WO2003072818A2

WO2003072818A2 - Method for sorting single-stranded nucleic acids

Info

Publication number: WO2003072818A2
Application number: PCT/EP2003/002025
Authority: WO
Inventors: Dirk BÖKENKAMP; Tom Heike Dieck; Hans-Ulrich Hoppe
Original assignee: Degussa Bioactives Gmbh
Priority date: 2002-02-28
Filing date: 2003-02-27
Publication date: 2003-09-04
Also published as: WO2003072818A3; DE10208794A1; AU2003214078A1

Abstract

The invention relates to a method for sorting single-stranded nucleic acids by isolating and hybridizing suitable nucleic acid pools followed by a selection. According to the invention, the single-stranded nucleic acids are selectively bound with the aid of capturing substances (capture), which are immobilized on a solid matrix surface and which have variable and invariable areas. This method is characterized in that the capturing substances are oligonucleotides having 5'-terminal invariable anchor sequences that, in complementary form, occur at least once in each single-stranded nucleic acid, and having 3'-terminal variable discriminating sequences consisting of every possible sequence of ≤ 10 nucleotides, which enable the formation of certain types of single-stranded nucleic acids. The invention also relates to the use of this method for analyzing single-stranded nucleic acid patterns, particularly in mucous membrane, hair root, blood, nerve or reproduction cells, and to the use for detecting the action of pharmaceutically and/or nutritional-physiologically active compounds. This novel method makes it possible, in a quick and economical manner, to depict reproducible differences in the single-stranded nucleic acid pool of cells.

Description

Method of sorting single-stranded nucleic acids

description

The present invention relates to a method for sorting single-stranded nucleic acids and the use thereof.

Usually, up to 30,000 genes with a defined nucleic acid sequence are active in a typical human cell, which always characterize the current state of the cell. The state of a cell, for example, indicates an increased cell division activity (reproductive cell;

Cancer cell) or generally altered metabolic activities as they occur in the event of illness or usually with aging. The genetic state of the cell also reacts to exogenous causes, such as changes in eating habits or intoxications. The activity of a specific gene can be, for example, via its mRNA

Transcription product or via the resulting protein as

Translation product can be determined. The mRNA or protein pattern of a cell therefore reflects its current state very precisely.

The characterization of the nucleic acid profile of cells has recently come to the fore, particularly in connection with the screening of pharmacologically active compounds. Numerous methods for characterizing the genetic state of a cell and its protein profile have already been developed.

Such methods for determining specific, different nucleic acid profiles of a cell usually use so-called arrays, ie special matrix systems. Arrays are defined by certain arrangements of immobilized recognition species, which in the meantime play an important role in diagnostics, among others, in the simultaneous one Play determination of so-called analytes. Examples are typical nucleic acid arrays as described in Southern et al., Genomics (1992) 13, 1 008, or in patents US 5,631, 1 34 or US 5,795,71 4.

From WO 96/01 836 an array of DNA molecules of different sequences is known, which is used for the diagnosis of pathogenic bacteria with the help of the detection of special gene segments.

No. 5,795,714 describes a method for replicating an array of nucleic acid samples that can be used on an industrial scale in the production of diagnostics for screening biological samples with special target sequences. Essentially, to create an array of probes, a first set of nucleic acids is first synthesized, which has a constant sequence of a certain length at the 3 'end and a random sequence of a certain length at the 5' end; then a second set of nucleic acids is synthesized with sequences complementary to the constant sequence of the first set of nucleic acids; finally, the first set of nucleic acids is hybridized with the second set of nucleic acids and thus the desired array of probes is obtained which, in addition to a double-stranded region, also have a single-stranded region which in turn has the random sequence.

The methods and arrays known from the prior art are in some cases very complex and designed for the isolation and identification of special nucleic acids or proteins, which is why they predominantly use several method steps.

The object of the present invention is therefore to provide a method for sorting single-stranded nucleic acids in which the disadvantages of the prior art are at least partially eliminated. With this method it is possible in particular With the help of an inexpensive and fast method and in a technically relatively simple way, the entire pattern of single-stranded nucleic acids of a cell can be reproduced.

This object was achieved with the aid of the method according to the invention, which is characterized in that the capture substances are oligonucleotides with 5'-terminal invariable anchor sequences which occur in complementary form at least once in each single-stranded nucleic acid, and with 3 ' -terminal variable discrimination sequences, consisting of several different sequences, preferably each possible sequence of <10 nucleotides, which enable the formation of certain types of single-stranded nucleic acids.

