DE19914808A1 - Information-bearing polymers - Google Patents

Abstract

The invention relates to methods for producing information-carrying polymers, information-carrying polymers obtained using these methods, and to methods for isolating, duplicating, and selecting such information-carrying polymers. The invention also relates to polymeric data memories and DNA computers which contain the information-carrying polymers, and to the use of information-carrying polymers for producing molecular weight standards, as markers or signatures, for encoding information, as molecular-scale adhesives, or for producing or processing the smallest molecular structures.

Description

The invention relates to methods for producing information-carrying polymers, according to these Process-available information-carrying polymers; Process for isolation, for Duplication and reading of such information-carrying polymers; polymers Data storage and DNA computers, which comprise information-carrying polymers, as well as the Use of information-bearing polymers for the production of Molecular weight standards, as markers or signatures, for the encryption of Information, as molecular glue or for the manufacture or processing of the smallest molecular structures.

It is known to use nucleic acids such as DNA (deoxiribonucleic acid = deoxyribonucleic acid) and RNA (Ribonucleic Acid = Ribonucleic Acid) also outside of organisms for Use information processing. So far there have been different approaches, DNA Using molecules to process information is presented. WO 97/07 440 and [L. M. Adleman, Molecular Computation of Solutions to Combinatorial Problems, Science, 266, 1021-1024, (1994)] describe a method, a graphene Algorithm ("Hamiltonian path problem") as a decision problem with the help of DNA To calculate molecules. The algorithm is implemented by the edges and nodes of the graph are represented as DNA sequences. The calculation of the algorithm will in that by hybridizing complementary DNA sequences Set of paths of the graph are generated.

This set of paths then becomes all using molecular biological methods Removed paths that are an incorrect or no solution to the algorithm. Were all steps successfully executed, at least one DNA strand must remain at the end, that contains the correct solution to the problem, or there is no DNA strand left, what means that the algorithm has no solution.

The method described assumes that enough molecules are available stand so that every possible solution to the respective problem instance is statistically at least occurs once in the initial set of paths. This can be due to the statistical However, the nature of the hybridization processes cannot be guaranteed. In addition, in First step also cyclic graphs are created, which is wrong from the start Solutions enlarged.

The approach described in WO 97/07 440 shows that symbol processing is in vitro at all can be done with DNA molecules. However, the approach described is in the Practically hardly usable: One problem is that the algorithm is not deterministic, but is only stochastic, the result found is therefore only with a certain one  Probability correct. Another problem is that the implementation of the Algorithm is not efficient (runtime optimal), so the calculation is slow. It is stated that the method used to solve NP-complete problems (a class of problems for which only deterministic solution algorithms with exponential term are known and are believed to be not efficient deterministic solution algorithms) are particularly well suited because the algorithm due to the parallelism of the executed operations only need linear runtime. This However, reasoning is flawed in that the linear term is exponential Number of molecules is compensated. The exponential problem size remains exist unchanged.

Overall, the method described in WO 97/07 440 is too restricted to be suitable for molecular information processing can be used beyond the described algorithm to be: The Hamiltonian path problem is hard-coded, other algorithms can use it are not calculated, every problem instance has to be recoded. Programming is not provided, all steps of the algorithm are carried out by hand. An input / Output system is not provided. Overall, the procedure for programming Algorithms or not suitable for the implementation of a computer. WO 97/29 117 and [Frank Guarnieri, Makiko Fliss, Carter Bancroft, Making DNA Add, Science, 273, 220-223, (1996)] describe a method of addition using DNA Molecules. The addition takes place as a shift operation with overflow carry in in vitro with DNA molecules and primer elongation.

The described method only describes additions. However, even that Use as an adder is unfavorable for at least two reasons: First, addition is with the method described is not implemented efficiently (optimal runtime). Second is the system used is formally incomplete because of the results generated by addition in turn, can no longer be used as numbers for calculations. About the No additional concepts are available. Overall, the procedure for programming algorithms or to implement a computer.

In [Qi Ouyang, Peter D. Kaplan, Shumao Liu, Albert Libchaber, DNA Solution of the Maximal Clique Problem, Science, 278, 446-449, (1997)] describes a method, a Implement algorithm for solving the "Max Clique problem". The algorithm comes from graph theory and falls under the NP-complete problems. As in [L. M. Adleman, Molecular Computation of Solutions to Combinatorial Problems, Science, 266, 1021-1024, (1994)] the method is only capable of a hard-coded Calculate solution algorithm. Again, the result is only with a certain one Get probability. The procedure described by the authors does not contain any advanced concepts (e.g. with regard to the implementation of other algorithms). Overall, the procedure for programming algorithms or for implementation not suitable for a computer.

In [Erik Winfree, Xiaoping Yang, Nadrian C. Seeman, Universal Computation via Self assembly of DNA: Some Theory and Experiments, Proceedings of the 2nd DIMACS Meeting  on DNA Based Computers, Princeton University, June 20-12, (1996)] is discussed, regular Grammars in DNA by linking oligonucleotides with "sticky ends" to implement. However, the explanations are purely theoretical in nature and fail essential, as yet unsolved problems. In particular, it is not explained how the Implementation of grammars required sequences must be constructed. But this is precisely the crucial problem, without which grammars cannot be solved can be implemented. The reason for this is that the sequences that the Represent variables and terminals of a grammar, both clearly and as well must be sufficiently dissimilar to one another. They also have to be certain meet structural, chemical and physical properties. Otherwise are Incorrect hybridizations between sequences inevitably result in the polymerization lead to unwanted chain extensions and chain breaks and thus the functioning make the procedure impossible. This problem worsens with the number of required sequences exponentially.

The described method is rejected by the authors themselves, or in so far as insufficiently designated as it does not allow any particularly interesting calculations (Erik Winfree, Xiaoping Yang, Nadrian C. Seeman, Universal Computation via Self-assembly of DNA: Some Theory and Experiments, Proceedinsrs of the 2nd DIMACS Meeting on DNA Based Computers, Princeton University, June 20-12, (1996), p. 8, 2nd paragraph, 1st line). Further experiments by the authors are therefore not based on any further regular grammars and linear polymers, but based on context-sensitive grammars Generation of DNA lattice structures [Erik Winfree, Furong Liu, Lisa A. Wenzler & Nadrian C. Seeman, Design and self-assembly of two-dimensional DNA crystals, Nature, 394, 539-544, (1998)]. The approach described by the authors may be to generate DNA based nanostructures (production of catalysts, etc.). For On the other hand, it is not suitable for information processing, here are the generated ones Lattice structures rather a hindrance.

Although the authors speak of the possibility of using the mathematical concept of "Wang tiles" through the generated lattice structures and thus potentially also for To use calculations, but they remain at the mere mention of such Possibility without describing how this is used in terms of information processing can be.

It is doubtful at all whether the approach described by the authors actually becomes one Information processing is suitable: The lattice structures prevent duplication information-carrying sequences (e.g. by cloning or PCR = polymerase chain Reaction = polymerase chain reaction) and make it impossible to generate the structures to be used as data because it can no longer be read out, for example by PCR. Accordingly, the chosen approach does not see any concept in its current form Possibility to use the generated structures in the sense of information processing, such as Data.  

To date, no method is known from the prior art which allows DNA molecules to be used for the implementation of efficient algorithms. No method is known for performing calculations automatically (for example in the manner of a molecular computer). There is also no known method for implementing programs in the form of regular grammars with molecular methods in such a way that

• a) different (ideally any) regular grammars are implemented can
• b) the words of a language generated with regular grammars are read out can
• c) the words of a language generated with regular grammars for technical purposes (e.g. information processing, polymer chemistry) can continue to be used.

The present invention is therefore based on the object of providing a method which allows information processing on a molecular basis using regular Make grammars without being limited to one or a few grammars his. The method is said to be compatible with conventional computers and one Allow information processing, partly on a molecular basis and partly on the Basis of conventional computers takes place. The process is said to be programmable and be largely automated. The polymers produced within the process are said to readable and usable for further applications and processes.

This object is achieved by a method for producing information-carrying polymers, which comprises

• A) a regular grammar G = (Σ, V, R, S) with a finite terminal alphabet Σ, a finite set of variables V, a finite rule set R and one Define start symbol S;
• B) the NFR process (Niehaus-Feldkamp-Rauhe process) for the production of Monomer sequences (oligo- or polymers);
• C) to implement a grammar defined in step I using the NFR method, by producing monomer sequences that the control set R one Display grammar G clearly;
• D) from the monomer sequences prepared in step III for each rule of Standard set R of G an oligomer (algomer) representing the standard assemble (algomer assembly);
• E) the oligomers (algomers) assembled in step IV to be information-carrying Linking polymers (symbol polymerization).

The following definitions and abbreviations are used in the context of the present invention:
Algomer: double-stranded oligomer that represents a rule of a given grammar. Algomers can be linked together to form logomers.
Readout PCR: PCR used to read out the information contained in logomers.
Bit polymerization: process of chaining algomers to logomers if there are only two different elongators.
Elongator: Algomer with two overhang sequences; can ligate with terminator or elongator and leads to a chain extension.
Grammar: formalism that describes languages. It is based on the formal theory of languages [Chomsky, N., Three models for the description of language, JACM, 2: 3, 113-124, (1956)], [Chomsky, N., On certain formal properties of grammars, lnf. and Control, 2: 2, 137-167, (1959)], [Chomsky, N., Formal properties of grammars, Handbook of Math. Psych., 2, 323-418, (1963)].
A grammar G describes a language L (G), the alphabet of this language and its syntax. All words of this language can be generated according to a grammar G.
A grammar G is a quadruple (Σ, V, R, S) with terminal alphabet Z, variable set V, start symbol S and rule set R.
Logomer: polymer that carries symbolic information and was created by the chaining of algomers. Accordingly, a logomer consists of repeating units of algomers. A logomer represents a word of a language L (G) generated by the corresponding grammar G.
Monomer: single molecule. Several monomers can be linked to form longer chains and thus form oligomers and polymers.
In the case of deoxyribonucleic acid, monomers are the nucleotides (adenine, cytosine, guanine, thymine as well as so-called base analogues such as hypoxanthine etc.).
Oligomer: Short-chain molecule made up of repeating units (monomers). Short double-stranded molecules are also called oligomers.
PCR: polymerase chain reaction = polymerase chain reaction; Exponential DNA amplification method.
The PCR requires a DNA template that is duplicated and two primers, each of which starts in the opposite direction in the template and acts as starting points for DNA polymerization. The DNA template is duplicated by iterative repetition of melt hybridization-polymerization cycles.
Polymer: Long-chain molecule made up of repeating units (monomers).
Rule: also: production rule, replacement rule or derivation rule.
Describes the replacement of symbols by others. By repeatedly applying rules, symbol chains can be created.
Sequence: computer science: sequence of characters;
Chemistry: sequence of covalently linked monomers;
Molecular biology: sequence of covalently linked nucleotides.
Complementarity: Two sequences are complementary if they can hybridize with each other. In the case of DNA, e.g. B. the sequences 5'attt3 'and 5'aaat3' complementary, the sequence 5'acgt3 'is complementary to itself.
Start symbol: Variable within a grammar, from which a symbol chain can be generated by applying the rules of the grammar.
Symbol polymerization: Process of chaining algomers to logomers.
Terminal: symbol of a grammar. A terminal can no longer be replaced. Terminals are the "letters" of the words of a language L (G).
Terminator: Algomer with an overhang sequence. Can only ligate with elongator. Leads to chain termination during symbol polymerization.
Variable: symbol of a grammar. A variable can be replaced according to the rule of a grammar of terminals, variables or combinations of terminals and variables.

Brief description of the pictures

Fig. 1: Algomers, as described in the explanation of step IV of the method according to the invention. The algomers each have a double-stranded core sequence that represents a terminal of a given grammar and at least one single-stranded overhang sequence that represents a variable of the given grammar, so that each algomer represents exactly one rule of the given grammar. X and Y are not variables, but overhang sequences that e.g. B. can be used for cloning. Letters with overlines indicate complementarity. By definition, A0A and A1A are elongators, XsA and AeY terminators. The algomers shown here represent the grammar described below for generating binary random numbers.

Fig. 2: Symbol polymerization, as described in the explanation of step V according to the invention. Algomers are linked to logomers by chaining (in the case of DNA: hybridization and ligation). The logomer Xs01010101eY contains a terminated bit sequence, which can be reproduced in a defined orientation by the overhangs X and Y.

Fig. 3: Pattern showing symbol polymerization for binary random numbers after gel electrophoresis and staining (lanes 1-3 ). Due to the random length of the binary random numbers, there is a regular conductor pattern. Lane 4 shows a 50 bp molecular weight standard (Gibco BRL, Life Technologies, Catalog No. 10416-014).

Fig. 4: Cloning of a logomer obtained by symbol polymerization in a vector, as described below for the amplification and isolation of logomers.

Fig. 5: Schematic representation of a band pattern which is obtained by reading out a binary logomer by PCR and subsequent gel electrophoresis (described below). In the example shown, the algomers have a length of 30 bp (overhang sequences calculated only ½). If the length of the logomer is known, it is sufficient to read out only 0s or only 1s. Reading out both bits is for control purposes. The process can also be used for multivalent logomers (more than 2 elongators).

Fig. 6: Band pattern of a gel electrophoresis after PCR for reading out three different logomers. The logomers were obtained according to the grammar described below for random numbers of any length. Read as random numbers from bottom to top is a = 262, b = 97, c = 329. M (lane 5 ) is a 50 bp molecular weight standard (Gibco BRL, Life Technologies, Catalog No. 10416-014).

Fig. 7: Schematic representation of reading out logomers by restriction digestion as described below. The elongators carry restriction sites which are arranged asymmetrically in such a way that the restriction digestion of logomers results in a clear cutting pattern of fragments. The pattern corresponds to bands of different lengths and can be made visible by gel electrophoresis. R1 and R2 are different restriction enzymes, x and y denote the length of the fragments obtained after restriction digestion. Appropriately z. B. a ratio x: y of 1: 2.

