Method for detecting cytosine methylations

ethylations method for detection of cytosine

The present invention relates to a method for the detection of cytosine methylations in DNA.

By the methodological developments of recent years in molecular biology well studied observation that the genes themselves, the translation of these genes into RNA and the resulting proteins. Which gene is turned on during the development of an individual and how the activation and inhibition of certain genes is controlled in certain cells and tissues, is correlated with the extent and nature of the methylation of the genes or of the genome. This regard, pathogenic states are also expressed by a modified methylation pattern of individual genes or of the genome.

5-methylcytosine is the most frequent covalently modified base in the DNA of eukaryotic cells. For example, to play a role in the regulation of transcription, in genetic imprinting and in tumorigenesis. The identification of 5-methylcytosine as a component of genetic information is thus of considerable interest. However, 5-methylcytosine positions can not be identified by sequencing, since 5-methylcytosine has the same base pairing behavior as cytosine. In addition comes the epigenetic information carried by 5-methylcytosine is completely lost during a PCR amplification.

A relatively new and now the most frequently used method for analyzing DNA for 5-methylcytosine is based on the specific reaction of

Bisulfite with cytosine which, upon subsequent alkaline Hydrolysis is converted into üracil, which corresponds in its base-pairing behavior to thymidine. 5- methylcytosine is not modified under these conditions. Thus the original DNA is converted so that methylcytosine, which originally can not be distinguished from cytosine by its hybridization behavior, now through "normal^'"Molecular biology techniques can be detected as the only remaining cytosine, for example, by amplification and hybridization or sequencing. All these techniques are based on base pairing, which can now be fully exploited. The prior art, which concerns sensitivity, is defined by a method to examined DNA in an agarose matrix, thus preventing the diffusion and renaturation of the DNA (bisulfite reacts only on single-stranded DNA) and all precipitation and purification steps by rapid dialysis (Olek, A. et al., Nucl. Acids. Res. 1996, 24, 5064-5066). Using this method, individual cells can be investigated, which illustrates the potential of the method. However, so far only individual regions of up to approximately 3000 base pairs long, a global analysis of cells for thousands of possible methylation analyzes is not possible. However, this method can analyze very small fragments from small sample quantities reliably. These are lost despite the protection from diffusion through the matrix.

An overview of other known possibilities for detecting 5-methylcytosine may be taken from the following review article: Rein, T., DePamphilis, ML, Zorbas, H., Nucleic Acids Res 1998, 26, 2255th.

The bisulfite technique has been, with few exceptions (eg. B. Zechnigk, M. et al., Eur. J. Hum. Gen. 1997, 5,

94-98) used only in research. But always be plifziert short, specific segments of a known gene after bisulfite treatment and either completely sequenced a (Olek, A. and Walter, J., Nät. Genet. 1997, 17, 275-276) or individual cytosine positions by a "primer -Extension reaction^Λλ (Gonzalgo, ML and Jones, PA, Nucl. Acids Res. 1997, 25, 2529-2531, WO Patent 9,500,669) or an enzyme cleavage (Xiong, Z. and Laird, PW, Nucl. Acids. Res. 1997, 25, 2532-2534) proved .. In addition, is also the detection been described by hybridization (Olek et al., WO 99 28498).

Other publications which deal with the application of the bisulfite technique for methylation detection in individual genes are: Xiong, Z. and Laird, PW (1997), Nucl. Acids Res. 25, 2532; Gonzalgo, ML and Jones, PA (1997), Nucl. Acids Res. 25, 2529; Grigg, P. and Clark, S. (1994) Bioassays 16, 431; Zeschnik, M. et al. (1997), Human Molecular Genetics 6, 387; Part, R. et al. (1994), Nucl. Acids Res. 22, 695; Martin, V. et al. (1995), Gene 157, 261; WO 97 46705, WO 95 15373 and WO 45560th

An overview of the prior art in oligomer array production can be taken from one in January 1999 achieved schienenen special edition of Nature Genetics (Nature Genetics Supplement, Volume 21, January 1999), the literature cited therein and US Patent 5,994,065 on methods for the production of solid supports for target molecules such as oligonucleotides remove background signal at reduced non-specific way.

fluorescently labeled probes are often used for scanning an immobilized DNA array frequently. Particularly suitable for fluorescent labels is the simple introduction of Cy3 and Cy5 dyes to the 5'-OH of the respective probe. The detection of the fluorescence of the hybridized probes for example, via a confocal microscope. Cy3 and Cy5 dyes, besides many others, are commercially available.