Surprisingly, when using this method, it has been found that the entire and thus complete pattern of all single-stranded nucleic acids in a cell can be reproduced reproducibly, and a diagnostic determination of the status of the cell under investigation is possible in this way. As a result, with the aid of a so-called "sorting chip", this method can not only be used to screen pharmaceutical and / or bioactive compounds, but also in the medical field the finest changes in the nucleic acid pool of cells, in particular of cells with a high division rate, can be shown become. The variety of these possibilities could not have been foreseen.

An essential aim of the method according to the present invention is the formation of certain types of single-stranded nucleic acids. According to the definition, the term “variety” is intended to encompass those single-stranded nucleic acids which occupy a defined distribution site on the respective matrix system (for example a chip). This type of nucleic acid is characterized by the fact that it is a shows selective hybridization with a specific capture substance under the chosen test conditions. The various types of single-stranded nucleic acids present in the sample, which are separated by the method according to the invention, preferably form a "pattern" in their entirety which is characteristic of the condition of a cell.

The method according to the invention should therefore not be used to determine specific nucleic acid sequences, e.g. Sequences of a cell transcriptome are identified and displayed, but rather different patterns are to be formed which reflect a certain (current) state of the transcriptome of a cell. The focus of the method according to the invention is therefore an assignment of nucleic acid patterns and not the mapping of individual genes, which is why matrix miniaturization is also possible.

With this technique e.g. In the context of an expression analysis, a complete distribution of the single-stranded nucleic acids present in the sample can be achieved on an array. For this purpose, capture substances (capture probes) in the form of oligonucleotides or nucleic acid analogs are used, which cover all potentially possible sequences. After completion of the array, the different nucleic acid pools are then hybridized on different matrix platforms (chips) and the resulting patterns are compared with one another. The resulting pattern of the nucleic acid hybridization event can be displayed via suitable detection systems (for example fluorescence).

As a preferred representative of the single-stranded nucleic acids which are to be sorted in a starting pool (analyte), the present method uses a transcriptome, which is in particular RNA and / or DNA. Of course, all types come here single-stranded conveniences, such as RNA in general or mRNA, cRNA, pRNA, DNA, such as cDNA, or nucleic acid analogues, such as PNA, LNA or combinations thereof.

A wide variation is also possible in the case of the capture substances (capture probes) within the claimed features. In principle, however, these capture molecules must have two sections in order to be suitable as such:

(i) an invariable, binding-dependent section with which all single-stranded substances occurring in the analyte (pool)

Nucleic acids are "captured", and (ii) a variable and distribution-dependent on the matrix

Section that has a discriminatory effect on single-stranded nucleic acids.

According to the present invention, the term “invariable sequence” means a single-stranded oligonucleotide section, but also a single-stranded sequence of oligonucleotide analogs, each with a defined nucleotide sequence.

In the context of the invention, the term "variable sequence" is understood to mean nucleic acid sequences, the sequence of which in complementary form has several different, e.g. > 10, preferably> 20, particularly preferably> 50 sequences and most preferably covers all potentially possible sequences of the single-stranded nucleic acids to be sorted.

It has been shown to be particularly advantageous for the present invention if nucleic acid sequences with a length of 10 to 50 nucleotides and in particular of 15 to 25 nucleotides are used as anchor sequences of the capture substances. An oligo-dT sequence, for example for immobilizing polyA-RNA molecules or an oligo-dA sequence, is particularly preferred as anchor sequences Immobilization of polyT-containing cDNA molecules. Other anchor sequences can be consensus sequences or conserved areas of certain gene classes.

For certain applications, it may be advantageous if the capture substances are preferably regularly distributed in nucleotide analogs, e.g. So-called "Locked Nucleic Acids" (LNA), whereby otherwise normal nucleotide sequences at mostly certain locations (e.g. every third position) contain a special nucleotide, which gives the capture substance high affinity properties. This can increase the binding capacity to the single-stranded nucleic acids in the sample to be examined.

As already described, the 3'-terminal, variable sequences of the catcher have the actual discrimination function for cultivar formation. In order to supply all the relevant nucleic acids of an analyte to a sorting pattern, the invention provides that combinations of the nucleic bases adenine, cytosine, guanine, thymine, uracil, inosine and their analogues are used as preferred 3'-discrimination sequences, the length of which 3 Discrimination sequences are advantageously from 3 to 8 nucleotides.