Fig. 8: Band patterns obtained by gel electrophoresis after reading out logomers by restriction digestion. Lanes 7 and 8 contain molecular weight standards (lane 7 : 50 pb ladder, Gibco BRL, Life Technologies, Catalog No. 10416-014; lane 8 : 10 bp ladder)

Fig. 9: Schematic representation of a polymeric data storage medium based on algomers and symbol polymerization as described in the following. Algomers can polymerize on anchor molecules (AM) bound to a solid support (C). The writing (W) is carried out by the repeated sequence (ReW) of hybridization-ligation restriction cycles (Hyb, Lig, Res). The logomers obtained can be separated and read out by denaturing or restriction (Den / Dig).

Fig. 10: Simplified representation of the components of a DNA desktop computer as described below. The details are:
A: oligo synthesizer, B: thermal cycler, C: reaction chambers, D: pipetting device, E: gel,
F: scanner, G: control computer

Characterize it:
1 : addition of oligonucleotides, 2 : addition of synthesized oligomers in reaction chambers of the thermal cycler, 3 : addition of solutions and molecules which are required for algomer assembly, symbol polymerization, for the isolation of logomers and for reading.

Fig. 11: Encryption of logomers as described below. If the terminators and the primers priming in them are unknown, the respective logomer is encrypted and cannot be read out (A). If, on the other hand, a primer priming in a terminator is available or the sequence of a terminator is known, the respective logomer can be read (B).

Fig. 12: Y-shaped molecule that can be used as a terminator for the asymmetric encryption of logomers. Elongators can be attached to the end marked with 2 , others, e.g. B. Y-shaped, molecules at the ends labeled 1 and 3 . If several Y-shaped molecules are linked together, tree-like structures are obtained.

Fig. 13: Logomer with terminators s and e, which are implemented as tree-like structures.

Fig. 14: Logomer L linked with a vector V with tree-like terminators. The terminators are designed so that only one branch of the terminator can be linked to the vector.

Fig. 15: Labeling of nucleic acids with logomers as described below: In order to label genetically engineered or modified products, a logomer is inserted into a non-transcribed area, e.g. B. before the promoter (P) of a gene to be labeled (G) used.

Fig. 16: Marking documents with logomeres as described below. Lane 1 shows the molecular weight standard used (50 bp, Gibco BRL, Life Technologies, catalog No. 10416-014), lane 2 (0-bits) and lane 3 (1-bits) the band pattern of reading out logomer No. 330 by PCR from aqueous solution, lanes 4 and 5 are the same as lanes 2 and 3 , but here logomer no. 330 is not dried from aqueous solution but in approx. 10 ⁹ molecules / µl dried on paper (3M Postlt, 1 hour dried) was used as a piece of paper (approx. 1 mm ² ) in the readout PCR.

Fig. 17: Preparation of molecular weight standards by readout PCR, which contains a unary logomer as a template, as described in the following. 1 shows the arrangement of nested primers in the readout PCR, FIG. 2 shows the band pattern obtained after gel electrophoresis of the PCR fragments.

Fig. 18: Gluing surfaces with nucleic acids as described below. C denotes the surface to be glued, Log the logomeres that are bound to the surfaces for gluing. 1 shows the behavior of surfaces with non-complementary, 2 the behavior of surfaces with complementary logomers.

Fig. 19: Gluing surfaces with logomers, antibodies and ligands as described below: C ₀ and C ₁ denote the surfaces to be glued, which can consist of the same or different material. The surfaces to be glued are offset with logomeres (log). Proteins, e.g. B. Bind antibodies of type Ab A and Ab B, where Ab A and Ab B can be the same or different. From A and Ab B are in turn connected to one another by a ligand (Li).

Fig. 20: Bonding surfaces with proteins e.g. B. Antibodies without the use of logomers. C ₀ and C ₁ denote the surfaces to be glued, which can consist of the same or different material. The proteins of type Ab A and Ab B bind directly to the surfaces to be glued and are in turn connected to one another by a ligand (Li).

Explanation to I Choice of grammars

A grammar is a quadruple G = (Σ, V, R, S) with a finite terminal alphabet Σ, a finite set of variables V, a finite set of rules R and a start symbol S. For a language L (G) described by G, all words from L can be words be formed over the terminal alphabet Σ by using the start symbol S accordingly derives the rules from R.

The definition of a grammar G after step I of the method according to the invention is free in that any finite sets of terminals and variables can be encoded. According to the invention, however, the definition of grammars which allows the binary representation of data, in particular of grammars with, is preferred

Σ: = {0, 1, s ₀ , s ₁ , s2,. . ., s _n-1 , s _n , e ₀ , e ₁ , e _2,. . ., e _m-1 , e _m } with n, m ∈ N and n, m ≧ 0.

These enable the production of binary logomers.

For illustration purposes, a regular grammar defined in accordance with the invention for generating random numbers of any finite length is shown in binary form below:
Let there be a grammar G = (Z, V, R, S) with terminal alphabet Σ: = {0, 1, s, e}, set of variables V: = {A}, start symbol S and rule set) R: =
{
S: = sA
A → 0A
A → 1A
A → e
},
in which
s: = start
e: = end
are.

With this grammar, words can be placed above the terminal alphabet

Σ: = {0, 1, s, e}

be formed. All words of the language L begin with "s" and end with "e", in between there is a random number (also 0) of random bits ("0" and "1"). Words from the language L, which are formed after R, are for example:

• a) S → sA → se  
• b) S → sA → s1A → s1e
• c) S → sA → s0A → s00A → s001A → s0010A → s0010e
• d) S → sA → s1A → s10A → s100A → s1000A → s10000A → s100000A → s100000e

The binary patterns generated as words of language L can be any, but unambiguous, representation of data types, e.g. B. read as letters, numbers, alphanumeric characters or strings. If you read them as the binary representation of numbers, the above words are in decimal notation:

• a) ∅ (empty)
• b) 1
• c) 2
• d) 32.

Explanation to II The NFR process for the production of monomer sequences

The NFR process (Niehaus-Feldkamp-Rauhe process) is a process for the production of monomer sequences (oligomers and polymers, e.g. nucleic acids), which ensures that the monomer sequences produced using the process:

• a) have a unique and unique sequence of monomers;
• b) are as similar as possible to one another and therefore, if possible, no incorrect hybridizations enter into one another;
• c) have certain structural properties, e.g. B. a certain ratio of different monomers to each other, being included or not certain partial sequences and maximum agreement (homology) among themselves;
• d) have certain chemical properties, e.g. B. the occurrence of certain chemically active partial sequences that influence chemical reactions, in the case of Nucleic acids in particular interact with proteins (Protein binding sites, restriction sites, stop codons);
• e) have certain physical properties, e.g. B. a specific Melting temperature.

The NFR process allows the production of clear, possibly dissimilar Monomer sequences with predefinable structural, chemical and physical Characteristics. It is suitable for the production of monomer sequences for controlled chemical reactions such as z. B. needed for molecular information processing become. It is in step III of the inventive method for implementation regular grammars are used.

The NFR method is a further development of the Niehaus method according to the invention Generation of clear, possibly dissimilar sequences, that in [Jens Niehaus, DNA  Computing: Evaluation and simulation, diploma thesis at the computer science department of the University of Dortmund, Chair XI, 116-123, (1998)], and what herewith in full reference is made.

The NFR process is a further development of the Niehaus process according to the invention, since only the NFR process allows the production of monomer sequences, such as those required for production in vitro, e.g. B. to implement grammars. The reasons are:

• a) The Niehaus method only describes the construction of sequences Base sequences of length 6. Since the length of base sequences is the maximum between different monomer sequences describes possible overlaps, therefore direct influence on the hybridization behavior of sequences and that Functioning of steps IV and V according to the invention, the method must, however, for Base sequences of any length can be generalized, which in the NFR method was made.
• b) The Niehaus method only allows the generation of sequences of the same length, whereas the NFR method sequences of different lengths and different Number can generate. The latter is for a correct hybridization behavior of Sequences and the functioning of steps IV and V according to the invention are essential.
• c) The Niehaus method ensures uniqueness and maximum contrast between Sequences only for sequences created by a single application of the procedure were generated. However, it is imperative to include existing sequences each compatible, d. H. generate clear and maximally dissimilar sequences can. In contrast to the Niehaus method, the NFR method can be too arbitrary predefined sequences compatible (i.e. unique and maximally dissimilar) new ones Generate sequences. The method can be applied to the same amount of as often as required Sequences can be applied.
• d) The Niehaus method limits the maximum length of common Partial sequences, but not their number. Therefore, any 2 can be Process constructed sequences contain several common partial sequences, what can unintentionally lead to high homology (sequence agreement). The Homology in turn has a direct influence on the hybridization behavior of Sequences and the functioning of steps IV and V according to the invention. By contrast, a method also leads to the construction of sequences Homology comparison and thereby avoids unwanted incorrect hybridizations.
• e) The Niehaus method does not allow the integration of certain, predeterminable sequences and partial sequences. However, such sequences are for execution and control certain chemical reactions, e.g. B. controlled enzymatic interactions, mandatory. Examples of such sequences are in the case of nucleic acids Protein binding sites, restriction sites and stop codons. In contrast, this allows NFR process the integration of any sequences and partial sequences in the production  of monomer sequences while ensuring uniqueness and maximum Dissimilarity.
• f) The Niehaus method does not allow for sequences with certain to generate structural, chemical or physical properties. On the other hand does the NFR method contain the possibility of sequences with certain structural, manufacture chemical and physical properties. These include u. a. the Ratio of different monomers to each other and the melting temperature. This too is an essential requirement for the implementation of grammars in vitro.
• g) The Niehaus method does not allow direct generation of rule-representative ones Sequences as needed to implement grammars. On the other hand enables the NFR method to generate symbol-representative sequences that to the rule-representative required for the implementation of a grammar Sequences can be linked.
• h) Sequences can only be constructed using the NFR method so that the Characteristics of uniqueness, maximum dissimilarity as well as structural, chemical and physical properties also apply to partial sequences. For example, in the case of nucleic acids to produce monomer sequences so that both the entire sequence and also z. B. the first third of the sequence have a 50% GC content. This Property is e.g. B. for the functioning of the concatenation of sequences per molecule are intended for multiple hybridization events (such as those below Algomers described), an unconditional requirement. This is the only way B. Construct sequences so that different hybridization events per molecule are equally likely.
• i) Only the NFR process allows the immediate, automatic production of Monomer sequences, e.g. B. by an oligonucleotide synthesizer.

To produce monomer sequences, these are constructed and synthesized using the NFR method. To prepare n monomer sequences, the NFR process is carried out as described below, the following abbreviations being used to describe the process:
A: = alphabet of thickness a ∈ N;
In the case of DNA, A: = {a, c, g, t} and a = 4.
S _i : = sequence.
Sequence of elements of the alphabet A, which corresponds to a sequence of monomers.
I _seq : = length of the sequences to be constructed per process cycle.
S _seq : = sequence of length I _seq .
S _seq.k : = (k-1) th monomer of the sequence S _seq (k ∈ N k <= I _seq ).
I _bas : = length of the base sequences used for the construction of sequences per process cycle.
The following applies: 0 <I _bas <= I _seq .
S _bas : = sequence of length I _bas base sequence; Sequences of length I _seq are constructed from basic sequences.
I _ov : = maximum length of the chain of successive monomers, each of which has two sequences generated by the process in common ("overlap").
I _bas - 1.
I _ov / I _seq = ratio of the maximum permitted sequence repetition to total sequence length;
1. Measure of the probability of incorrect hybridization.
I _bas / I _seq = ratio of base sequence length to total sequence length;
2. Measure of the probability of incorrect hybridization.
M _seq : = set of sequences.
M _bas : = set of all base sequences of length I _bas .
M _nobas : = subset of the set of base sequences of length I _bas that would be obtained from M _seq by decomposition.
| M _nobas |: = thickness of M _nobas = number of base sequences in M _nobas .
n: = number of sequences to be constructed per process cycle.
n _tot : = total number of sequences produced in all traversing cycles.
n _max : = maximum number of sequences that can be constructed per process cycle.
h: = homology. A measure of the match between two sequences.
The following applies: 0 <= h <= 1.
t _m : = melting temperature. For a DNA sequence, the melting temperature is determined by the nearest neighbor method (see Breslauer, KJ, Frank, R., Blocker, H., Marky, LA, Proc. Natl. Acad. Sci., 83, 3746-3750, 1989) certainly:
t _m = ΔH / (ΔS + R × In (C / 4)) - 273.15 ° C
ΔH and ΔS are the enthalpy and entropy of the DNA helix, R the molar gas constant and C the concentration of the DNA sequence.

The production process is carried out as a sequence of any, but finitely many, process cycles in which n _dead sequences are constructed and a subsequent synthesis in which all n _dead sequences are generated in vitro.

The maximum number n _{max of} the sequences that can be constructed per process cycle is calculated according to the definitions given above

n _max = (a, I _bas , I _seq ): = ∟ (½. ((a ^Ibas - (a ^{Ibas / 2} - | M _nobas |)) / (I _seq - I _ov ) ┘.

That is, a maximum of n _max sequences of length I _{seq can be constructed} from elements of an alphabet A of size a (in the case of DNA a is: - 4), which correspond in chains of monomers that are connected in a maximum of I _ov . The number of sequences actually obtained per process cycle can be lower if they are to have certain physical, chemical or structural properties, or if additional sequences are produced in addition to existing sequences.