Matrix-assisted laser desorption / ionization

Mass spectrometry (MALDI-TOF) is a very powerful development for the analysis of biomolecules (Karas, M. and Hillenkamp, ​​F. (1988), laser desorption ionic zation of proteins with molecular masses exeeding 10,000 daltons. Anal. Chem. 60: 2299-2301). An analyte is embedded in a light-absorbing matrix. A short laser pulse, the matrix is ​​evaporated and the analog lytmolekül transported unfragmented into the gas phase. By collisions with matrix molecules the ionization of analytes is achieved. An applied voltage accelerates the ions into a field-free flight tube. Due to their different masses, the ions are accelerated at different rates. Smaller ions reach the detector sooner than bigger ones.

MALDI-TOF spectroscopy is excellently suitable for the analysis of peptides and proteins. The analysis of nucleic acids is somewhat more difficult (Gut, IG and Beck, S. (1995)), DNA and Matrix Assisted Laser Desorption Mass Spectrometry Ioniza- tion. Molecular Biology: Current innovation vation and Future Trends 1: 147-157) For nucleic acids, the sensitivity is approximately 100 times poorer than for peptides and decreases with increasing fragment size from disproportionately.. For nucleic acids having a multiply negatively charged backbone, the ionization by the matrix is ​​considerably less efficient. In MALDI-TOF spectroscopy, the choice of matrix plays an eminently important role. For the desorption of peptides, several very efficient matrixes have been found which produce a very fine crystallization. For DNA, there are indeed mittlererweile several effective matrices, however, by the difference in sensitivity has not been reduced. The difference in sensitivity can be reduced by modifying the DNA chemically in such a way that it is a peptide similar. P osphorothioatnuklein- acids, in which the usual phosphates of the backbone are substituted by thiophosphates, can be prepared by simple alkylation in a charge-neutral DNA convert (Gut, IG and Beck, S. (1995), A procedu- re for selective DNA alkylation and detection by mass spectrometry Nucleic Acids Res. 23:. 1367-1373). The

Coupling of a "charge tag" to this modified DNA results in an increase in sensitivity by the same amount as is found for peptides. Another advantage of "charge tagging" is the increased stability of the analysis against impurities which the detection of unmodified substrates greatly complicate.

Genomic DNA is obtained by standard methods from DNA of cell, tissue or other test samples. This standard methodology is found in references such as

Fritsch and Maniatis eds. , Molecular Cloning: A laborato- ry Manual., 1989

Urea improves the efficiency of the bisulfite treatment prior to the sequencing of 5-methylcytosine in genomic DNA (Paulin R, Grigg GW, Davey MW, Piper AA (1998), Nucleic Acids Res. 26:. 5009-5010).

The object of the present invention is therefore a procedure for the detection of cytosine methylation in DNA to make available, which overcomes the disadvantages of the prior art.

The object is achieved by providing a method for the detection of cytosine methylation in DNA is provided to give off the following steps resulting: a) a genomic DNA sample is incubated with a solution of a bisulfite (= hydrogen sulfite, disulfite) in the concentration range between 0.1 and 6 mol / 1, wherein a de-naturierendes reagent and / or solvent and at least one radical scavenger is present ; b) the treated DNA sample is diluted with water or an aqueous solution; c) the DNA sample is amplitude-fied in a polymerase reaction; d) one detects how far the sequence has changed by the treatment according to step a) with respect to the genomic DNA sample and concludes the methylation state of at least one locus in the genomic DNA sample.

According to the invention, there is the fact that the denaturing reagent and / or solvent is selected from the following list of compounds or classes of compounds:

Polyethylene glycol dialkyl, dioxane and substituted derivatives, urea or derivatives, acetonitrile, primary alcohols, secondary alcohols, tertiary alcohols, diethylene lenglykoldialkylether, triethylene, Tet raethylenglykol dialkyl, Pentaethylenglykoldiaky- ether, Hexaethylenglykoldialkylether, DMSO, THF.