The number of discriminating nucleotides (x) and the number of available bases (usually 4, namely A, G, C, T) determine the size of the matrix that carries the imaging pattern at the end of the sorting process. If, for example, the discriminating sequence is x = 3 nucleotides long, the maximum possible number of capture substances is 64 with the 4 common bases adenine, guanine, cytosine and thymine (4 ³ ). Correspondingly, 256 (4 ⁴ ) are obtained with a 4 nucleotide long discrimination sequence and with a 5 nucleotide long one Discrimination sequence 1 024 (4 ⁵ ) different catcher substances etc.

If there is a large number of occupancy positions available on a corresponding matrix, the length of the discriminating sequence can also be longer, which of course also results in a higher resolution of the distribution pattern.

For the purposes of the present invention, the term “matrix” is understood to mean materials in solid or gel-like form. In particular, plastics, ceramics, glasses, metals, such as e.g. Precious metals, crystalline materials and / or membranes, each with semiconductor properties, are suitable. These can also be in the form of thin layers. A matrix in chip form is to be regarded as particularly preferred.

As also already described, the single-stranded nucleic acids are selectively bound with the aid of the capture substance, which itself is immobilized, that is to say bound, on the matrix surface.

For this purpose, the capture substances are preferred according to the invention via a bioaffine interaction between two partners of a binding pair, e.g. Biotin / streptavidin or avidin or hapten / antibodies etc., bound to the matrix surface. The capture substances in biotinylated form are particularly preferably bound to the matrix surface by means of streptavidin, which preferably represents a layer of (bio) molecular filaments, such as cellulose and agarose, framework proteins, hydrogels or polyacrylamide.

The capture substances (capture probes) are immobilized in a site-specific manner, for example concentrated and immobilized by means of tension at the positions of the array intended for them. This definition happens in a known manner by binding the biotin residues, which are at the 5 'end, with streptavidin, which is fixed in the matrix surface.

Carrier systems in the form of semiconductor chips are known to be very well suited to electronically controllable specific binding reactions of nucleic acids at fixed, addressable locations in the form of an array.

For this reason, the present invention also provides that the capture substances are bound to the matrix surface covalently, quasi-covalently, supramolecularly, physically, in an electrical field or by means of a molecular sieve.

In this way, the nucleic acid pool (analyte) of a sample to be sorted on a nucleic acid array, for example on a semiconductor chip, can be hybridized with the aid of an electrical field in a particularly advantageous manner, and then the bound nucleic acids by simply reversing the polarity of the electrical field be removed. Electronic chip variants are therefore particularly suitable.

In a preferred method variant, the final readout of the fixed single-stranded nucleic acids provided for the present method is carried out by displaying the now sorted nucleic acid fractions as reproducible patterns.

Different patterns that are obtained from different starting analytes can thus be reproducibly compared with one another.

For this reason, the present invention also sees the actual method for sorting single-stranded nucleic acids Use of this method before, the analysis of single-stranded nucleic acid patterns, in particular in mucous membrane, hair root, blood, nerve or reproductive cells, to be regarded as preferred.

These cell types are particularly suitable because they have high division rates and also react relatively quickly to exogenous influences.

Finally, the present invention also provides the use of the claimed method for the detection of the activity of pharmaceutically and / or nutritionally active compounds, such as food additives or supplements, in particular of minerals, trace elements, organic acids, such as amino acids, carboxylic acids and fatty acids, and their derivatives, salts and mixtures.

With this new method it is possible to quickly and inexpensively reproduce differences in the single-stranded nucleic acid pool of different cells within an individual or comparable cells of different individuals, which are due to diseases, malnutrition or malnutrition or other exogenous factors (stress, Medication) and which are reflected in the manifestation (phenotype), for example in the physical and mental state, in behavior and in the psyche.

The following example and the illustrations illustrate the advantages of the claimed method for sorting single-stranded nucleic acids. example

250 ng of polyA-RNA from rat liver were used as analyte

»Untreated and treated animals.