The following procedural steps are necessary:

• 1. n _tot sequences are constructed in z process cycles and collected in a quantity M _seq . You start with the empty sequence set M _seq .
• 2. Any, but unambiguous, sequences can be added once to the sequence set M _seq before the start of the process cycle. This can be useful to add certain sequences that should be included in the amount of monomer sequences produced for further use. It is advisable not to add too many and not too long sequences, since the procedure does not guarantee the same characteristics for these sequences as for the sequences constructed below.
• 3. Beginning of the process cycle: The structural, chemical and physical properties that the sequences to be produced must fulfill are specified. Structural properties are at least the presence or absence of certain partial sequences (possibly also the positions at which the partial sequences are or should not be contained in the sequences to be generated) and the ratio of the number of different monomers to one another (in the case of DNA: GC / AT ratio). Chemical properties are at least the occurrence of certain partial sequences which have an influence on the chemical interaction with own or other substances (in the case of DNA e.g. certain protein binding sites, restriction sites such as 5'gatatc3 'for EcoRV, the stop codons 5' tca3 ', 5'tta3', 5'cta3 'etc.). The physical properties to be determined include at least the melting temperature t _{m of} the sequences to be produced.
• 4. The number of sequences n <= n _max desired per cycle is now determined, according to the above formula I _{seq of} the sequences to be constructed and I _bas of the basic sequences required for the construction. I _seq and I _bas are chosen according to the formula given above so that the desired number n sequences to be constructed can be achieved. Since I _bas / I _{seq is} proportional to the probability of incorrect hybridization, I _bas and I _seq are chosen so that the ratio I _bas / I _{seq is} as low as possible (values below 0.3 are recommended) and values for I _seq guarantee good hybridization conditions. Depending on the temperature, any values for I _seq <0 are possible for DNA.
• 5. The set M _{bas of} all sequences of the alphabet A of length I _{bas is} generated. The sequences generated in this way are referred to as base sequences. There are always a ^Ibas base sequences. These basic sequences serve to construct the sequences to be produced. Each base sequence is assigned a status of "used" or "unused". First, all are marked as unused.
• 6. Self-complementary base sequences from M _bas are now marked as used. There are exactly a ^{Ibas / 2} basic sequences complementary to themselves.
• 7. If sequences already exist in the set M _seq , new compatible sequences can either be constructed or the sequences of a subset of M _{seq can be} extended in a compatible manner. For this purpose, a set M _{nobas of} all partial sequences of the length I _{bas of} the sequences from M _seq specified in step 4 is formed from the already existing sequences from M _seq within a decomposition process:
  Set M _nobas = {}.
  Each sequence S _seq from M _seq is now broken down into (I _seq - I _ov ) decomposition steps:
  Start with i = 0 with the decomposition step:
  As long as i <(I _seq - I _ov ):
  Form as new sequence S _new as a sequence of length I _bas consisting of the monomers S _seg.i to S _{seq, i + Ibas} : S _new : = S _{seq, i ,.} . ., S _{seq.i + Ibas} . Add S _to the set M _nobas .
  Set i: = i + 1.
• 8. The set M _nobas obtained in this way is by definition a subset of M _bas .
• 9. All base sequences of the set M _bas , which also occur in M _nobas , are marked as used, so that exactly unused base sequences are left from which to construct sequences (a ^Ibas - a ^{Ibas / 2} - | M _nobas |) now maximum
  Various new sequences can be constructed that do not have any base sequences or their complements in common with each other and with the existing sequences.
• 10. A directed graph is constructed from the base sequences, the nodes of which represent certain base sequences: The graph contains exactly a ^Ibas nodes, of which (a ^{Ibas / 2} + | M _nobas |) nodes are already marked as used. Each node is associated with a base sequence S _{b (i)} that is not complementary to itself (furthermore, there will be no distinction between nodes and associated base sequence). There is an edge from S _{b (i)} to S _{b (k)} if the I _ov last letters of S _{b (i )} correspond to the I _ov first letters of S _{b (k)} , so if:
  S _{b (i), 2,.} . ., S _{b (i), Ibas} = S _{b (k), 1 ,.} . ., S _{b (k), Ibas-1} . The node Sb ~ (k) is called the successor node of Sb (i). The node associated with the complement of the base sequence encoded by node S _{b (i)} is called the complement node to node S _{b (i)} . Sequences of length I _seq are found by searching for a path with (I _seq - I _ov ) nodes. Each node can only be used once. In addition, for each node that lies in one of the paths, the complement node must not appear in any path. Like the sequence, the start node of the path has a status of "used" or "unused".
• 11. Sequences are constructed from the graph using the following procedure:
  Mark all unused nodes in the graph as unused starting nodes. For every s between 1 and n:
  As long as node S _{b (k)} exists that has not yet been marked as the used starting node:
  Select an unused node S _{b (k)} .
  Mark node S _{b (k)} as the used starting node; Moreover:
  Mark sequence S _{b (k)} and its complement as used.
  Now a new sequence S is _newly constructed, the new element for M _seq or an existing _sub sequence S of M _seq extended:
  Set S _{new, o} : = S _{b (k), 1} .
  If it is constructed as a new sequence, set i: = 0.
  If it is constructed as an extension of an existing sequence S _sub , set i: = I _sub .
  As long as i <I _seq - (I _ov -1) and i <= 0:
  If there is no unused successor node S _{b (m)} of node S _{b (k)} , then mark node S _{b (k)} and its complement node as unused and set i: = i-1.
  Otherwise, randomly select an unused successor node S _{b (m)} from node S _{b (k)} and mark b _m and its complement node as used. Also set: i: = i + 1, k: = m, S _{new, i} : = S _{b (m), 1} .
  If i = I _seq - (I _ov -1), the sequence S is _newly completed: it consists of the letter S _{new, 0} to S _{new (ISEQ - ov).}
• 12. For each of the sequences obtained in step 10 , the respective structural, chemical and physical properties are determined in comparison to the conditions specified in step 3 . If the respective sequence does not meet the specified conditions, it is deleted and the nodes and complement nodes used by it are again marked as unused.
• 13. If the number of sequences constructed is insufficient, the process cycle from step 3 or 4 can be repeated any number of times. In particular, it is possible to reset the structural, chemical and physical criteria and the values for n, I _seq , I _{bas for} each process cycle. In this way it is possible to construct sequences with different structural, chemical and physical properties, as well as different lengths, which are nonetheless compatible, i.e. unambiguous and maximally dissimilar. Otherwise you go to the next step, the in vitro synthesis.
• 14. The constructed sequences are preferred in vitro, in the case of nucleic acids by means of an oligonucleotide synthesizer (e.g. ABI 392, ABI 398, ABI 3948 from Perkin- Elmer Applied Biosystems), synthesized. The sequence data of the Control unit of the oligonucleotide synthesizer transmitted and the sequences as single-stranded nucleic acids. The sequences can also be commercial be ordered. PAGE-purified oligonucleotides (e.g. from ARK Scientific GmbH Biosystems, 64293 Darmstadt, available).

Explanation to III Implementation of regular grammars with the NFR method

The production of monomer sequences to represent the regular set of grammars in step III of the method according to the invention is carried out using the NFR method (Niehaus-Feldkamp-Rauhe method) after step II of the method according to the invention. For the implementation of grammars according to the NFR procedure for r rules Grammar G produced exactly 2r monomer sequences, so that the monomer sequences for the s symbols shown (terminals and variables) and the rules R of the grammar G clearly and. are as different as possible from each other, as well as the required structural, have chemical and physical properties.

The monomer sequences are prepared according to the INFR process so that they are like described below to form algomers. It will be there each composed 2 monomer sequences to an algomer, which exactly one rule R represented a grammar G. Both contain monomer sequences  Sequences containing the related symbols required by R rule (Terminals or variables) included.

The design of the oligomers after step III of the process according to the invention depends on the chemical nature of the monomers used. In the preferred case of the oligomers constructed from nucleotides, the procedure is as follows:
Algomers are double-stranded and have a 5 ″ strand (top strand) and a 3′-strand (bottom strand). The top strand and the bottom strand can be the linkage of a terminal sequence and a variable sequence.

Let X, Z be any variables, S be a variable that acts as a start symbol, y, z be any terminal symbols. Then for each rule the form is:

• a) S: = yX constructed an algomer that contains a unique double-stranded core sequence y to which a unique single-stranded overhang sequence X is attached to the 3 'end; ": =" means in this case that S and yX are identical, i. H. yX acts as a starter molecule.
• b) X → yZ constructed an algomer that contains a unique double-stranded core sequence y to which a unique single-stranded overhang sequence X at the 5 'end and one at the 3' end unique single-stranded overhang sequence Z is attached. X can be identical to Z his.
• c) X → z constructed an algomer that contains a unique double-stranded core sequence z to which a unique single-stranded overhang sequence X is attached to the 5 'end.

Algomer of the form S: = yX and X → z are called term forums ("start", "end"), because they are for Chain termination during the polymerization in linking step V according to the invention lead, which is described below; Algomers of the form X → yZ are called elongators, because they lead to a chain extension during the polymerization in the invention Link step V lead.

Preferably include those in steps II and III of the method according to the invention prepared monomer sequences nucleotides, especially ribonucleotides, particularly preferably deoxyribonucleotides. The in step II of the method according to the invention Constructed monomer sequences can advantageously certain sequences, e.g. B. Stop codons, recognition sequences for enzymes that cleave nucleic acids (Restriction nucleases), recognition sequences for nucleic acid binding proteins and the like. a. contained, as for example in [J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning, A Laboratory Manual, (1989)], [Rolf Knippers, Molecular Genetics, Georg Thieme Verlag, (1997)] and [Benjamin Lewin, Genes V, Oxford University Press, (1994)] become.  

Explanation to IV Algomer assembly

The sequences synthesized in step III are assembled in step IV of the method according to the invention. In the context of the present invention, all of the techniques known to those skilled in the art of assembling oligomer sequences can be used to carry out step IV. In the preferred case according to the invention of using nucleotide sequences, it is advisable to proceed as follows:

• a) The single-stranded sequences belonging to elongators are phosphorylated. Various protocols known to the person skilled in the art are possible for this purpose, e.g. B. the following:
  In a 20 µl batch, 16 µl of a synthesized 100 µM sequence, 2 µl ligation buffer (e.g. 50 mM Tris-HCl pH 7.5, 10 mM MgCl ₂ , 10 mM dithiothreitol, 1 mM ATP, 25 µg / ml BSA Bovine Serum Albumin, New England Biolabs) and 2 μl PNK (polynucleotide kinase, e.g. from New England Biolabs, catalog number # 201 S or # 201 L) were incubated at 37 ° C. for 1 hour. Volumes, incubation time and temperature can be varied in a manner known to the person skilled in the art.
• b) In one batch, the single strands (upper and lower strand) belonging to an algomer are hybridized to a finished algomer. The phosphorylation batches from the upper and uniler strands can simply be used for elongators. Several protocols are possible for hybridization; Preferred is a denaturation at about 95 ° C at the beginning (this deactivates the PNK in the approaches of the elongators at the same time) and a slow hybridization. For example, the following protocol can be used for oligomers of length 30, which can be varied according to the knowledge of the person skilled in the art:
  In a 40 µl batch, 20 µl of the top strand (100 µM) and 20 µl of the bottom strand (100 µM) are heated in a thermocycler for 5 minutes at 95 ° C, incubated for 5 minutes at 72 ° C and then 25 ° C for 1 minute cooled down per minute.

Explanation to V Production of logomers by chaining algomers Symbol polymerization

In step V of the process according to the invention, the algomers become longer-chain, linked information-carrying polymers (logomers). Since algomers are the rule set R represent a grammar G, all logomer words generated thereby correspond to Language L (G), which is described by the grammar G.

The regulated process of linking algomers to logomers is referred to as "symbol polymerization" if the terminal symbols represent bits, that is, if:

Σ: = {0, 1, s ₀ , s ₁ , s2,. . ., s _n-1 , s _n , e ₀ , e ₁ , e2,. . ., e _m-1 , e _m } with n, m ∈ e N and n, m <0

the process is referred to as "bit polymerization".  

In the context of the present invention, all techniques known to those skilled in the art to link oligomers to polymers can be used to carry out step V. In the preferred case according to the invention when using oligonucleotides, it is expedient to incubate the algomers to be linked in the presence of ligase. For example, the following protocol can be used, which can be varied according to the knowledge of the person skilled in the art:
In a 27 µl batch, 1 µl 50 µM "start" algomer, 1 µl 50 µM "end" algomer and 2 × 10 µl 40 µM elongator algomers (obtained from step IV of the method according to the invention), 1.5 µl 10 mM rATP and 3.5 µl T4 DNA ligase with 400 NEB units / µl (e.g. catalog numbers # 202S and # 202L NEB) incubated between 4 ° C and 25 ° C for 2 to 24 hours. Other reaction volumes work analogously. Incubation time and temperature can be varied in a manner known to those skilled in the art.

Fig. 3 shows the result of such symbol polymerization.

In principle, any number of algomers that are not terminators can be linked. The polymerization process then occurs for each individual logomer a standstill when the respective molecule carries an algomer at its ends, that acts as a terminator molecule ("start", "end").

Separation and duplication of logomers by cloning

Another object of the present invention is a method for isolation and Reproduction of information-bearing polymers according to one of the preceding Claims were obtained which are characterized in that obtained in step V. information-carrying polymers ligated into cloning vectors, competent cells with them Transformed vectors and the successfully transformed bacteria using Selection markers selected.

The from step V of the method according to the invention described above (Symbol polymerization) obtained logomers can be used for the purpose of separation and Duplication to be cloned.

For this purpose, the cloning vector used is cut open with restriction enzymes and a Logomer ligated into it. The process ensures that only fully polymerized (with Terminators) can be ligated to the target vector. Correct in the Logomers ligated to the target vector form a ring-shaped molecule again. The separation of Logomeres occur through the transformation of bacteria with the mixture of molecules that the The result of the ligation is. The vectors carry selection markers (preferably antibiotic Resistance e.g. B. ampicillin resistance) with which successfully transformed bacteria can be selected. Since each bacterium expresses only one plasmid, each is successfully transformed bacterium carrier of exactly one logomer. Such cloned Logomers can then be used easily and in large quantities (on solid or reproduced in liquid medium, fermenters).  