Preference is further is that the scavenger from the following group is selected from compounds: di-, trihydroxybenzenes, green tea extract (green tea extract), Pycnogenol (pine bark extract), Ginkgo Biloba extract (EGb 761), flavonoid mixture of different fruit - and vegetable extracts (GNLD), Bio-Normalizer (Sun-0 Corp), DPPH (1, l-diphenyl-2-picrylhydrazyl), NDGA (Nordihydroguajaret acid), Trolox (6-hydroxy-2, 5, 7, 8 - tetramethylchroman-2-carboxylic acid), 2,6-di-tert- butylphenol, 4-methyl-di-tert-butylphenol, 4-methoxy-di-tert-butylphenol, 2, 6-di-tert-butyl-p-cresol, 3,4-dihydroxybenzoic acid, vitamin C, vitamin E, vitamin Q , hydroquinone, ubiquinone, lignans, Hydroxyterpene, flavonoids, curcumin, tannins, Retinsäureverbindungen, Ge-132 Bisbetacarboxyethyl germanium sesquioxide, superoxide dismutase (SOD), superoxide catalase, alpha naphthoflavone, di (2-methyl 5-chlorophenyl) dithionate and Cu (II) - derivatives, mebendazole, CS (Chloroformlöslicher) Älkaloid- extract,

4- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-hydroxy-l, 2-naphthoquinone,

4- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-methoxy-l, 2-naphthoquinone, 4- (3, 5-di-tert-butyl-4-hydroxyphenyl) -1, 2- naphthoquinone, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-bromo-l, 4- naphthoquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-chloro-l, 4- naphthoquinone, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-methoxy- l, 4-naphthoquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-hydroxy-l, 4- naphthoquinone, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -1, 4- naphthoquinone, 4- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-hydroxy-5, 5,8,8- tetramethyl-5, 6, 7, 8-tetrahydro-l, 2-anthraquinone, 4- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-methoxy-5, 5,8,8- tetramethyl-5, 6, 7, 8-tetrahydro-l, 2-anthraquinone, 4- (3,5-di-tert-butyl-4-hydroxyphenyl) -5,5, 8,8- tetramethyl-5, 6, 7, 8-tetrahydro-l, 2-anthraquinone,

3-bromo-4- (3, 5-di-tert-butyl-4-hydroxyphenyl) -5, 5,8,8- tetramethyl-5, 6, 7, 8-tetrahydro-l, 2-anthraquinone, 2- (3, 5-di-tert-butyl-4-oxocyclohexa-2, 5-dienylidene) - indan-1, 3-dione, 2- (3, 5-di-tert-butyl-4-oxocyclohexa-2, 5 -dienyliden) -3, 4-epoxy-3-hydroxy-4-methoxy-3, 4-dihydro-2H-naphthalen-l-one, 2- (3, 5-di-tert-butyl-4-oxocyclohexa-2, 5-dienylidene) -3, 4-epoxy-3, 4-dimethoxy-3, 4-dihydro-2H-naphthalen-l-one, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -indan-1-one, 3, 3-bi- [2- (3, 5-di-tert-butyl-4-hydroxyphenyl) indene -1-one] -3-yl, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-bromo-5, 5,8,8- tetramethyl-5, 6, 7, 8- tetrahydro-l, 4-anthraquinone, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-chloro-5, 5,8,8- tetramethyl-5, 6, 7, 8-tetrahydro- l, 4-anthraquinone, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-methoxy-5, 5,8,8- tetramethyl-5, 6, 7, 8-tetrahydro-l, 4-anthraquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-hydroxy-5, 5,8,8- tetramethyl-5, 6, 7, 8-tetrahydro-l, 4-anthraquinone, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -5, 5,8,8- tetramethyl-5, 6, 7, 8-tetrahydro-l, 4-anthraquinone, 2-bromo-3- (3 -bromo-5-tert-butyl-4-hydroxyphenyl) -5,5,8,8- tetramethyl-5, 6, 7, 8-tetrahydro-l, 4-anthraquinone, 2-bromo-3- (3, 5 -dibromo-4-hydroxyphenyl) -5,5,8,8- tetramethyl-5, 6, 7, 8-tetrahydro-l, 4-anthraquinone, 2-bromo-3- (3-bromo-5-tert-butyl -4-hydroxyphenyl) -3-hydroxy- 5,5,8, 8-tetramethyl-5, 6, 7, 8-tetrahydro-l, 4-anthraquinone, 3-bromo-2- (3, 5-di-tert -butyl-4-hydroxyphenyl) -1, 4-anthraquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-methoxy-l, 4-anthraquinone, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-hydroxy- l, 4-anthraquinone,

5,5,8, 8-tetramethyl-5, 6, 7, 8-tetrahydronaphthalene-l, 3- diol, 3-methoxy-5, 5, 8, 8-tetramethyl-5, 6,7,8-tetrahydronaphthalene 1-ol,

