequence-specific DNA strand cleavage by the ene diyne neocarzinostatin chromophore. Structure of the post activated chromophore-DNA complex. Biochemistry 34 (1): 40-9.
Garner, MM, and Revzin, A. (1981) A gel elektrophoresis method for quantifiying the binding of proteins to specific DNA regions: application to components of the E. coli lactose operon regulatory system. Nucleic Acids Res. 9: 3047-3060.
Harford, C., S. Narindrasorasak, et al. (1996). "The designed protein M (II) -Gly-Lys-His-Fos (138-211) specifically cleaves the AP-1 binding site containing DNA. Biochemistry 35 (14): 4271-8.
Kane, SA, SM Hecht, et al. (1995). "Specific cleavage of a DNA triple helix by FeII-bleomycin. Biochemistry 34 (51): 16715-24.
Mauch L., Bichler V., and Brandisch R (1990) Functional analysis of the 5 'regulatory region and the UUG translation initiation codon of the Arthrobacter oxidans 6-hydroxynicotine oxidase gene. Mol Gen Genet 221: 427-434.
Norton, JC, JH Waggenspack, et al. (1995). "Targeting peptide nucleic acid-protein conjugates to structural features within duplex DNA. Bioorg Med Chem 3 (4): 437-45.
Oikawa, S., M. Kurasaki, et al. (1995). "Oxidative and non-oxidative mechanisms of site-specific DNA cleavage induced by copper-containing metallothioneins. Biochemistry 34 (27): 8763-70.
Sambrook, J. Fritsch, EF, and Maniatis, T. (1989) Molecular cloning a laboratory manual. Cold Spring Harbor, Cold Spring Harbor Laboratory Press 1: 1.25-1.28.
Sergeev, D. 5., T. 5. Godovikova, et al. (1995). "Cleavage of a doubled-stranded DNA target by bleomycin derivatives of oligonucleotides, forming a ternary complex. Bioorg Khim 21 (3): 188-96.
Vesely Z., Müller A, Schmitz G, Kaluza K, Jarsch M, Kessler C (1990) RIeAI: a novel class - IIS restriction endo nuclease from Rhizobium leguminosarum recognizing 5'-CC- CACA (N12 / 9) -3 ', Gene 95: 129-131.
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Claims (22)

1. A method of synthesizing nucleic acid molecules comprising the following steps:

• a) provision of a nucleic acid molecule which has at least one end which permits attachment and / or linkage of or with a further nucleic acid molecule;
• b) attachment and / or linkage of at least one further nucleic acid molecule to or with the nucleic acid molecule, the one end of the at least one further nucleic acid molecule being attached and / or linked to the or with the at least one end (s) of the nucleic acid molecule and the other end of the at least one further nucleic acid molecule is masked in the event of a link;
• c) masking the at least one end of the nucleic acid molecule to or to which no further nucleic acid molecule has been attached and / or linked;
• d) cleavage of the at least one further attached and / or linked nucleic acid molecule at a predetermined location, the masking being removed and an end being generated which permits attachment and / or linkage of or with a further nucleic acid molecule; and
• e) repeating steps (b) to (d) at least once, possibly several times, in each case suitable nucleic acid molecules being used in step (b).

2. The method of claim 1, wherein the further nucleic acid remolecule is a single-stranded nucleic acid molecule. 3. The method of claim 2, which after step (b) comprises the step of:

• a) Filling the second strand of nucleic acid complementary to the single strand in its sequence by a polymerase activity, the masking being removed if necessary beforehand.

4. The method according to claim 2, which comprises the following step after step (d) or (e):

• 1. (d / ea) Filling in of the second strand of nucleic acid complementary to the single strand in its sequence by a polymerase activity.

5. The method according to any one of claims 1 to 4, wherein not incorporated into the provided nucleic acid molecule further nucleic acid molecules, fragments thereof and / or Nucleotides after step (b), (ba), (c), (d), (da), (e) and / or (ea) are separated. 6. The method according to any one of claims 1 to 5, wherein the predetermined position of the nucleic acid molecule by In corporation of an artificial or modified Nucleotide, a base analog, a chemical Group or a "miss match" in an artificial Hairpin structure is generated, which by means of an egg physical, chemical or enzymatic ver driving can be split. 7. The method according to claim 6, wherein the artificial or modified nucleotide 5-hydroxy-2-deoxycytidine, 5-Hy droxy-2-deoxyuridine or 5-hydroxy-2'-deoxyuridine. 8. The method according to any one of claims 1 to 7, wherein the Linkage via a 3'-hydroxy and a 5'-phosphate End of two terminal nucleotides using one Ligase activity, and attachment via hybridization complementary sequences.   9. The method according to any one of claims 1 to 8, wherein the Nucleic acid is DNA or RNA. 10. The method according to any one of claims 1 to 9, wherein the Masking in step (c) additively or subtractively Addition or removal of a chemical group or of a chemical molecule. 11. The method of claim 10, wherein the masking by a phosphatase, a terminal transferase, a Po lymerase or an exonuclease reaction or chemical he follows. 12. The method according to any one of claims 1 to 11, wherein the Masking of a 5 'end by removing the phos phat group (s) or the incorporation of a 5'-modified Nucleotide. 13. The method according to any one of claims 1 to 11, wherein a masked 3 'end by the presence of a Amino blocks, a dideoxynucleotide, a 3'-Phos distinguishes phats or an artificial 5 'end. 14. The method according to any one of claims 1 to 13, wherein the further nucleic acid molecule after attachment and / or Linking to the nucleic acid mo provided by forms a hairpin loop at the distal end, which serves as a primer for the polymerase activity. 15. The method according to any one of claims 1 to 14, wherein the Cleavage at a predetermined location in step (d) through a sequence-specific splitting triple helical DNA or by a type II S restriction endonuclease he follows.   16. The method of claim 15, wherein the type II S restriction tion endonuclease the Rle AI enzyme from Rhizobium legumi is nosarum. 17. The method according to any one of claims 1 to 16, wherein the provided nucleic acid molecule and / or the wide one ren nucleic acid molecules synthetic or semisynthetic are of table origin. 18. The method according to any one of claims 1 to 17, wherein the Synthesis is at least partially automated. 19. The method according to any one of claims 1 to 18, wherein the Synthesis is carried out matrix-bound. 20. The method of claim 19, wherein the matrix of nylon, Glass, silicate, latex, polystyrene, epoxy or silicon is. 21. The method according to any one of claims 1 to 20, wherein the synthesized nucleic acid molecule after synthesis is isolated. 22. Kit at least

• a) a ligase;
• b) a polymerase; and
• c) a type II S restriction enzyme; and or
• d) a uracil DNA glycosylase and an apyrimidase and / or an endonuclease III and a formamidopy rimidin DNA glycosylase and / or a "miss match repair"enzyme;
• e) optionally a phosphatase, a terminal transferase and / or an exonuclease;
• f) optionally a washing buffer for the elution of reaction by-products and material not incorporated into the product of the synthesis according to the invention;
• g) if appropriate, a synthesis matrix with a nucleic acid molecule which may already be bound to it as a starter molecule; and
• h) optionally suitable reaction buffers for the enzymes listed in (a) to (e).