The logomers obtained from step V of the procedure according to the invention become Purpose of isolation and duplication in any cloning vector (Plasmid) cloned, which carries restriction sites which depend on the sequence of the Terminators of the logomers are compatible (e.g. HindIII, BamHI recognition sequences in the Cloning vector pBluescript II KS +/-, Stratagene, catalog no. # 212207). The cloning takes place according to usual laboratory protocols, such as. B. in [J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning, A Laboratory Manual, (1989)]. For example, the following protocol can be used, which can be varied according to the knowledge of the person skilled in the art:

a) Restriction and preparation of the cloning vector

Arbitrary cloning vectors can be used for cloning, as are common for molecular biological methods. (The plasmid pBluescript II KS +/-, Stratagene, catalog no. # 212207 is suitable, for example). The plasmid is subjected to a restriction digest with two restriction enzymes that produce overhangs that are compatible with the terminators of the logomers. As restriction enzymes z. B. BamHI and HindIII (e.g. from NEB, New England Biolabs, Catalog #: # 136S and # 104S) can be used. A conventional restriction protocol can be used for this, which can be varied according to the knowledge of the person skilled in the art, e.g. B. the following:
In a 50 µl batch, 30 µl plasmid (0.7 µg / µl), 5 µl 10 × buffer (e.g. NEB2, New England Biolabs), 5 µl BamHI (100 units), 5 µl HindIII (100 units) and Spl BSA incubated 10 × for 1-2 hours at 37 ° C. As a control, 1 μl of the mixture and a corresponding amount of uncut plasmid are electrophoresed on a 1% agarose gel (eg UItraPure ™ from Gibco BRL, Life Technologies, catalog no .: 15510-027). To prepare the DNA, the restriction mixture is phenolized, the DNA is precipitated and resumed:

• - Mix to 200 µl with distilled water. fill up
• - Add 200 µl phenol, vortex, centrifuge at 10000 g for 3 minutes
• - Take up the supernatant and add 200 µl chloroform, vortex, at 10000 g for 3 minutes centrifuge
• - Absorb the supernatant, acidify with 1/10 vol. 3M NaAc and with 2.5 × volume 100% Add EtOH, vortex, for at least 15 minutes at -70 ° C
• - Centrifuge at 10,000 g for at least 15 minutes, remove and discard the supernatant
• - Wash with approx. 500 µl 70% EtOH, centrifuge at 10,000 g for 5-10 minutes, supernatant remove and discard
• - Dry the pellet and soak in aqua dest. resume so that the cut plasmid with 100 ng / µl to 1 µg / µl is present (higher concentrations are also possible). Plasmid can be used for further ligations are diluted if necessary.

b) Ligation of the logomer into the cloning vector

The logomers obtained in step V of the method according to the invention and the prepared cloning vectors are ligated. The manner in which the preparation is carried out ensures that, if possible, only the desired ligations take place: the terminators of the logomers carry two different overhang sequences which are compatible with the overhang sequences of the cloning vector which were created by restriction. As a result, the logomers can only ligate into the cloning vector in a defined direction. In addition, the terminators of the logomers are not phosphorylated, which is why logomers can only ligate with the overhang sequences of the cloning vector. The ligation is carried out according to a customary ligation protocol such as e.g. B. in [J. Sambrook, EF Fritsch, T. Maniatis, Molecular Cloning, A Laboratory Manual, (1989)], which can be varied according to the knowledge of the person skilled in the art, for example as follows:
The molar ratio of logomers / cloning vector can e.g. B. 500, in 10 µl total volume:

1 µl plasmid (10 ng / µl = 5 nM)
0.5 µl 4 µM logomers from step V of the method according to the invention
1 µl 10 × ligation buffer
0.5 µl T4 DNA ligase (400 u / µl)
7 µl H ₂ O

ligated to 16 ° C for 12 hours.

The protocol can be varied according to the knowledge of the person skilled in the art. For example, it is possible a protocol for TCL (Temperature Cycle Ligation, [Lund, A. H., Duch, M., Pedersen, F. S. Increased cloning efficiency by temperature-cycle ligation, Nucleic Acids Research, 24: (4), 800-801, (1996)]).

The ligation mixture obtained is now used for the transformation of competent cells, as shown below as an example:

c) transformation of competent cells

The transformation of bacteria (competent cells, host strain e.g. dH5α, GIBCO BRL, catalog no .: 18258-012) with the ligation approach from b) is carried out according to one of the usual protocols, such as B. in [J. Sambrook, E. F, Fritsch, T. Maniatis, Molecular Cloning, A Laboratory Manual, (1989)] and which can be varied according to the knowledge of the person skilled in the art, e.g. B. as follows:

• - 200 µl of competent cells (∿ 5 × 10 ⁷ CFU = Colony Forming Units, stored at -70 ° C) are thawed and placed on ice
• - About 1 ng of the ligation mixture from b) is pipetted into this and placed on ice for 20-30 minutes let stand, mix gently every 5 minutes
• - Heat shock: batch at 42 ° C for 2 minutes, back on ice
• - Add 0.8 ml LB medium (+ 0.02 M MgSO ₄ + 0.01 M KCl)
• - Roll the batch in a test tube at 37 ° C for 20-60 minutes
• - Plate 0.1-1 ml on an agar plate (with antibiotic, e.g. ampicillin) and overnight to 37 ° C

The transformation acts as a selection process for successfully cloned logomers and as an isolation process for individual logomers, because each bacterial cell has exactly one plasmid expressed.

In addition to the mentioned cloning method, which is preferred according to the invention, can the information-bearing ones obtained in step V of the method according to the invention Polymers (logomers) can be isolated and reproduced using the following methods:

Isolation through hybridization and chromatographic methods

Here, the molecular mixture obtained from a symbol polymerization is passed over a carrier material to which oligomers ("anchor sequences") are bound, which in turn can bind to the terminators of the logomers. The bound logomers are then taken up individually and can be reproduced as required. Various methods can be used for this:

• a) affinity chromatography; thereby anchor sequences become the overhanging ones Ends of terminators have compatible ends, e.g. B. on a hydroxyapatite column bound. The logomers obtained from step V of the process according to the invention are passed over the column, whereby the logomers are hybridized to the Can tie anchor sequences. The logomers bound in this way then become recorded individually.
• b) anchor sequences are attached to a membrane (e.g. Gene Screen Plus, DuPont, Biotechnology systems; Hybond-N, Amersharn Life Sciences) covalently bound. The Membrane is rasterized, i. H. divided into individual fields. Exactly one is per field Anchor sequence covalently bound to the membrane. Then the from step V of the Logomers obtained according to the invention given over the membrane and hybridized with the anchor sequences. The membrane is then in the individual fields of the grid cut. The individual fields, which are now a maximum of one for each Anchor sequence containing bound logomer can now be placed in separate vessels (e.g. Eppendorf Tubes) are transferred. The logomers can then be denatured be separated from the membrane. When denaturing, please note that the Denaturation temperature is chosen so that it is high enough to logomer and Separate anchor sequence, but not so high that the logomer itself is complete melts.

Isolation by dilution

In the case of isolation by dilution, the logomers, which are obtained as a mixture from a symbol polymerization, are diluted to such an extent that exactly one molecule is statistically contained in a respective target volume. This can then be detected and reproduced using the PCR. The dilution of the initial volume in target volumes can take place simultaneously, so that an initial volume is diluted to n target volumes. To this end, the procedure according to the invention is as follows:
Obtained at a, from step V of the method according to the invention, mixture of Logomeren in an initial volume V _a is determined a dilution factor of n in V _a given Logomere so diluted to n target volumes V _z, that in each target volume V _z statistically exactly one Logomer contain is. To determine the dilution factor, an aliquot of the initial volume (e.g. 1 µl) is titrated in a dilution series. An aliquot (eg 1 µl) of each dilution in the series is then used as a template for a PCR reaction in order to determine the dilutions in which logomers can still be detected. If the last dilution which still contains logomers is obtained in this way, and the dilution which already contains no more, an approximate dilution factor can be specified which just about still contains a logomer. If necessary, this dilution factor can be determined more precisely by titrating out the last dilution, which still contains logomers. Correspondingly smaller dilution steps are used (e.g. if you dilute 1:10 in the first series of dilutions, you can dilute 1: 2 in the second series of dilutions; the method of determining the dilution factor more precisely can also be used arbitrary accuracy can be applied).

Since the PCR of the dilutions serves to determine whether there are any logomers in the dilution, the PCR can be carried out in at least two ways:

• a) Two opposing primers, z. B. the 5'- Strand of the start terminator and the 3-strand of the end terminator. Otherwise are the PCR conditions as below with amplification of logomers by PCR described.
• b) The PCR is carried out like that for reading out the logomers, but it is sufficient Approach with a pair of primers as with reading out logomers by means of PCR in Described below.

The DNA fragments obtained by PCR are checked by gel electrophoresis made visible. For this, z. B. a 2-4% agarose gel (e.g. UltraPure ™ from Gibco BRL, Life Technologies, catalog no .: 15510-027) and as a molecular weight standard e.g. Legs 50 bp conductor (Gibco BRL, Life Technologies, catalog INr. 10416-014) is used. The received Gel is stained in 0.001% ethidium bromide for 5 minutes and visualized under UV.

Duplication of logomeres

The logomers obtained from a symbol polymerization can - depending on their Intended use - may be reproduced. This is for example for the Visualization of the stored information or the further use of the logomer as Markers are necessary to identify substances and objects.

Duplication of logomeres by PCR

In this process, logomers are reproduced by FCR. That to be reproduced Logomer serves as a template, the required primers prime in opposite directions in the Terminators (either 5'-start and 3'-end, or 5'-end and 3-start) so that the logomer is completely reproduced. Alternatively, the primers, provided that the material to be reproduced The logomer is surrounded by further DNA, also priming further outside the logomer.

For the PCR approaches, which can be varied in a manner known to the person skilled in the art, e.g. B. The following reagents and conditions are used:
dNTPs (e.g. Pharmacia Biotech, catalog no .: 27-2035-01 / 2/3), Taq polymerase (e.g. Gibco- BRL, catalog no .: 18038-034, 18038-042, 18038- 067), PCR buffer, MgCl ₂ (e.g. GIBCO-BRL, catalog no .: 18067-017)

For example, the following protocol (primer: 30-mer) can be used as the PCR program:

Another object of the invention are information-carrying polymers which according to the The inventive methods described above are available.

Reading out the information contained in logomeres

Finished logomers can either be by means of PCR (Polymerase Chain Reaction = Polymerase chain reaction), which is preferred according to the invention, or by means of Restriction digest can be read. The PCR method has the advantage, already to be able to reproduce the smallest amounts of DNA in such a way that the logomers directly after PCR and gel separation can be read. In the case of binary logomers can the band pattern obtained by the PCR method on a gel immediately as Binary code can be read.

The invention therefore furthermore relates to a method for reading out information from information-carrying polymers which have been obtained and / or isolated and reproduced as described above, characterized in that

• a) n solutions containing the information-carrying polymer, each with a pair opposed primer, where n for the number of elongators in the Polymer-containing oligomers;
• b) carries out at least n-1 PCR batches, n being the number of elongators in the polymer containing oligomers, Lind is a primer of each pair primes in the terminator opposite the elongator and the other primer primes in the elongator itself;
• c) the length of the polymer fragments obtained from the PCR Electrophoresis and d) optically reads the pattern obtained from electrophoresis.

The method can also be done using different colored fluorescent labels Primers or nucleotides can be performed. This makes it possible to have multiple approaches to mark different colors and to gelelectrophoretically several approaches in one track read. Furthermore, the reading process can be carried out with modern sequencing machines (e.g. available from ABI).

Reading out logomers using PCR

To make the information contained in logomeres visible, the logomers can can be read out using the PCR. The method of reading binary patterns with PCR was already used as DNA typing by [Jeffreys, Minisatellite reapeat coding as a digital approach to DNA typing, Nature, 354, 204- 209, (1991)]. The method described there is modified here Form used as readout PCR for reading out the logomeres. Differences to that in [Jeffreys, Minisatellite reapeat coding as a digital approach to DNA typing, Nature, 354, 204-209, (1991)] The method described is that synthetically generated templates are used here that do not necessarily contain binary patterns. In addition, PCR and gel Electrophoresis conditions are chosen so that the result can be read directly from the gel can, without the signals obtained as a band pattern being additionally amplified by blotting Need to become.  

a) PCR

To read out a logomer, at least n-1, but mostly n PCR approaches are used for n elongators of the underlying grammar. Each PCR approach contains the logomer to be read as a template and also a pair of opposing primers, one of which primes in the elongator, the other in the terminator opposite the elongator (e.g. 5 '- "Start" and 3'-0 , see Fig. 5). Any commercially available thermal cycler (e.g. PTC-100, MJ Research) can be used for the PCR. 20-30 bp have proven to be expedient for the length of the primers, but other lengths can also be used. Depending on the primer length, the annealing temperature must be selected accordingly (can be determined using the Oligo 5.0 program, for example). Appropriate annealing temperatures are approx. 55 ° C for 20-mers and approx. 65 ° C to 74 ° C for 30-mers. For the PCR approaches, e.g. B. uses the following reagents and protocols, which can be varied in a manner known to those skilled in the art:
dNTPs (Pharmacia Biotech, catalog no .: 27-2035-01 / 213), Taq polymerase (Gibco-BRL, catalog no .: 18038-034, 18038-042, 18038-067), PCR buffer, MgCl ₂ ( GIBCO-BRL, catalog no .: 18067-017)

In order to obtain enough DNA so that the band pattern obtained after the Gel electrophoresis can be read directly from the gel, any PCR approach m times can be scheduled. For example, m = 4.  

The following protocol (primer: 30-mer) can be used as the PCR program:

After the PCR has been carried out, it is for the readability of the band patterns of the following ones Gel electrophoresis may be useful, as much amplified DNA as possible in as little as possible Hold volume that can then be loaded onto a gel. To do this, the volumes in a suitable manner (e.g. with a SpeedVac, e.g. SpeedVac Concentrator, Savant) be restricted.

b) Gel electrophoresis

The DNA fragments obtained from the PCR for each elongator have different lengths. If they are separated by electrophoresis, the different length fragments result in a specific pattern, on the basis of which the logomer to be read can be identified (see Fig. 5 and Fig. 6). For gel electrophoresis, a 4% agarose gel (e.g. UltraPure ™ from Gibco BRL, Life Technologies, catalog no .: 15510-027) can be used as the molecular weight standard e.g. B. a 50 bp conductor (Gibco BRL, Life Technologies, catalog no .: 10416-014) can be used. The DNA is separated in a gel chamber in a suitable manner, e.g. B. 1: 45 hours at 60 V. Here, however, the parameters can be selected relatively freely, in particular the gel runtime can be shortened further.