4- (3-chloro-5, 5, 8, 8-tetramethyl-l, 4-dioxo-l, 4, 5, 6, 7, 8-hexahydroanthracene-2-yl) benzoic acid, methyl-4- (3 -chloro-5, 5, 8, 8-tetramethyl-l, 4-dioxo-1,4,5,6,7, 8-hexahydroanthracene-2-yl) benzoate, 4- (3-hydroxy-l, 4 -dioxo-l, 4-dihydronaphthalene-2-yl) - benzoic acid, Methyl (3-methoxy-l, 4-dioxo-l, 4-dihydronaphthalene-2-yl) - benzoic acid,

4- (3-hydroxy-5, 5, 8, 8-tetramethyl-l, 4-dioxo-l, 4, 5, 6,7,8-hexahydroanthracene-2-yl) benzoic acid, methyl-4- (3 -hydroxy-l, 4-dioxo-l, 4-dihydronaphthalene-2-yl-azo) benzoate, 4- (3-hydroxy-5, 5, 8, 8-tetramethyl-l, 4-dioxo-l, 4 , 5,6,7,8-hexahydroanthracene-2-yl-azo) benzoic acid,

3- (3, 5-di-tert-butyl-4-oxocyclohexa-2, 5-dienylidene) - 5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-cyclopenta [b] naphthalene-1, 2-dione,

3- (3, 5-di-tert-butyl-4-oxocyclohexa-2, 5-dienylidene) -

5,5,8, 8-tetramethyl-5, 6, 7, 8-tetrahydroanthracene-3H-l, 2, 4- trione, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl -3-methoxy -5, 8- dimethyl-1, 4-naphthoquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl -3-methoxy-6, 7-dimethyl-1, 4-naphthoquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl -3-methoxy-5-methyl-1, 4-naphthoquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl -2-methoxy-5-methyl-

1, 4-naphthoquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl -3-methoxy-6-methyl-

1, 4-naphthoquinone, 3- (3, 5-di-tert-butyl-4-hydroxyphenyl -2-methoxy-6-methyl-

1, 4-naphthoquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl -3-methoxy-5, 6- dimethyl-1, 4-naphthoquinone,

3- (3, 5-di-tert-butyl-4-hydroxyphenyl -2-methoxy-5, 6- dimethyl-1, 4-naphthoquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl -3-methoxy-5, 7- dimethyl-1, 4-naphthoquinone,

3- (3, 5-di-tert-butyl-4-hydroxyphenyl -2-methoxy-5, 7- dimethyl-1, 4-naphthoquinone, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl - 3-ethylthio-5-methyl-1, 4-naphthoquinone, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-ethylthio-6-methyl-1, 4-naphthoquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-hydroxy-5, 8-dimethyl-1, 4-naphthoquinone, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl ) -3-hydroxy-6, 7-dimethyl-1, 4-naphthoquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-hydroxy-5-methyl-

1, 4-naphthoquinone,

3- (3, 5-di-tert-butyl-4-hydroxyphenyl) -2-hydroxy-5-methyl-1, 4-naphthoquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-hydroxy-6-methyl-

1, 4-naphthoquinone,

3- (3, 5-di-tert-butyl-4-hydroxyphenyl) -2-hydroxy-6-methyl-

1, 4-naphthoquinone, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-hydroxy-5, 6- dimethyl-1, 4-naphthoquinone,

2- (3-bromo-5-tert-butyl-4-hydroxyphenyl) -3-hydroxy-5, 6- dimethyl-1, 4-naphthoquinone,

3- (3, 5-di-tert-butyl-4-hydroxyphenyl) -2-hydroxy-5, 6- dimethyl-1, 4-naphthoquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-hydroxy-5, 7- dimethyl-1, 4-naphthoquinone,

3- (3, 5-di-tert-butyl-4-hydroxyphenyl) -2-hydroxy-5, 7- dimethyl-1, 4-naphthoquinone.

According to the invention preference is that one thermally denatured genomic DNA sample prior to treatment.

Particularly preferred according to it, that you c) performs the step in two steps as follows: a) a PCR preamplification with at least one pair of primers of different sequence which hybridizes to a pretreated according to claim 1 DNA sample nonspecific and therefore the PCR step over an amplicon yield; b) a PCR amplification of the product formed in the preamplification with primers of different sequence, each to a portion of the pretreated according to claim 1 DNA sample [(+) strand or (-) strand] are identical or inversely complementary to and hybridize specifically amplified DNA ornamental.

It is according to the invention further preferred that by carrying out the amplification of several DNA segments in one reaction vessel.