The animals were 24 male Sprague-Dawley rats with an average weight of. 1 21 g (± 9.5), which were divided into two groups with 1 2 animals each. All animals received a semi-synthetic diet (AIN 93G) four times a day by gavage. The diet of the control group ("untreated") contained 25 mg Zn / kg diet, that of the deficient group ("treated") 1.3 mg Zn / kg diet. After 11 days, the animals were decapitated after light anesthesia and the blood was collected and preserved in heparinized tubes for biochemical analysis. The tissue samples taken were snap frozen and stored at -80 ^° C. The total RNA from the liver was isolated using a modified guanidinothiocyanate / phenol / chloroform extraction. A NanoChip ™ cartridge from Nanogen, San Diego (USA) was used as the micro-array, the electronic semiconductor chip of which has 100 (1 x 10) occupied spaces and 100 individually controllable electrodes (maximum charge density: approx. 10 ⁹ fragments per Area; chip size: 0.7 cm ² ; array size: 2 mm ² ).

A NanoChip ™ Molecular Biology Workstation from Nanogen, San Diego (USA), which has a highly sensitive laser-based fluorescence scanner, was used to read out the nucleic acid patterns. It has two lasers, with excitation wavelengths of 635 nm (Laser I) and 532 nm (Laser II).

Detection was carried out using a photomultiplier method (radiation wavelength I: 660 to 720 nm (Cy 5); emission wavelength II: 550 to 600 nm (Cy 3)). Process Description:

The 64 capture substances in the form of oligonucleotides, consisting of 1 8mer dT and 3 discriminating bases (see Table 1), which had a so-called Locked Nucleic Acid (LNA) at every third T base site instead of a normal deoxyribonucleotide Concentrated and immobilized for 1 min at the intended positions of the array using voltage (2.0 volts) (for distribution see Table 2). The biotinylated capture substances were immobilized by binding via streptavidin in the agarose layer of 2 chips as the carrier matrix. After immobilization of the capture probes, the electronic hybridization took place. For this purpose, the unlabelled polyA-RNA samples of the treated and untreated animals were hybridized on the two chips for 2 minutes and under 2.1 volts. After the hybridization, a series of discrimination was carried out in the device using a counter voltage of -0.5 to -1.08 volt in order to remove non-specifically bound molecules. After the discrimination, a so-called Enzymatic Reporting was carried out with the incorporation of fluorescence-labeled nucleotides (Cy 5-CTP) for labeling. The pattern was then fluorescence-optically displayed by laser excitation in the reader (see Figure 3).

On the basis of the fluorescence signals shown, different patterns of single-stranded nucleic acid (RNA) from untreated animals (control group A) and treated deficient animals (test group B) can be reproduced optically.

Table 1: Sequences of the 64 capture oligonucleotides

1.5 'Biotin-ττrττrττ7ττrττrττr-AAA (SEQ ID NO.1)

2.5 'Biotin-TTr TTr TT7 ^" TTr TTr TTr -AAG (SEQ ID NO. 2)

3.5 'Biotin-ττrττrττrττ7ττrττr-AAC (SEQ ID NO.3) , 5'Biot n-ττrττrττrττrττrττr-AAT (SEQ ID NO.4). 5'Biot n-ττrττrττrττrττrττr-AGA (SEQ ID NO. 5). 5'Biot n-ττrττrττrττrττrττr-AGG (SEQ ID NO.6). 5'Biot n-ττrττ7-ττrττrττrττr-AGC (SEQ ID NO.7). 5'Biot n-ττrττrττrττrττrττr-AGT (SEQ ID NO. 8)

9.5'Biot n-ττrττrττ7 ^" ττrττrττr-ACA (SEQ ID NO. 9)

10. 5'Biot n-ττrττrττrττrττrττr-ACG (SEQ ID NO.10)

11. 5'Biot n-TTr TTT TTT TTT TTT TTT -ACC (SEQ ID NO.11)

12. 5'Bioti n-ττrττrττrττrττrττr-ACτ (SEQ ID NO.12)

13. 5'Biot n-ττrττrττrττrττrττr-ATA (SEQ ID NO.13)

14. 5'Biot n-ττrττrττrττrττrττr-ATG (SEQ ID NO.14)

15. 5'Biot n-ττrττrττrττrττrττr-Aτc (SEQ ID NO.15)

16. 5'Biot n-ττrττrττrττ7-ττrττr-ATT (SEQ ID NO.16)

17. 5'Biot n-ττrττrττrττrττrττr-GAA (SEQ ID NO.17)

18.5'Biot n-TT7 TTT TTT TTT TTT TTT -GAG (SEQ ID NO.18)