The gel obtained is stained in 0.001% ethidlium bromide for 5 minutes and visible under UV made.

An example of a gel obtained in this way can be found in Fig. 6.

Reading out logomers by restriction digestion

Logomers can be read out by restriction digestion. To do this, the Elongators should be designed to have asymmetrically offset restriction sites wear. Each elongator has a specific restriction interface. For example the restriction enzyme EcoRV (e.g. NEB, catalog no. # 195S) for 0-elongators and Smal (e.g. NEB, Catalog No. # 141S) can be used for 1-elongators.

For reading, the logomer to be read is cut in various restriction approaches. For this purpose, a restriction approach with the respective restriction enzyme is formed for each elongator. The DNA fragments obtained from the restriction are of different lengths. Become  separated by electrophoresis, they result in different length fragments specific pattern on the basis of which the logomer to be read can be identified.

The following example shows the DNA fragment lengths of all binary logomers with 4 bits without terminators with elongator length = 30 bp; Restriction interface per elongator after 10 bp.

In the above example (4 bits) you can see that the length pattern of the numbers 0010 and 0100 at Restriction of the 0 bit is not unique. 0010 and 0100 can, however, be based on the Differentiate length pattern with restriction of the 1 bit.

In detail, the following example was used to carry out the above example: the protocols can be varied according to the knowledge of the person skilled in the art:

a) DNA extraction

A z. B. obtained by isolation and duplication of logomers by cloning Bacterial colony is used to inoculate 2-5 ml of a 37 ° C overnight culture (e.g. LB medium with 10 µg / l ampicillin as in [J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning, A Laboratory Manual, (1989)]). As a cloning vector z. B. pGEM-T Easy (Promega, Catalog No. A1360) and binary logomers also without terminators be used. The overnight culture becomes the plasmid DNA containing the logomer isolated (e.g. using the Qiagen Plasmid Miniprep Kit, Qiagen, Catalog No. 12123, or by alkaline lysis as in [J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning, A Laboratory Manual, (1989)]).

b) restriction

The plasmid DNA obtained from a) is cut in n restriction batches for n elongators. Each restriction approach contains a restriction enzyme that cuts in a specific elongator and a restriction enzyme that cuts out the logomer from the cloning site. The following approaches were used in the example used:

The indicated batches are incubated at 37 ° C. for 1 hour.

c) Gel electrophoresis

The DNA fragments obtained from b) are separated electrophoretically (for example 4% Agarose UltraPure ™ from Gibco BRL, Life Technologies, catalog no .: 15510-027), stained with ethidium bromide (0.001%) and made visible under UV (see Fig. 8).

The methods described above enable the implementation of binary logomers, for example:
Binary logomers are generated with grammars in which:

Σ: = {0, 1, s ₀ , s ₁ , s _2,. . ., s _n-1 , s _n , e ₀ , e ₁ , e _2,. . ., e _m-1 , e _m } with n, m ∈ e N and n, m ≧ 0.

They enable the simplest and most universal representation of data, like that of conventional data processing machines is used. Binary logomers are Result of bit polymerization. Because almost depending on the grammar chosen Any symbols and rules can be encoded, so can any data and data types are generated.  

The methods described above enable the implementation of character alphabets, for example:
Logomeres can be used to encode the characters of a chosen alphabet. The character length can be selected differently, but it makes sense to use the character lengths (nibble, 1-byte, 2-byte, 4-byte, 8-byte, etc.) that are also used for conventional data processing systems. The convention for the interpretation of the characters shown is also optional. Characters can be numbers, letters, alphanumeric characters or any other data structure.

For example, the methods described above enable the implementation of a 1-byte alphabet:
Let there be a grammar G = (Σ, V, R, S) with terminal alphabet Σ: = {0, 1, s, e}, set of variables

V: = {S ₀ , S ₁ , S ₂ , S ₃ , S ₄ , S ₅ , S ₆ , S ₇ , S ₈ }, start symbol S and rule set) R: =

{
S: = sS ₀
S ₀ → 0S ₁
S ₀ → 1S ₁
S ₁ → 0S ₂
S ₁ → 1S ₂
S ₂ → 0S ₃
S ₂ → 1S ₃
S ₃ → 0S ₄
S ₃ → 1S ₄
S ₄ → 0S ₅
S ₄ → 1S ₅
S ₅ → 0S ₆
S ₅ → 1S ₆
S ₆ → 0S ₇
S ₆ → 1S ₇
S ₇ → 0S ₈
S ₇ → 1S ₈
S ₈ → e
}

in which:
s: = start
e: = end example:
All 8-bit characters can be generated according to the rules of the specified grammar:
e.g. E.g .: generation of 8-bit 0, 00000000:

S → sS ₀ → s0S ₁ → s00S ₂ → s000S ₃ → s0000S ₄ → s00000S ₅ → s000000S ₆ → s0000000S ₇ → s00000000S ₈ → s00000000e

e.g. E.g .: generation of 8-bit 255, 11111111:

S → sS ₁ → s1S ₁ → s11S ₂ → s111S ₃ → s1111S ₄ → s11111S ₅ → s111111S ₆ → s1111111S ₇ → s11111111S ₈ - <s11111111e

e.g. E.g .: generation of 8-bit 85, 01010101:

S → sS ₀ → s0S ₁ → s01S ₂ → s010S ₃ → s0101S ₄ → s01010S ₅ → s010101S ₆ → s0101010S ₇ → s01010101S8 → s01010101e

The sequences required for the implementation in vitro are according to the NFR method, as described in steps II and III according to the invention. Algomers and Logomers are prepared as described in steps IV-V according to the invention and the logomers obtained are preferably separated by means of and Duplication of logomers isolated by cloning and described method reproduced. The alphanumeric characters created in this way can now be used for various technical purposes (e.g. marking and identification of substances) are used and, possibly after additional technical steps, in accordance with Method according to the invention for reading out information from information carriers Polymers (as described above) can be read out.

Since the binary patterns generated here can be read optionally as data and symbols, can you e.g. B. interpreted as alphanumeric characters. They are numbers interpreted, the specified grammar generates 3-bit random numbers. All 8-bit patterns can be made from a sufficiently large amount of random 8-bit binary patterns isolated and created as a library (e.g. to display all alphanumeric characters) become.

The methods described above enable the display of strings, for example:
Since algomers can be produced in such a way that logomers of different lengths can be generated, it is also possible to display character strings. For practical reasons, however, it is advisable to provide only a few characters per logomer. For example, a single logomer can contain 4 bits (nibbles) or 8 bits (1 byte). (If more bits are required, multiples of 8 bits = 1 byte should always be used.) In binary representation, any alphanumeric characters can be displayed in any coding (e.g. ASCII, ANSI, ISO 8859-x, Unicode). A character can also span several logomers (e.g. half-byte representation, in which a 1-byte character extends over two logomers, or Unicode, in which a 16-bit character spans two 8-bit or four 4-bit logomer stretches). To display character strings, it is then necessary to provide the individual characters with position information. To do this, it is sufficient to use different terminators (eg different "start" terminators) in the respective grammars, the sequences of which each represent the position information. Example: Let us have a grammar G = (Σ, V, R, S) with terminal alphabet Σ: = (0, 1, e, s ₀ , s ₁ , s ₂ ,..., S _n-1 , s _n } , Variable set V: = (S ₀ , S ₁ , S ₂ , S ₃ , S ₄ , S ₅ , S ₆ , S ₇ , S ₈ }, start symbol S and rule set R: =

{
S: = s _i S ₀
S0 → 0S ₁
S0 → 1S ₁
S1 → 0S ₂
S1 → 1S ₂
S2 → 0S ₃
S2 → 1S ₃
S3 → 0S ₄
S3 → 15 ₄
S4 → 0S ₅
S4 → 1S ₅
S5 → 0S ₆
S5 → 1S ₆
S6 → 0S ₇
S6 → 1S ₇
S7 → 0S ₈
S7 → 1S ₈
S8 → e
}

in which:
s; : = Start, with i = {0,. . ., n}
e: = end.

The 1-byte alphabet generated with this grammar is sufficient e.g. B. for all alphanumeric characters of the ASCII, ANSI or ISO 8859-x standards. N different terminators are required for a string of lengths, so that the strings generated with this grammar are structured as follows:
s ₀ xe, s ₁ xe,. . ., s _n-1 xe, s _n xe
where x is any binary representation with, in this case, 8 bits.

It then means:
s ₀ xe: = character x at position 0 (0th character)
s ₁ xe: = character x at position 1 (1st character)
s ₂ xe: = character x at position 2 (2nd character)
etc.

With the grammar, for example, the character string "Elisabeth" can be represented in ASCII code:

s ₀ 01000101e, s ₁ 01101100e, s ₂ 01101001e, s ₃ 01110011e, s ₄ 01100001e, s ₅ 01100010e, s ₆ 01100101e, s ₇ 01110100e, s ₈ 01101000e

Alternatively, Σ: = {0, 1, s, e ₀ , e ₁ , e _2,. . ., e _n-1 , e _n } can be selected, whereby the character string "Elisabeth" in ASCII code as:

s01000101e ₀ , s01101100e ₁ , s01101001e ₂ , s01110011e ₃ , s01100001e ₄ , s01100010e ₅ , s01100101e ₆ , s01110100e ₇ , s01101000e ₈

is pictured. Another possibility is Σ: = {0, 1, s ₀ , s ₁ , s _2,. . ., s _n-1 , s _n , e ₀ , e ₁ , e _2,. . ., e _n-1 , e _n }, with which the string "Elisabeth" in ASCII-Cade as:

s ₀ 01000101e ₀ , s ₁ 01101100e ₀ , s ₂ 01101001e ₂ , s ₃ 01110011e ₃ , s ₄ 01100001e ₄ , s ₅ 01100010e ₅ , s ₆ 01100101e ₆ , s ₇ 01110100e ₇ , s ₈ 01101000e ₈

is pictured.

In a half-byte representation with position information, the character string "Elisabeth" would be displayed in ASCII code as:

s ₀ 100e, s ₁ 0101e, s ₂ 0110e, s ₃ 1100e, s ₄ 0110e, s ₅ 1001e, s ₆ 0111e, s ₇ 0011e, s ₈ 0110e, s ₉ 0001e, s ₁₀ 0110e, s ₁₁ 0010e, s ₁₂ 0110e, s ₁₃ 0101e, s ₁₄ 0111e, s ₁₅ 0100e, s ₁₆ 0110e, s ₁₇ 1000e

Due to the position information represented by the sequence of the terminators the individual characters can be processed completely independently of each other and e.g. B. read separately. With such an alphabet, the representation of any data types is possible. For example, you can e.g. B. two bytes each (0. + 1., 2. + 3.,..., N. + N + 1.) To a 2-byte code (e.g. Unicode) be summarized. Alphanumeric strings can be used to display any identifier (name, number, date, etc.).

The sequences required for the implementation in vitro are according to the NFR method, as described in steps II and III according to the invention. Algomers and Logomers are produced as described in the documents IV-V according to the invention, the logomers obtained preferably by means of separation and duplication isolated from logomers by cloning and reproduced. The alphanumeric characters and strings generated in this way can now be used for various technical purposes (e.g. marking and identification of substances) are used and, possibly after additional technical steps, in accordance with Method according to the invention for reading out information from information carriers Polymers (as described above) can be read out.  

The methods described above enable, for example, the implementation of a random number generator:
Random numbers are required for certain problems in computer science (e.g. simulations) and mathematics. These are generally generated with the aid of algorithms. However, since these algorithms are deterministic, the generated random numbers are only pseudo-random numbers. In addition, due to the different quality of the algorithms, the number series generated are randomized to different extents. Real random numbers are therefore required for some applications. If this is the case, a physical process must be included in the generation of random numbers. One such process is the noise of the sound card in a PC, which is processed by appropriate software.
Using the methods described here, a real random number generator can be implemented that generates random numbers much faster than is possible with conventional methods.
The random number generator is implemented with the following grammar (grammar for binary random numbers of any length):

G = (Σ, V, R, S) with Σ: = {0, 1, s, e}, V: = {S},
R: =
{
S: = sA
A → 0A
A → 1A
A → e
}

in which
s: = start
e: = end

With this grammar, words can be placed above the terminal alphabet given above L = {0, 1, s, e} be formed. All L language words begin with "s" and end with "e", in between is a random number (also 0) of random bits ("0" and "1").

Example: Words from the language L that are formed after R:

S → sA → se
S → sA → s1A → s1e
S → sA → s0A → s00A → s001A → s0010A → s0010e
S → sA → s1A → s10 62137 00070 552 001000280000000200012000285916202600040 0002019914808 00004 62018A → s100A → s1000A → s10000A → s100000A → s100000e

The binary patterns generated as words of language L can be read as letters, numbers, alphanumeric characters or character strings. If you read them as the binary representation of numbers, these binary patterns are in decimal representation:

∅3 (empty)
1
2nd
32

and can be used as random numbers. In principle, any sequences are suitable for in vitro implementation, as long as they are clear and maximally dissimilar to one another. For example, the following algomers can be used:

The sequences required for this are commercially available. You can e.g. B. ordered as 40 nmol, PAGE cleaned (ARK-Scientific, Darmstadt) and z. B. 100 uM in aqua dest. be included (recommended storage at -20 ° C). As described in step IV according to the invention, algomers are prepared from the sequences. As described in step V according to the invention, these algomers are polymerized to logomers and can be read out according to the method according to the invention for reading information from information-carrying polymers (as described above). Random numbers generated in this way can be seen in Fig. 6.

The methods described above enable, for example, the encryption of logomers:
The information contained in logomers can be encrypted. For this purpose, at least one of the primers required for the readout PCR acts as a secret key (see Fig. 11). This is preferably one of the primers priming in the terminators. If the sequence of this primer (key sequence) and the primer itself are only known to authorized accesses, the information contained in the logomers encrypted in this way is also only accessible to authorized accesses.