It is preferred according to the invention also there is used a heat-resistant DNA polymerase for the polymerase reaction.

Particularly, it is preferable in the invention that prior to step c) of the inventive method a Desulfo- ordination of DNA performs.

It is also preferred that hybridizes to the detection of the pretreated DNA, the PCR products on a Oligonukle- otid array and one then the following sub-steps: a) the amplified genomic DNA is hybridized to at least one oligonucleotide, forming a duplex, wherein said hybridized oligonucleotides with their 3 'end directly or at a distance of up to 10

Bases adjacent to the positions with respect to their

Methylation must be examined in the genomic DNA sample;

(B) to the oligonucleotide with known sequence of n nucleotides by means of a polymerase by at least one

Nucleotide extended, wherein the nucleotide bears a detectable label and the extension depends on methylation state of the respective cytosine in the genomic DNA sample. It is preferable in the invention that for the detection of the pretreated DNA tion, the PCR products on an oligonucleotide array hybridized and you then run the following substeps: (a) hybridizing a set of oligonucleotides to the amplified genomic DNA, forming a duplex , whereby this set of oligonucleotides from two different species and whereby the hybridized oligonucleotides of the first species with its 3 'end directly or at a distance of up to 10 bases to the

Positions adjacent, which are to be investigated relative to their methylation in the genomic DNA sample, and wherein the second oligonucleotide of the second species hybridizes to a second region of the target molecule, so that the 5 'end of the oligonucleotide of the second species through a gap the size of a Einzelnukleotides or up to 10 nucleotides is isolated from the 3 'end of the hybridized oligonucleotide of the first species at the site of said selected position; (B) is at most extended, the oligonucleotide of the first species with known sequence of n nucleotides by a polymerase to the number of nucleotides. Between the 3 'end of the oligonucleotide of the first species and the 5' end of the oligonucleotide of 2 species are, the extension of the methylation state of the respective cytosine depends in the genomic DNA sample; (C) incubating the oligonucleotides in the presence of a ligase, whereby the adjacent, tion by the Polymerasereak- extended oligonucleotide of the first species and the oligonucleotide of the second species are joined and one thereby obtains a ligation product if the preceding step, an extension of the oligonucleotide of first species such carried out that now the 3 'end is contiguous with existing 3' -Hydroxyfunktion the extended oligonucleotide immediately adjacent to the 5 'end of the oligonucleotide of the second species. ω ω M> H¹

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In particular, it is also preferred that the PCR products and / or extension products and / or ligation to modulation products in total evidence in the mass spectrometer and are thus clearly characterized by their mass. It is preferred according to the invention also means that you can prove each a fragment of the PCR products and / or extension products and / or ligation products in the mass spectrometer.

The inventive method is preferably also characterized in that it generates the fragment of the PCR product and / or extension product and / or ligation product by digestion with one or more exo- or endonucleases.

It is further preferred that one tierbarkeit provides for improved detection in the mass spectrometer, the fragments generated with a single positive or negative net charge.

With very particular preference, that detects and visualizes the PCR products and / or extension products and / or ligation products means of matrix assisted laser desorption ons / ionization mass spectrometry (MALDI-TOF) or by means of electrospray mass spectrometry (ESI) is.

The inventive method is also such an advantage, in the obtained genomic DNA from a DNA sample, whereby sources for DNA z. B. cell lines, blood, sputum,

Stool, urine, cerebrospinal fluid, tissue embedded in paraffin tissue, for example, tissue from eyes, intestine, kidney, brain, heart, prostate, lung, breast or liver, histological slides and all possible combinations thereof include. oo M tv>> cπ o o Cπ Cπ o cπ

<Ö Φ CL 3 rt CQ M li rt φ o π d CN P "5 d rt ^ ω σ iQ Ω CQ iQ li d rt PJ CL t φ μ- μ- Φ μ- Φ Ω μ- φ ^ Φ ii od φ φ li P φ φ Ω φ φ tr μ- φ φ μ- μ- μ- μ- iQ li φ d H d rt tr P d * d Holy iQ ^•d li CO PJ CL 3 tr tr J 3 doodd co φ d φ d

Hi Φ Φ rt HN φ μ- Φ Hi O d \ CD PJ: d d Φ • • • • od 0

PJ <! or φ d μ- μ- -z ddd CQ 3 S i- o •• Hi fr d O CQ Φ rt CQ <s: o tr § Φ φ φ CL CQ Ω Φ ß ß tr CL μ- tr N d iQ CQ O ß Φ φ Φ Φ