19. 5'Biot n-ττr TTr TTΓ TTΓ TTΓ TTΓ -GAC (SEQ ID NO.19)

20. 5'Biot Π-TTΓ TTΓ TTΓ TTΓ TTΓ TTΓ -GAT (SEQ ID NO.20)

21. 5'Biot n-ττrττrττrττrττrττr-GGA (SEQ ID NO.21)

22. 5'Biot n-ττrττrττ7ττrττrττr-GGG (SEQ ID NO.22)

23. 5'Biot n-TT7 TTT TTT TTT TTT TTT -GGC (SEQ ID NO.23)

24. 5'Biot n-ττrττ7ττrττrττrττr-GGT (SEQ ID NO.24)

25. 5'Biot n-ττrττrττrττrττrττr-GCA (SEQ ID NO.25)

26. 5'Biot n-TT7 TTT TT7 TTr TT7 TTr -GCG (SEQ ID NO.26)

27. 5'Biot n-TTrTTrTTrTT7-TT7TTr-GCC (SEQ ID NO.27)

28. 5'Biot n-ττrττrττrττrττrττr-GCτ (SEQ ID NO.28)

29. 5'Biot n-ττrττrττ7ττrττrττr-GTA (SEQ ID NO.29)

30. 5'Biot n-ττrττrττ7 ^" ττrττrττr-GTG (SEQ ID NO.30)

31. 5'Biot _n -ττrττrττ7ττrττrττr-Gτc (SEQ ID NO.31)

32.5'Biot n-TTrTT7TT7-TTrTT7TTr-GTT (SEQ ID NO.32)

33.5'Biot n-TTrTT7TT7TTrTT7TTr-CAA (SEQ ID NO.33)

34.5'Biot n-TT7 TT7 TT7 TT7 TT7 TT7 -CAG (SEQ ID NO.34)

35.5'Biot n-TT7TT7TT7TT7TT7TT7-CAC (SEQ ID NO.35) 36.5 ^' Biotin-TT7TT7TT7TT7TT7TT7-CAT (SEQ ID NO.36)

37.5'Biotin-TT7TT7TT7TT7TT7TT7-CGA (SEQ ID NO.37)

38.5 'Biotin-TT7 TT7 TT7 TT7 TT7 TT7 -CGG (SEQ ID NO.38)

39.5 'Biotin-TT7 TT7 TT7 TT7 TT7 TT7 -CGC (SEQ ID NO.39) 40.5 ^' Biotin-TT7TT7TT7TT7TT7TT7-CGT (SEQ ID NO.40)

41.5 'Biotin-TT7 TT7 TT7 TT7 TT7 TT7 -CCA (SEQ ID NO.41)

42.5 'Biotin-TT7 TT7 TT7 TT7 TT7 TT7 -CCG (SEQ ID NO.42)

43.5 'Biotin-TT7 TT7 TT7 TT7 TT7 TT7 -CCC (SEQ ID NO.43)

44.5 'Biotin-TT7 TT7 TT7 TT7 TT7 TT7 -CCT (SEQ ID NO.44) 45.5''Biotin-TT7TT7TT7TT7TT7TT7-CTA (SEQ ID NO.45)

46.5 'Biotin-TT7 TT7 TT7 TT7 TT7 TT7 -CTG (SEQ ID NO.46)

47.5'Biotin-TT7TT7TT7TT7TT7TT7-CTC (SEQ ID NO.47)

48.5'Biotin-TT7TT7TT7TT7TT7TT7-CTT (SEQ ID NO.48)

49.5'Biotin-TT7TT7TT7TT7TT7TT7-TAA (SEQ ID NO.49) 50.5''Biotin-TT7TT7TT7TT7TT7TT7-TAG (SEQ ID NO.50)

51.5'Biotin-TT7TT7TT7TT7TT7TT7-TAC (SEQ ID NO.51)

52.5 'Biotin-TT7 TT7 TT7 TT7 TT7 TT7 -TAT (SEQ ID NO.52)

53.5 ^' Biotin-TT7TT7TT7TT7TT7TT7-TGA (SEQ ID NO.53)

54.5 'Biotin-TT7 TT7 TT7 TT7 TT7 TT7 -TGG (SEQ ID NO.54) 55.5 ^' Biotin-TT7TT7TT7TT7TT7TT7-TGC (SEQ ID NO.55)