To read out the information contained in the logomer without this key prevent further precautions must be taken. Try encryption to break can also be based on duplicating non-secret sequences to read the logomeres. If bacterial vectors are used to clone logomers a potential attacker could e.g. B. try using the entire cloning site Reproduce PCR and then read with sequencing or through the for selection "read" the required resistance genes into the cloning site. They could also Elongators themselves as starting points for PCR-based reading of the secret sequence (s) serve.

Common to such attempts to attack is that they attempt over non-secret sequences could decrypt the key sequence and use it to encrypt the sequence read. A defense against such attacks can be done in that Information transport via logomers only ever uses completely secret sequences become. However, this may be too complex and costly. Instead or in addition the information-bearing logomers with sequences (sham sequences) that have the same potential points of attack (e.g. bits, resistance genes) included, but different key sequences. Run through these sham sequences potential attempts at attack in vain, because all for the unauthorized attacker Individual sequences indistinguishable and therefore the encrypted information is hidden are. The more fake sequences a logomer is mixed with, the more difficult it becomes Key sequence to break.

The procedure corresponds to a molecular steganography and is shown as an example using the encryption of characters from a 1-byte alphabet:
Let the grammar used be G = (Σ, V, R, S) with terminal alphabet Σ: = {0, 1, s _key , s ₀ , s ₁ , s ₂ ,. . ., s _f-1 , s _f , e}, where f ∈ N, f ≧ 1. s _key is the secret key. For a key length of I, the maximum theoretical encryption Key _max = a ^I , the maximum usable encryption Key _eff = a ^Id , where d indicates the number of bases in which a dummy key must differ from the correct key, so that in the Read out PCR no dummy key read the correct sequence and vice versa the correct key cannot read a dummy logomer (only the target logomer). For highly specific PCR conditions, d ≧ 1 can become. Key _imp = number of bogus keys + 1 indicates the encryption actually used and Key _min = 1 + x the minimum encryption. Here x is the number of dummy logomers required for minimal encryption. If n logomers are used as information carriers, this can be chosen to be greater than n.

Additional encryption automatically results for character strings, because here the for additionally encrypting a potential attacker unknown sequence of characters works.  

Example: The following grammar G is used for a grammar with strings of n 1-byte characters:
Let G = (Σ, V, R, S) with terminal _alphabet Σ: = {0, 1, s _key0 , s _key1 , s _key2,. . ., s _keyn-1 , s _keyn , s ₀ , s ₁ , s _{2 ,.} . ., s _f-1 , s _f , e}, where n, f ∈ N, n ≧ 1. n indicates the number of real keys used, f the number of dummy keys used; the set of variables V used and the set of rules R used are optional and depend on the information to be encoded.

Grammars with other character encodings work analogously. The sequences required for the implementation in vitro are according to the NFR method, as described in steps II and III according to the invention. Algomers and Logomers are prepared as described in steps IV-V according to the invention, the logomers obtained preferably by means of separation and duplication isolated from logomers by cloning and reproduced. The logomers generated in this way, as already described as readout PCR, can be read out, with - as described - one of the primers required for reading out, preferably one of the primers priming in the terminators acts as a secret key. With the grammar described above, in addition to the information-bearing Logomeric "sham sequences" ("sham logomers") that generate an unauthorized Read access to the information contained in the logomer should prevent. The Process can be used for all logomer applications and has u. a. the Advantage, very effective due to the specificity of the primer used as a secret key his.

Based on the method described, symmetrical and asymmetrical Encryption methods are implemented. For a symmetrical Encryption methods suffice for participants A and B to communicate agree on a secret key sequence. Subscriber B encrypts one as Logomer saved message to A by B logomer only with the secret Terminators produces and its message with additional logomeres, the other Wear terminators. Only A is then able to read the primer pair required for reading to be provided, since only A knows the terminator sequence required.

An asymmetrical encryption method, on the other hand, requires additional effort, e.g. B. the use of molecules with irregular shapes (see e.g. Fig. 12 and Fig. 13): If a communication subscriber B wants to send an encrypted message to A, he receives a public key from A, which he uses to encrypt the as Logomer represented message can use. A's public key consists of a pair of terminators and a pool of additional dummy terminators with similar properties, but different sequences and other 3 'overhang sequences. Of the real terminators, only the 3 'overhang sequence is known, with which it can be linked to the - publicly known - elongators. To encrypt a message, B creates logomers, using the key (containing terminators and dummy terminators) made publicly available by A in the symbol polymerization reaction. The nature of the terminators now ensures that only A is able to read the message encrypted in this way: since only A knows the actual sequence of the real terminators, only A is able to read the message encrypted as logomers -PCR read again.

Because the public key is potentially vulnerable to the elongators via the known overhang sequence (because the fake terminators must not have this overhang sequence, a potential attacker can link any sequence to the real terminators, whereby the sequence of the terminators can be identified) are termed terminators Molecules based on irregular shapes, e.g. B. used on the basis of the Y-shaped molecules shown in Fig. 12. The Y-shaped molecules shown can be assembled into further branched tree-like molecules. The root of such a terminator molecule implemented as a tree then contains the overhang sequence compatible with the elongators, while only one of the branches of the tree contains the real terminator sequence. Since the encrypted message is also supplemented with dummy logomers as described above, only A who knows the real terminator sequence can filter out the correct message from the set of molecules by linking it to the compatible vector, whereas a potential attacker without knowledge of the real terminator sequence can can not.

The methods described above enable encryption with real-time identification, for example:
Without reading out the information contained in encrypted logomers, the presence of the key sequence itself can provide information as to whether a labeled product is genuine or not. The information about the authenticity of the labeled product can be verified or falsified using a process in which the key primer serves as a hybridization probe for a fluorescence detection reaction.

Another object of the present invention is therefore the use information-carrying polymers, in particular those obtainable according to the invention information-carrying polymers, for encrypting information.

The methods described above allow, for example, a test of the quality of oligonucleotides:
A typical problem in the production of oligonucleotides, e.g. B. for molecular biological purposes, the quality control is the same, which is problematic due to the manufacturing process and the fluctuating quality of the chemicals used.

The method described in steps IV according to the invention can be used to test the quality of oligonucleotides. For this purpose, a binary grammar such as that for the random number generator (see above) or a unary grammar such as that for the production of molecular weight standards (see below) is used to generate logomers of any length. Under otherwise identical conditions, the length of the logomers obtained from a symbol polymerization (step V of the method according to the invention) is directly proportional to the quality of the oligonucleotides used: the longer the logomers made electrophoretically visible, the better the quality of the oligonucleotides used. An example of the conductors of different lengths of logomer obtained from symbol polymerization can be found in Fig. 3.

Another object of the present invention is therefore the use information-carrying polymers, in particular those obtainable according to the invention information-bearing polymers, for quality control of synthetically manufactured Oligonucleotides.

The methods described above enable, for example, the production of logomers as markers and signatures:
Due to the ability to display any information in principle, the logomers described here can be used as markers to identify products, products, substances and devices.

Another object of the present invention is therefore the use information-carrying polymers, in particular those obtainable according to the invention information-bearing polymers, as markers or signatures.

The logomers can be used as a "binary label" that is added to a product and contains information about the product so labeled. The logomers can z. B. Information about

• a) Manufacturer
• b) Product (e.g. serial number)
• c) Intended use
• d) substance class
• e) Hazard class
• f) quality
• g) purity
• h) Date of production and expiry

contain. It can be used with almost any product and product any information. Since the logomers are non-toxic and biodegradable,  They can also be used for highly sensitive products such as food, medication and pharmaceutical products are used.

Labeled products can e.g. B. be:

• a) chemical products, e.g. B. paints, paints, oils, lubricants and fuels, Solvents, ink, etc.
• b) liquid materials: solutions, suspensions, emulsions
• c) genetic engineering products
• d) food, e.g. B. to identify genetically modified components or the Monitoring of food during the production process
• e) Medicines and pharmaceutical products
• f) Paper products, documents, money
• g) devices

The need to label products exists in a wide variety of areas and due to different requirements. Reasons for labeling can e.g. B. be: quality assurance in the production process, monitoring of product purity and Avoidance of contamination, protection against counterfeiting and product piracy, proof certain product components, product labeling with any product information. This need also arises in particularly sensitive areas, such as labeling of food, medical and pharmaceutical products and genetic engineering manufactured or modified products. Here it is desirable that information are available directly on the product via the respective product, especially if Confusion or falsification should be avoided and also identification different components of a product is desired.

There are numerous examples of the need for product labeling. For example, you can Logomeres can be used as serial numbers, which are mixed in car paints, thereby the vehicle owner can be identified in the event of an insurance claim (e.g. accident or theft). Product labeling could improve the quality and purity of chemical products monitored and contamination avoided. This is a permanent problem Falsification of documents, signatures and money, the most sophisticated Labeling process necessary.

Especially against the background of animal diseases and diseases (BSE, swine fever, Salmonella poisoning, etc.) raises the problem of quality assurance Food and labeling of end products to contaminated products from Keep consumption away. Another problem is food that is genetically engineered manufactured ingredients included. These components can be used without one Labeling process in the end product not at all or only with great difficulty be detected. The problem arises in particular because many Foods contain ingredients of different origins and the production routes are partially obscure.  

The problem of labeling arises e.g. B. also in the field of blood products, where, for. B. Contamination can occur through pooling. It would also be z. T. desirable Information about identity, origin, production and expiry date directly with the product to have available. The same applies to medicines and pharmaceutical products.

So far, one used for the marking and marking of paints, varnishes, fuels, etc. various carbon compounds, polyanilines, liquid crystals or others chemical compounds. These substances and compounds have different Weaknesses: they are z. T. toxic, difficult to biodegrade, the available Information capacity is severely limited, they interact chemically with certain ones Fabrics or are only expensive and expensive to manufacture.

For use in labeling sensitive and highly sensitive products such as Food or medication are the substances and compounds mentioned due to of the disadvantages mentioned not suitable at all.

The requirements for a labeling process for any product include at least:

• - The label should be available directly in or on the product;
• - the labeling should be clearly demonstrable;
• - The labeling must be harmless to health.

The logomers described here have the following advantages over the previously used compounds:

• - coding of any information,
• - high storage capacity,
• - no risk to health,
• - chemically, biologically, pharmacologically and genetically neutral,
• - readily biodegradable (biomolecules),
• - no environmental pollution,
• - very good traceability,
• - High compatibility with existing techniques in computer science and molecular biology (PCR),
• - Information can be encrypted and is also suitable for authentication and encryption,
• - Identification of individual components in product mixtures,
• - cheap to produce in large quantities (cloning, reproduction with bacteria in Fermenters).

These properties make logomeres much better for the tasks mentioned suitable as the existing techniques. In addition, areas of application open up with the previous techniques were not possible.  

Due to the properties of nucleic acid-based logomers, these can also be used for Labeling of particularly sensitive products such as food and pharmaceutical products are used.

The reasons for this lie on the one hand in the chemical nature of nucleic acid-based logomers and on the other hand in the coding of information in logomers:

• 1. As basic building blocks of life "nucleic acids are elementary building blocks of all so far known biological life forms. Therefore, due to their chemical nature No harmful biological condition ("biochemical compatibility"). Based on the genetic information it contains (the contained programs), however, nucleic acids can very well be potentially harmful if they are read in an organism: if they are for toxic proteins encode or (as in the case of viral genetic material) a respective target organism can "reprogram". However, the logomers used here can be derived from the im The following reasons are not harmful to an organism contain.
• 2. Logomeres do not contain any genetic, i.e. H. biologically relevant information ("Semantic incompatibility"): Correspond to the information contained Logomeric letters, numbers or sequences of letters and numbers in Binary encoding for us or a computer, but not for genetic Apparatus of an organism are legible. They are for a biological organism simply "nonsense code".
• 3. Logomers, especially the algomers from which they are built, are too short to contain biologically relevant information by chance.
• 4. Any eventuality (the unlikely event that the binary information of the Logomers in any way from any organism as genetically relevant Information is interpreted) to exclude those for coding symbols used sequences in the case of sensitive areas of application with stop codons provided so that they are never read by a biological organism can.
• 5. Nucleic acids are part of all biological organisms in ours Environment omnipresent. They will be dismantled in no time.

In order to be used as a marker, logomers according to the in the invention Steps I-V generated method and according to the invention according to the above described procedures are reproduced. Depending on the area of application Duplication of logomers different procedures and different Refurbishment processes are carried out. For marking petrochemical Products e.g. For example, logomers can be replicated in bacteria by cloning. Around The bacteria obtained can be lysed to avoid unnecessary contamination. A special purification of the logomer DNA is generally not necessary.  

For the production of logomeres for sensitive and highly sensitive products, however, is a more complex duplication and purification process necessary. The goal is preservation as pure a logomer as possible. To avoid biological intermediate steps, the Duplication - as described according to the invention - can be carried out using PCR. Should if a duplication is carried out by cloning, the purification of the DNA obtained from bacteria is necessary. If the logomers are cloned in bacterial vectors, so is a targeted breakdown of these resistance genes (e.g. by restriction enzymes) component a further workup.

Logomers can be directly connected to a product to identify it become. You can e.g. B. mixed in liquid substances or applied to solid substances become. In the case of genetic engineering products, the logomers can be identified using Recombination and cloning techniques covalently with the product to be labeled get connected.

The labeling of substances and products by logomers enables u. a. the Monitoring of production processes, quality control, the identification of individual Components and the quantitative and qualitative detection of contamination.

The methods described above enable, for example, the labeling of genetically engineered or modified products:
The logomers described here can be used to label genetically engineered or modified products.

So far, genetically engineered or modified ones have been used to identify them Products in food "classic" molecular biological methods such as PCR, Restriction digestion, Southern blot hybridization and sequencing were used. With these However, methods is a characterization of genetic engineering products and components still very complex and requires its own for each product to be characterized Methods and procedures.