HH> dd 50 P μ- li O ß rt tr rt co * -ι φ φ <d ιQ φ CQ μ- _*<; 3 d 3 d li μ-

Φ H o Φ H rt d CL d PJ 3 J φ μ- li Φ fr d μ- ß * <; Cfl 3 ω Φ μ- ß 2, rt d H o μ- μ- PJ Φ d φ φ d iQ he Φ tr rt rt 1-5 et d d Ω Ω 13 1 H d 0 d Φ φ

Φ PJ Hi co iQ Ω ü iQ li CQ o H- ta I-¹ μ- iQ -. CL tr tr et ^ 1 ~ t CL CL d H

N iQ << μ- o Φ d φ d -QNH Hi CL Φ Φ o \ o O Φ Φ ß: Φ Φ \ CL φ d Φ CQ xs: d H Ω φ α Φ μ- ß Φ rt d O ^ o rt 3 tr d Q li od H

3 d J μ- N φ Φ 3 Φ d co d co o N CL Φ CL μ- ß Φ O rt d α d rt Φ μ- Ώ st PJ: H Ω α fr d B Φ tr Φ Q d Hi Ω Φ H Φ ιΩ O

3 φ J Φ P Φ Φ EP o tr Φ rt ^ <! d H co li Ω CL O ß tr dd H Φ

PJ Ω ** "ΪΛ CL iQ he Φ t Φ o μ- O: PJ iQ Ω Holy tr CL P rt CQ CL Φ LQ

Ω f tr PJ Φ «Φ Φ Φ d H o li H¹ -> Tr ^ ß J Φ Q Φ Φ for μ- πd H- Φ tr li CQ CL d μ- dd Hi H ^ ω d • d Φ pj: H d co OH rt φ <! H dd

~ Hi φ O μ- PJ rt CQ <μ- φ H Φ CO t? CL PJ fr co Λ N μ-> μ- μ- O Φ co

Φ μ- μ- fr rt •_^ rt o Φ P CQ P^" d li N d μ- d rt O rt μ- <o H d CL iQ CQ rt μ- μ- PP P> d P> CL CL H φ α CΛ CQ φ rt -Q fr μ- NO: J Φ PN φ ß d PJ

CQ CL φ φ H he d Φ Hi ß X Hi Φ Ω P INI dto μ- H li φ d is Φ o Φ dd 3 Hi μ- Φ li PJ P dd iQ tr PJ N ß: d Φ d μ- d φ tr P Φ d CQ li CL

<! d φ PJ: Φ CQ tr iQ Φ d Φ PJ: I-¹ , d VQ μ- φ P PJ_^ μ- CL _^"Hi Φ

O iQ Φ μ- P li rt CL NH fr s: CL CL co d rt H iQ i H "3 Φ μ- CL

P is d Q φ φ φ φ μ- d rt Φ μ- ^ d O d rt Φ o et CΛ μ- H s; dd φ co Hi Φ φ d tr li HP co Φ μ- he ιQ> Φ d CL L CL Φ dd Φ Ω rt o PJ CL li

S rt μ- li CL φ CQ et o Φ ß he tr iQ Φ Φ CQ d tr rt Hi He Ω ß φ Φ d> P φ P <! α ^ d CQ d ~ t "li Q CΛ CO d Φ CQ PJ: Φ O Φ tr d <rt CL dd CL O d N PJ Φ iQ Φ Ω • Holy <d Λ CO CL μ- μ- rt iQ O tr dd ι-i rt ß d tr o μ- d H t iQ tr d d CL et Φ ≤ Φ tQ CQ H

^»<Rt d Φ d IΛ JH φ Ω H CΛ CL o Ω dd li J PJ: CL φ O: d μ- Φ α μ- iQ l-¹

H tQ μ- CL φ ß μ- H tr Hi Ω φ CL tr fr Hi PJ co H πd d HH d μ- H Φ μ-

Ω Φ co rt J CQ Φ co α ß tr li φ d rt Φ d rt μ- o Φ N do for CL φ φ 3 μ-

^ μ- ^•Q d πd iQ iQ ddd PJ: H d μ- fr fr li li ιQ 3 Φ d CL ß φ ω iQ JU: Q rt d Φ d H Φ φ φ Ω rt μ- fr CL CL U3 o rt tr O ß φ CO d iQ Φ dd φ Φ Φ EP