56.5 'Biotin-TT7 TT7 TT7 TT7 TT7 TT7 -TGT (SEQ ID NO.56)

57.5'Biotin-TT7TT7TT7TT7TT7TT7-TCA (SEQ ID NO.57)

58.5 'Biotin-TT7 TT7 TT7 TT7 TT7 TT7 -TCG (SEQ ID NO.58)

59.5 'Biotin-TT7 TT7 TT7 TT7 TT7 TT7 -TCC (SEQ ID NO.59) 60.5''Biotin-TT7TT7TT7TT7TT7TT7-TCT (SEQ ID NO.60)

61.5'Biotin-TT7TT7TT7TT7TT7TT7-TTA (SEQ ID NO.61)

62.5'Biotin-TT7TT7TT7TT7TT7TT7-TTG (SEQ ID NO.62)

63.5'Biotin-TT7TT7TT7TT7TT7TT7-TTC (SEQ ID NO.63)

64.5'Biotin-TT7TT7TT7TT7TT7TT7-TTT (SEQ ID NO.64) Table 2: Distribution of capture substances (capture probes) on a chip

The distribution of the capture substances is shown on a chip with 1 0 x 10 filling positions; columns 1 and 2 contain sequence-specific control gene oligonucleotides; empty fields mean vacant chip spaces.

The read out patterns are shown in Figure 1: (A) control and (B) manganese RNA. Significant differences can be seen in the patterns of both groups of animals, which enable a diagnostically relevant statement.

Claims

Expectations

Method for sorting single-stranded nucleic acids by isolating and hybridizing suitable nucleic acid pools and then reading them out, the single-stranded nucleic acids being selectively bound with the aid of capture substances immobilized on a solid matrix surface which have variable and invariable regions, characterized in that they are the capture substances are oligonucleotides, comprising

(i) 5'-terminal invariable anchor sequences, which occur in complementary form at least once in each single-stranded nucleic acid, and

(ii) 3'-terminal variable discrimination sequences, consisting of several different sequences of ≤ 1 0 nucleotides, which enable the single-stranded nucleic acids to be formed.

A method according to claim 1, characterized in that the single-stranded nucleic acids are a transcriptome, in particular RNA and / or DNA.

Method according to one of claims 1 or 2, characterized in that nucleotide base T sequences with a length of 10 to 50 nucleotides and in particular of 1 5 to 25 nucleotides are used as anchor sequences. -r. v cnαi ii ci i nα n uer πsprucπe i DIS, characterized in that the capture substances preferably have "Locked Nucleic Acids" (LNA) in a regular distribution.

5. The method according to any one of claims 1 to 4, characterized in that 3'-discrimination sequences are used which contain combinations of the nucleic bases adenine, cytosine, guanine, thymine, uracil and inosine and / or their analogs.

6. The method according to any one of claims 1 to 5, characterized in that the 3'-discrimination sequences have a length of 3 to 8 nucleotides.

7. The method according to any one of claims 1 to 6, characterized in that as a matrix plastics, ceramics, glasses, metals, resins, gels, crystalline materials and / or membranes, each with

Semiconductor properties, and are preferably used in chip form.

8. The method according to any one of claims 1 to 7, characterized in that the capture substances, preferably in biotinylated form, are bound to the matrix surface by means of streptavidin, which preferably comprises a layer of (bio) molecular filaments, such as cellulose and agarose, Framework proteins, hydrogels or polyacrylamide.

, Method according to one of claims 1 to 8, characterized in that the capture substances are bound to the matrix surface covalently, quasi-covalently, supramolecularly, physically, in an electrical field or by means of a molecular sieve.

10. The method according to any one of claims 1 to 9, characterized in that the reading comprises a representation of the sorted nucleic acid fractions as reproducible patterns.

11. Use of the method according to one of claims 1 to 10 for the analysis of single-stranded nucleic acid patterns, in particular for the determination of an expression profile of cells in mucous membrane, hair roots, blood, nerve or reproductive cells.

12. Use according to claim 11 for the analysis of nucleic acid patterns.

13. Use of the method according to one of claims 1 to 10 for the detection of the activity of pharmaceutically and / or nutritionally active compounds, such as food additives or supplements, in particular of minerals, trace elements, organic acids, such as amino acids, carboxylic acids and fatty acids, and their derivatives,

Salts and mixtures.