So far there is no universal labeling method for genetically engineered products. Such a labeling process must meet several criteria:

• - the labeling must be absolutely harmless to health,
• - the label should be inextricably linked to the labeled product,
• - the labeling should be tamper-proof,
• - the label should be able to store enough information,
• - The labeling should be detectable and legible in small quantities.

The use of nucleic acid-based logomers for labeling genetic engineering products fulfills these criteria and also has the following advantages:

• - Logomers can store almost any information;
• - Individual components can be detected quickly and with high sensitivity;  
• - A detailed method can be used to read out the information are used (readout method according to the inventive method, thereby standardized primers can be used);
• - The sequence of the genes sought can be completely unknown;
• - The sequence of the genes detected can be secret without the identification or authentication is restricted;
• - Additional security mechanisms can additionally by encryption be implemented.

Specifically, the procedure for labeling genetically engineered or modified products is as follows:
Any suitable grammar, e.g. B. as that for the random number generator, the 1-byte alphabet or for character strings already described used. The characters required for the representation of markers are taken from the characters generated with the grammar and added to the product to be labeled. For this purpose, the characters produced as logomers can be introduced into the product to be labeled in the following way:

• a) by admixture; Here, logomers are used, as in the method according to the invention described, produced, isolated and reproduced.
• b) by cloning, as described in the process according to the invention; Fig. 6 shows e.g. B. the identification of bacterial clones by logomers, which were produced by using the grammar already described for the random number generator.
• c) through the use of recombinant techniques; Here, logomers are produced, isolated and reproduced according to the invention. The logomers obtained in this way are now introduced into the product to be labeled using recombinant techniques. For this, e.g. B. the methods of gene targeting by homologous recombination [Capecchi MR, Altering the genome by homologous recombination. Science, 244 (4910), 1288-1292, (1989)] or e.g. B. the Cre-loxP system [Kilby, NJ, Snaith, MR & Murray, JA, Site-specific recombinases: tools for genome engineering, Trends Genet., 9, 413-421, (1993)] can be used. For example, logomers obtained according to the invention can be placed with the aid of these techniques in front of or behind a specific nucleic acid sequence (gene) which they are intended to denote. In this case, a logomer is "attached" to a genetic product as a kind of "label" and can provide information about the manufacturer, product group, hazard class, expiry date or similar (see Fig. 15).

The method is suitable for. B. for all organisms (microorganisms, plants, animals). E.g. clones of microorganisms or plants can be labeled. The procedure is suitable z. B. also for the labeling of cattle to combat BSE.  

For example, the methods described above enable food to be labeled:
Due to illnesses, epidemics or contamination, food can be harmful or dangerous. Examples are diseases such as BSE, swine fever, salmonella poisoning. It may be desirable to be able to identify the product and manufacturer in order to be able to immediately take certain products and product series out of the market, to examine them and to take appropriate countermeasures.

For highly sensitive products such as food, the criteria for Labeling of genetic engineering products in particular (see: The above described For example, methods enable the labeling of genetically engineered or modified products).

Overall, there is still no universal one for genetically engineered foods Labeling process. So far, the identification is genetically engineered or modified components in food therefore smelled difficult and expensive. It will "classic" molecular biological methods such as PCR, restriction digestion, Southern blot Hybridization and sequencing are used to characterize these products.

Due to the BSE epidemic was specially designed for the identification of cattle and their Products (beef and milk) an immunological labeling process proposed that on the immunization of the animals to be labeled with specific Proteins based ("Ear tag in the blood should detect BSE cattle", Süddeutsche Zeitung Nr. 283, 08.12.1998, page v2 / 9). Through the production caused thereby more specific Antibodies in the identified target animal, which are presumably in all tissues can be verified, the label can be read again using an immunoassay (ELISA) become. In principle, however, such a method is limited to higher vertebrates. In addition, the amount of information available for the process is very limited Information-carrying proteins are not heat-resistant and the process requires them comparatively high amounts of substance for immunization and detection reaction.

In the case of food, too, logomers based on nucleic acids can be used, as already mentioned under "For example, the methods described above enable labeling genetically engineered or modified products "for labeling be used. Logomers can provide information about the manufacturer, expiry date, Serial number u. a. contain. The procedure for labeling food is doing the same as already possible under "The methods described above for example the labeling of genetically engineered or modified products " described. With the help of the recombinant and Cloning techniques can be used to identify individual organisms, so that a individual organism and all of its descendants are identifiable.

The labeling process using logomers enables:

• - Monitoring of the target product across the entire manufacturing and Processing to the end user,
• - Uniform, quick and easy identification of individual components.

As an admixture, logomers based on nucleic acids are suitable for labeling Drinks.

The methods described above enable, for example, the labeling of medical and pharmaceutical products:
Due to the non-toxicity, logomers based on nucleic acids are suitable for labeling sensitive and highly sensitive products. In addition to food, these are also medical and pharmaceutical products. In order to rule out mix-ups, contamination and counterfeiting, logomers can e.g. B. medical and pharmaceutical products.

The advantage of labeling with logomers is that the labeling is immediate is associated with the marked product and that products z. B. individually have it marked. In this way it is possible to monitor the manufacturing process, Products can be clearly identified even after repeated decanting Contamination and pooling of samples can be detected. With the help of Logomeres can e.g. B. in the case of preserved blood, date of manufacture and expiry, Identify blood group and manufacturer.

The methods described above enable authentication and counterfeit protection, for example:
Logomeres can be used to authenticate products, objects and devices. For this purpose, logomers are preferably generated and reproduced according to the invention.

If logomers obtained in this way are applied to products, objects and devices applied or admixed, so that contained in logomers according to the invention can be read out again at any time. Should be used to generate the logomeres Encryption techniques are used, as already described, so it requires Read out authorized access.

For example, logomers obtained according to the invention can be applied in aqueous solution (preferably at 10 ³ to 10 ¹⁸ molecules / μl) directly to documents for identification. As such, they can serve as a certificate of authenticity for the document marked in this way. To read out the logomers used as identifiers, a small snippet of paper (1 mm ² is sufficient) is used as a template for a PCR readout reaction according to the invention. An example can be found in Fig. 16.

In the underlying readout experiment, a logomer in aqueous solution in approx. 10 ⁹ molecules / µl on 3M Postlt paper was dried for approx. 1 hour and 1 mm ^{2 of} the Post-lts was used as a template for a readout PCR to test the principle. Documents of any paper quality can be used, e.g. B. 80 g / m ² standard copy paper.

The same procedure is also suitable for marking money or banknotes, with the following advantages being that

• - to be able to mark already printed notes,
• - to be able to assign serial numbers,
• - Use encryption.

In order to be able to check documents for authenticity in real time, one of the Reading out in the PCR also used primers as a hybridization probe for a subsequent one Staining detection reaction (e.g. with an intercalating dye, e.g. ethidium bromide) be used.

Another example is the authentication of liquid solutions, suspensions and Emulsions with logomeres. For example, ink can be individually marked with logomers, so that signatures can be checked for authenticity.

Another example is the marking of automobiles by moving the Car paints with logomeres. Logomeres are used in car paint (or others Components of the vehicle) added as an admixture. The added logomers can Information about serial number, manufacturer, vehicle type etc. Information included. Because of This information can be used in the event of an accident, theft or illegal disposal Vehicle can be identified. An advantage of the method described here is that traces of paint are sufficient to identify the vehicle, which is particularly the case with Accidents may be of interest. Another advantage is that the identity of a vehicle is difficult to fake. To do this, first of all would have to have all the labeled components removed, secondly a fake identity can be established. This endeavor is alone very difficult due to the mechanical effort, and can also be used cryptographic methods of copying and imitating identifiers as desired complicate. To read out the information contained as logomers, the Logomers isolated from the paint and can then be read out as described in the description information contained in logomers, preferably by PCR, can be read out. To a sample of the lacquer is taken with a suitable solvent (turpentine or similar) dissolved and mixed with the same volume of water. By subsequent centrifugation hydrophilic and hydrophobic components separated. Then you take the aqueous phase on (where the hydrophilic nucleic acid based logomers are) and the contained logomers by the usual methods (e.g. as in [J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular Cloning, A Laboratory Manual, (1989)]). After the precipitation the resulting logomers can be obtained by the processes according to the invention, e.g. B. by Readout PCR to be read out.  

Therefore, further objects of the present invention are the uses information-carrying polymers, in particular those obtainable according to the invention information-bearing polymers, for the purpose of quality assurance, the Anti-counterfeiting, food labeling, labeling genetic engineering, chemical, medical and pharmaceutical products, the Labeling organisms, labeling documents, labeling of money, the marking of objects and machines, the marking of Solutions, suspensions and emulsions as well as the authentication of people and Objects.

The methods described above enable, for example, the production of molecular weight standards:
The typical methods of molecular biological analysis include the electrophoretic length separation of DNA and RNA fragments. In order to determine the length of such fragments in electrophoresis, a molecular weight standard (“ladder”) is usually used, which contains fragments of known length and is used for comparison with the fragments of the samples to be analyzed.

So far there have been molecular weight standards that either contain irregular conductors (e.g. Boehringer V, Boehringer Mannheim, catalog no .: 821 705) or regular ladders with regular fragment length spacings (e.g. 50 bp conductor Gibco BRL, Life Technologies, Catalog No. 10416-014). None of the previous conductors has shorter lengths than 10 bp (e.g. 10 bp head of Gibco BRL, Life Technologies, Catalog No. 10821-015).

With the methods described here, DNA fragments of different lengths can be targeted and defined distances between lengths. These fragments can be used as Molecular weight standards are used, the method allowing, in principle, any Realize lengths and any length distances. In particular, length distances are up to down to 1 bp distance, that is, molecular weight standards of the highest resolution.

The method is based on using logomers as a template for a readout PCR, the result of which is a mixture of DNA fragments of defined length and defined Length distances is. It is based on the inventive method already described for generating and reading out logomers.

DNA fragments of different lengths at very short, regular intervals can be obtained if a unary polymer acts as a template for a readout PCR with nested primers. The 5 'primer primes in the start sequence of the polymer. A 3 'primer acts as an opposing 3' primer, which is nested in the elongator (shown in Fig. 17 as 3 levels of primers offset against each other). Since the polymer consists of repetitions of only one elongator molecule, nm bands are obtained for n repetitions and m nested 3 'primers. The process also works in mirror image with a primer priming in the end terminator and correspondingly opposite 5 'primers. The bands obtained can be used as molecular weight standards e.g. B. used in electrophoresis.

For high resolution molecular weight standards, the use of unary logomers and nested primer most appropriate. However, the procedure is not on that limited, since in principle all grammars described in step I according to the invention can be used.

Unary logomers of any length can be written with a grammar G = (Σ, V, R, S) with terminal alphabet Σ: = {0, s, e}, set of variables V: = {A}, start symbol S and rule set
R: =
{
S: = sA
A → aA
A → e
}
getting produced. If it is necessary to produce unary logomers of a defined length, the number of variables and thus the number of elongators must be selected accordingly (as already described in the grammar for implementing a 1-byte alphabet). Depending on the desired length of the DNA fragments to be generated, the length of the algomers can also be varied.

The logomers produced in this way can be separated and be reproduced.

DNA fragments of defined length and defined length spacings are generated preferably with PCR analogous to the method according to the invention for reading out Logomeres. For high-resolution molecular weight standards at least one elongator one primer, mostly even a number of several primers used against each other are moved. The number of primers per elongator depends on the desired Length distances. For example, it is desirable to use algomers of length 30 bp the lengths of the DNA fragments generated each have a 10 bp difference, so are pro Elongator three primers, each exactly 10 bp shifted, used become.

For the synthesis of sequences for the production of algomers and logomers, the Requirements are met that already under step II of the method according to the invention have been described. In particular, the NFR method used ensures that that the partial sequences serving as templates each have the same GC component can, the use of nested primers and the balance of the AT / GC Ratio of DNA fragments generated. The latter is especially so given this crucial that the running behavior of DNA fragments in electrophoresis not only depends on the length, but also on the composition of the fragments. this applies  especially for high resolution molecular weight standards in which there are only very low There are length distances.

Another object of the present invention is therefore the use information-carrying polymers, in particular those obtainable according to the invention information-bearing polymers, for the production of molecular weight standards.

The methods described above enable, for example, the production of polymeric data storage media:
The described logomers can be used as RAM (read and write) and ROM (read only) data storage of high capacity and low energy consumption. To ensure the highest possible density of information and to make the handling of the memory as easy as possible, the logomers are bound to a solid support. This ensures that the logomers can be addressed individually, ie written, read and deleted individually (see Fig. 9).

The write and read operations required are based on the im Step V according to the invention and the step of reading out those contained in logomers Information described enzymatic reactions carried out by themselves Temperature changes can be controlled. The temperature changes can either be per Laser or by heating and cooling the solid support z. B. in a thermal cycler be performed.

For writing, a modification of the above-described method of Symbol polymerization used. The polymerization takes place on a solid support, the Algomers are linked together by repeated restriction ligation cycles.

In order to manufacture individual, addressable memory cells, at certain intervals single-stranded "anchor molecules" irreversibly (by UV radiation or in the oven) to the Carrier tied. The carrier thus provided with anchor molecules can now be reversed be described with logomeres that are removed in a delete process can be.

In a writing process, specific algomers ("starter algomers") are carried out first Hybridization bound to the anchor molecules. These starter algomers are in turn Starting point for a symbol polymerization in which further algomers are linked together become. The algomers are linked, in a manner different from that in step V of the Procedure described inventive procedure, by repeated cycles of Restriction and ligation. In each cycle, the last algomer becomes one polymerizing chain cut with a restriction enzyme so that a single-stranded overhang sequence (elongation point), to which in turn the next algomer can be bound by ligation.  