O 3 iQ μ- μ- d d tr φ φ et μ- Φ "d μ- Φ μ- d O: Φ H among others li Φ d

CQ PJ P>: 3 N d d H CL μ- μ- -QHH *_* o μ- d iQ d <! d Φ ^ dd CL μ- d EP N φ μ- Φ φ do <o ß PJ 3 d rt rt • fr h-¹ O • K d JP⁾ M Φ

P rt Φ d li Φ 3 d iQ Ω φ dd ^ d CΛ _^ Φ J μ- d H rt Ω lS <d

? O d H H ddt • iQ d fr M Ω CL CO ^ li Ω r J Φ tr Φ it Φ 3 CD H CL φ s: O tr tr d H φ rt Φ CL tr Q d ^ iQ rt μ- li M

Φ J <σ N o μ- ι-i φ μ- φ Ω Φ O rt PJ: ^ o tr μ- μ- μ- fr Φ fr li O Φ iQ Hi H ii rt Φ dd LO Hi li d CL CL p - μ- s: CL § P o Φ tr tr P Φ ι-i μ- d J Hi

Φ μ- H li H Φ ß ß μ- μ- μ- CL Φ Φ r d> PJ Hi <l μ- μ- μ- Φ he μ- H "μ- μ- μ- tr CQ Hi Ω li d H tsι dd H d Ω ιQ d rt M d PJ et H CQ μ- μ- O φ CQ li d rt μ- J tr JE Hi φ iQ d> • CL fr ß H 3 φ d CQ Q d Ω rt φ d • ιQ o φ

* Φ tr Holy φ i → μ- d -Q "φ d rt ß P fr fr CQ co tr • H Φ Φ dd ι-i li d = li O: rt d <! H Φ f ä ß iQ PJ: P rt ß et Ω Φ fr J d d CQ he st Φ tr CQ CQ iQ o φ PJ PP et -QH μ- O: tr "*> HP Hi iQ

Φ PJ d li d rt φ μ- μ- d d CL tc iQ O CQ H o ​​PJ: li P): iQ iQ PJ ß: N

HH d Φ dd μ- Hi d fr o PJ CQ CQ CL PJ d li d α ^•d d Φ iQ ti li ß μ-

Ω Φ PH¹ iQ rt CQ Hi ιsι φ tr X ß d Φ • <: fr Φ d μ- CQ H fr tr Φ ι-i tr CQ CQ iQ CO tr rt φ Φ Q φ li rt HH CQ et iQ ιQ • <; Φ d O: μ- π rt

Φ d 1 J 1 (_. Μ- μ- • * O 1 Φ φ d 1 d 1 CO ιQ PJ σ co d r-¹ rt d 1 co dd 1 1 1 1 1 1 H Φ • 1 1

only pipetting contains. This increases the efficiency of existing procedures relating to improving the ease of handling, the quality, the cost and especially the throughput.

is described in an automatable method for the detection of methylcytosine in genomic DNA samples:

To be analyzed genomic DNA is preferably obtained from the usual sources of DNA such. Eg, cell lines, blood, sputum, stool, urine, cerebrospinal fluid, tissue embedded in paraffin, for example tissue from eyes, intestine, kidney, brain, heart, prostate, lung, breast or liver, histological slides and all possible combinations thereof.

In the first step of the process, the DNA used preferably treated with bisulfite (= disulfite, hydrogen sulfite) such that all non se at the 5-position of the base methylated cytosine are changed so that a different regarding the base pairing behavior Base incurs while methylated in the 5-position cytosines remain unchanged.

The genomic DNA sample is denatured thermally particularly preferably before treatment.

If the reaction bisulfite in the concentration range between 0.1 and 6 mol / 1, as an addition occurs on the unmethylated cytosine bases. also need to be present, a denaturing reagent or solvent as well as a scavenger for the inventive method. Here come as denaturing reagents or solvents preferably the following compounds or classes of compounds in question:

Polyethylene glycol dialkyl, dioxane and substituted derivatives, urea or derivatives, acetonitrile, primary alcohols, secondary alcohols, tertiary alcohols, diethylene lenglykoldialkylether, triethylene, Tet raethylenglykol dialkyl, Pentaethylenglykoldiaky- ether, Hexaethylenglykoldialkylether, DMSO or THF.

As radical scavenger the group of compounds listed in Schedule 1 or derivatives thereof is preferably suitable. The subsequent alkaline hydrolysis then leads to the conversion of unmethylated cytosine nucleobases to uracil.

In the second step diluting the treated DNA sample with water or an aqueous solution. Preference given to desulfonation of DNA (10-30 min, 90-100 ° C) is then carried out at alkaline pH.