For the writing process described in this way, the algomers must have a specific design:

• a) Each algomer to be written has a single-stranded overhang sequence with which it can bind to the elongation point of a logomer.
• b) An algomer that has only a single-stranded overhang sequence with which it is attached to one Can bind the elongation point, but does not itself have a restriction interface which it can be recognized by restriction enzymes and therefore not as Elongation point can be called the end Algomer.
• c) Every algomer to be written that is not the end algomer has one double-stranded end, in which the recognition sequence for the selected one Restriction enzyme is located. When restricted, the enzyme cleaves the algomer so that a single-stranded overhang sequence is created, which in turn is again as Elongation point can serve.
• d) Each finished logomer has exactly one starter and one end algomer. Starter and End algomers are, according to the definition given above, terminators.
• e) The overhang sequences of the algomers are compatible with a selected one Restriction enzyme, so that an algomer to be linked to the elongation point of a Can bind logomers and the subsequent one that results from restriction Elongation point is identical to its predecessor.
• f) The sequence following the overhang sequence in the algomer is chosen such that a chained algomer cannot be restored by a subsequent restriction process can be split off.

Membranes such as Gene Screen Plus (DuPont, Biotechnology Systems, catalog no .: NEF-986 or NEF-987), Hybond-N (Amersham Life Sciences, catalog No .: RPN 82N or RPN 137 N), silicon, silicate surfaces or glass. The anchor molecules are each covalently bound to it (UV radiation or heat).

So that the molecules can be addressed individually, they have to be opened at regular intervals be attached to the carrier.

Suitable restriction enzymes are commercially available enzymes, preferably those which are not be deactivated at 65 ° C (e.g. HindIII).

The write operations are based on the fact that the enzymes used for writing have different temperature optima. So is the temperature optimum for Chain of symbols used ligase at 16 ° C, whereas the required Restriction enzymes only work at 37 ° C. This is the timed writing of Symbols possible in cycles of restriction and ligation.

Since all read and write operations are performed using enzymes, it is necessary to to be able to temper the carrier. Thereby, when using a thermal cycler and thermo-manipulable material, all memory cells simultaneously in the same operation be subjected. Alternatively, memory cells can be used individually and independently of one another be accessed if a correspondingly small read / write head is used. This  The read / write head must be used as a pipetting device for the required molecules (Enzymes, algomers) and on the other hand those required for a specific manipulation Generate temperature (e.g. using a laser).

During the deletion process, a logomer is created by denaturing the respective anchor molecule solved. The anchor molecule is then available again for a writing process. For During a reading process, a logomer is detached from an anchor molecule by denaturation. It can then be read out using the method according to the invention.

The operations described writing, erasing, reading are carried out by enzymes different temperatures. Here, when using a material that can be manipulated thermally, all storage cells simultaneously in the same operation be subjected. Alternatively, memory cells can be used individually and independently of one another be accessed if a correspondingly small read / write head is used. This Read / write head must serve on the one hand as a pipetting device and on the other hand that for one generate certain manipulation required temperature.

The advantage of the polymer memory presented here is that compared to the materials previously used, it has a higher spoke density (DNA molecules have a size of 10 ^-10 m) and a much lower energy consumption (for the calculation of the energy required in enzymatic reactions, see [LM Adleman, Molecular Computation of Solutions to Combinatorial Problems, Science, 266, 1021-1024, (1994)]). In addition, there is a considerably improved environmental compatibility because the polymers described here are biomolecules without any toxicity.

The approach described here has compared to those previously used for DNA computing aqueous solutions the advantage of higher spoke density and the addressability of individual Polymers.

Another object of the present invention is therefore a polymeric data storage device, comprising information-carrying polymers according to the invention, methods according to the invention for linking molecules, readout method according to the invention or Isolation and duplication methods according to the invention.  

The methods described above enable the production of a DNA computer, for example:
A DNA computer can be constructed using the information representation by algomers and logomers already described. The DNA computer includes (see Fig. 10):

• a) Oligonucleotide synthesizer
• b) Thermocycler
• c) pipetting device
• d) Device for isolating polymers
• e) Device for the separation of nucleic acids, eg. B. electrophoresis system
• f) scanner
• g) control computer (workstation, e.g. PC)

The DNA computer is constructed as a hybrid system in which simple, computation-intensive Algorithms or the storage of data using DNA and the necessary Devices (devices a) to e)) are made and a conventional computer (preferably workstation, e.g. PC) serves as host and control computer.

The DNA computer contains at least one thermal cycler, which acts as an "information reactor" serves and can be controlled via the control computer (e.g. MJ Research PTC-100, Eppendorf Mastercycler). Located in the tubes or microtiter plates of the thermal cycler Molecular mixtures of nucleic acids, enzymes and other chemical substances (e.g. buffers, nucleotides) which are necessary for the algomer assembly (step IV of the inventive method) and the symbol polymerization (step V of inventive method) are required. The DNA computer is programmed by implementing algorithms as grammars (step I of the inventive method). The one required for each grammar Monomer sequences are generated by the NFR method and the use of a Oligonucleotide synthesizers (e.g. ABI 392, ABI 398, ABI 3948 from Perkin-Elmer Applied Biosystems) produced (steps II and III of the method according to the invention). This Monomer sequences are used as input in the thermocycler in which the algomer Assembly (step IV of the method according to the invention) takes place.

The algomers thus obtained are placed in one or more reaction chambers of the Thermocycler transferred where they are linked to logomeres (symbol polymerization, Step V of the method according to the invention). The logomers obtained in this way are stored in one own device isolated and reproduced. This device can operate on the principles work that have been described in the inventive method. After isolation and reproduction, the logomeres can be read. To do this, they will turn in read out a thermocycler according to the invention. Those from the selection process resulting nucleic acid fragments can, as already described, by Gel electrophoresis can be read. Here the gel electrophoresis device serves as an output device of the DNA computer, the band patterns obtained in this way are scanned in by a scanner read the control computer.  

The control computer in turn now takes on the basis of the results obtained in this way further calculations and controls and, if necessary, implements further molecular reactions in Gear. For example, the control computer can use the results obtained New implementation of grammars (production of oligomers with the NFR- Procedure). In this way, the by molecular methods as Logomeres receive information not only as passive data but also as Instructions (and addresses) serve. This is a self-governing process different algorithms possible, which is necessary for universal data processing.

The DNA computer processes information as oligo- and polymers. As Data storage cells are used for cloning vectons (plasmids) that are in liquid solution or are on a solid, addressable carrier.

The DNA computer is programmable as described above using grammars and can be controlled by a conventional computer, which acts as a control computer and host system. Since the DNA computer can perform certain operations in molar orders of magnitude (e.g. symbol polymerization, as described in step V of the method according to the invention, in 10 ¹² -10 ²⁰ elementary operations per second), one possible application is the calculation of simple, computationally intensive algorithms. However, the DNA computer can also be used for other applications, e.g. B. the generation of certain, regular DNA structures z. B. are interesting within nanotechnology can be used.

Another object of the present invention is therefore a DNA computer comprising Information-bearing polymers according to the invention. Another subject of The present invention is a DNA computer in which an inventive Selection method and / or isolation and duplication method according to the invention be used.

The processes described above enable, for example, the production of the smallest molecular structures with logomers:
With the logomers described here, the smallest molecular structures (nanostructures) can be produced. The building blocks of such structures are algomers, which can be combined to form regular patterns of logomers. For example, different algomers can be doped with different foreign molecules (ligands) in order to obtain regular, high molecular weight patterns of the respective ligands. The logomers act as a "skeleton" of molecular assemblies of ligands.

For example, a binary logomer could alternate patterns more conductive and non-conductive Contain ligands. By arranging many logomers on a solid support this creates conductor tracks in the nanometer range.  

Biomolecules, antibodies, optically active molecules or the like can also be used as ligands. be used.

Logomeres can be used as an "intelligent" adhesive (see Fig. 18). The hybridization of complementary nucleotides is used here. If logomers are applied to the surfaces to be bonded, which are covalently bound to the surfaces to be bonded, otherwise completely identical surfaces can only be bonded if they have complementary sequences, since only surfaces of complementary nucleic acids form hydrogen bonds. The "intelligence" of the adhesive therefore consists in a highly selective adhesive effect due to the sequences used in each case. The adhesive strength for surfaces to be bonded can also be set precisely: on the one hand, the density of the logomers on the respective surfaces can be used to set their adhesive strength, and on the other hand, the melting point can be varied via the AT / GC ratio of the logomers, so that bonded surfaces lose their adhesion at different temperatures. Due to the fact that harmless, easily degradable biomolecules are used as an adhesive, such adhesives can also be used for medical use (e.g. surgery, micro-surgery). Another use is the use of the described method in high-precision and authentication applications.

If the algomers used for the production of logomers are mixed with sequences for binding ligands, stronger adhesion effects can be achieved. Such ligands can in the case of DNA DNA binding proteins, e.g. B. specific antibodies against DNA sequences. These can e.g. B. can be connected to each other via another ligand (see Fig. 19). A simple, non-programmable adhesive effect can also be achieved without logomers if the proteins used (e.g. antibodies) can in turn bind to the surfaces to be bonded (such as antibodies if the surface to be bonded is their antigen). See also Fig. 20. Simpler adhesives are possible if proteins that bind specifically to certain surfaces are genetically engineered.

With logomeres, surfaces of the most varied structure and most varied can be created physical properties are produced. The applications have in common that with let the logomer form almost any pattern in the nanometer range that the skeleton smallest molecular devices can be.

Another object of the present invention is therefore the use information-carrying polymers, in particular those obtainable according to the invention information-bearing polymers, for the manufacture or processing of the smallest molecular Structures or as molecular glue.  

credentials

Patents

Publications

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Breslauer, KJ, Frank, R., Blocker, H., Marky, LA, Proc. Natl. Acad. Sci., 83, 3746-3750, 1989
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Claims (31)

1. A process for the preparation of information-bearing polymers which comprises

• A) to define a regular grammar G = (Σ, V, R, S) with a finite terminal alphabet Σ, a finite set of variables V, a finite set of rules R and a start symbol S;
• B) the NFR process (Niehaus-Feldkamp-Rauhe process) for the production of monomer sequences;
• C) using the NFR method to implement a grammar defined in step I by producing monomer sequences which uniquely represent the rule set R of a grammar G;
• D) from the monomer sequences prepared in step III for each rule of the rule set R of G an oligomer representing the rule;
• E) to link the oligomers assembled in step IV to form information-carrying polymers.

2. The method according to claim 1, characterized in that the terminal alphabet Σ a grammar G, the terminals 0, 1, n start terminals s (s ₀ , s ₁ ,..., S _n ) and m end terminals e (e ₀ , e ₁ ,..., e _m ), where n and m are each greater than or equal to 0 and represent a natural number. 3. The method according to claim 1 or 2, characterized in that in step III constructed monomer sequence nucleotides, especially ribonucleotides, especially preferably comprises deoxyribonucleotides. 4. The method according to claim 3, characterized in that the constructed in step III Monomer sequence Protein recognition sequences (e.g. restriction sites, Protein binding sites, stop codons). 5. The method according to any one of the preceding claims, characterized in that the synthesis of the monomer sequences in step II and III in vitro, preferably by means of a Oligonucleotide synthesizer.   6. Process for the isolation and duplication of information-carrying polymers, the were obtained according to one of the preceding claims, characterized in that ligating information-carrying polymers obtained in step V into cloning vectors, competent cells transformed with these vectors and the successfully transformed Bacteria selected using selection markers. 7. A method for reading information from information-carrying polymers, which have been obtained or isolated and reproduced according to one of the preceding claims, characterized in that

• a) at least n-1 solutions containing the information-carrying polymer are each mixed with a pair of opposing primers, where n stands for the number of oligomers contained in the polymer as elongators;
• b) carries out at least n-1 PCR runs, where n stands for the number of oligomers contained as elongators in the polymer and one primer of each pair primes in the terminator opposite the elongator and the other primer primes in the elongator itself;
• c) the polymer fragments obtained from the PCR are separated according to their length by electrophoresis and
• d) optically reads the pattern obtained from electrophoresis.

8. The method according to claim 7, characterized in that the reading in step d) automated by a scanner or a sequencing machine. 9. Information-carrying polymer, obtainable according to one of claims 1 to 6. 10. random number generator, comprising information-carrying polymers, in particular Polymers according to claim 9. 11. A polymer data storage device comprising information-carrying polymers according to claim 9. 12. DNA computer, comprising information-carrying polymers according to claim 9. 13. Use of information-carrying polymers according to claim 9 for the production of Molecular weight standards.   14. Use of information-carrying polymers according to claim 9 for the representation of Data structures. 15. Use of information-bearing polymers, in particular of polymers Claim 9 as markers or signatures. 16. Use of information-bearing polymers, in particular of polymers Claim 9 for the purpose of quality assurance. 17. Use of information-bearing polymers, in particular of polymers Claim 9 for the purpose of preventing counterfeiting. 18. Use of information-carrying polymers, in particular of polymers Claim 9 for the purpose of labeling genetic engineering products. 19. Use of information-bearing polymers, in particular polymers according to Claim 9 for the purpose of labeling food. 20. Use of information-bearing polymers, in particular of polymers Claim 9 for the purpose of labeling organisms. 21. Use of information-bearing polymers, in particular of polymers Claim 9 for the purpose of labeling chemical products. 22. Use of information-bearing polymers, in particular of polymers Claim 9 for the purpose of labeling medical and pharmaceutical Products.   23. Use of information-bearing polymers, in particular of polymers Claim 9 for the purpose of marking documents. 24. Use of information-bearing polymers, in particular of polymers Claim 9 for the purpose of labeling money. 25. Use of information-bearing polymers, in particular of polymers Claim 9 for the purpose of labeling objects and machines. 26. Use of information-bearing polymers, in particular of polymers Claim 9 for the purpose of labeling liquids, solutions, suspensions or emulsions. 27. Use of information-carrying polymers, in particular of polymers Claim 9 for encryption of information. 28. Use of information-bearing polymers, in particular of polymers Claim 9 for the purpose of authenticating people and objects. 29. Use of information-carrying polyesters, in particular polymers Claim 9 as a molecular adhesive. 30. Use of information-carrying polymers according to claim 9 for the production or processing of the smallest molecular structures. 31. Use of information-carrying polymers according to claim 9 for Quality control of synthetically produced oligonucleotides.