In the third step of the method of amplifying the DNA sample in a polymerase chain reaction, preferably using a heat-resistant DNA polymerase. The amplification of several DNA segments is preferably made in one reaction vessel.

The process step is preferably carried out in two steps. You start with a PCR preamplification with at least one pair of primers of different sequence, which non-specifically hybridize the pretreated DNA sample, and thus constituting step over a Amplifkat in PCR. It is then fed by a PCR amplification on the product formed in the preamplification with primers of different sequence, each to a portion of the pretreated DNA sample [(+) strand or (-) strand] are identical or inversely complementary and the DNA to be amplified specifically hybridize.

It is clear that such Präamplifikationen are frequently performed not as PCR reactions, but as Primerextensi- onsreaktionen, - which do not require heat-resistant polymerase.

In the final step is detected, the extent to which the sequence has changed by the treatment with a bisulfite reagent contained compared to the genomic DNA sample and concludes the methylation status of at least one locus in the genomic DNA sample.

For the detection of the PCR products are particularly preferably hybridized to an oligonucleotide array.

In a preferred variant of the process is carried out after the hybridization to an oligonucleotide array by the following sub steps:

a) the amplified genomic DNA is hybridized to at least one oligonucleotide, forming a duplex Siert hybridized, said hybridized oligonucleotides are adjacent with their 3 'end directly or at a distance of up to 10 bases to the positions that respect ih rer methylation in genomic DNA sample should be examined;

(B) the oligonucleotide with known sequence of n nucleotides is extended by means of a polymerase by at least one nucleotide, whereby the nucleotide bears a detectable label and the extension depends on methylation state of the respective cytosine in the genomic DNA sample. co co M IV)

Cπ o o Cπ cπ o π

rt <! d CL rt t^"1 ι_ι- KQ ß ß N ω PJ d N d N cπ he μ- πd ß rt <rt lo Hi H μ- μ- μ- Ω od PJ ~ Φ φ d St 3 he Φ ß ß ß Φ - ≤ Φ d O d d Φ Φ pT μ- μ-: d

CL HL co o ιQ - - s: d CL μ- ώ - 'CQ 1 Φ μ- Q 3 PH 13 Φ α tr

CQ PJ d J Φ co tr ß -Q t-¹ M μ- μ- μ- ω CL li Φ d 3 o co 3 μ- CL Ω O: Φ Φ Φ CL O ß: d rt rt rt CL Φ O Ω Φ J 3 rt μ- iQ J φ s PJ Φ tr Ω P PJ to PJ Ω Φ H μ- rt tr! x! 3 PJ H d

Φ Φ d μ- φ *_* d μ- O 3 φ tr N CO PJ: et 2 fr Φ oo Φ μ- μ- dd H 3 φ

H tr VQ li ιQ he d CQ tr μ- ß φ ta iQ P ιQ Φ PJ PJ ι-i φ α O s: μ- φ Φ Cπ et <o CL CL fr Φ N φ φ he o CL s; μ- tr <d

Φ d J d PJ: odd H- \ CL Φ o H rt Q Φ iQ s: dd PJ d φ o hi "<st

H CL α ß d he x 1 φ φ φ dd μ- o CQ μ- Φ o H d P he μ- he α d Φ

CQ φ ß fr iQ Φ ß o CL M 3 CQ iQ d CL odo μ- 3 PJ fr φ Φ NH μ- μ- PJ rt d H Φ he μ- μ- ^ ddo φ rt rt od μ- do (-^■ P μ- μ- μ- Φ Ω rt

Φ o Φ K μ- et φ CL CL ω d πd iJ μ- CL Φ co -Q CL Φ Φ CL tr φ d co tr O rt L Φ Φ O \ μ- 3 do CL φ μ- CL Ω li φ oo L li μ- Hi H

Ω rt Φ PJ li o 1 Φ d fr CQ φ Φ H VQ Φ O tr Φ H rt rt 13 μ- CQ o Φ

CΛ tr Φ μ- Q et μ- Φ CL M fr μ- li d O CQ r-¹ φ d μ- Φ φ Φ μ- Φ d

13 H μ- CL O d H d Φ Φ rt o Q d d N μ- μ- CL NQ φ φ H iQ μ- d M PJ CL co M CQ CL d Φ o rt μ- <! H d N iQ Φ 3 Φ μ- φ iQ H Φ he

N rt μ- μ- d PJ: φ N o rt o Φ o O: O fr μ- σ d φ Φ H rt d tc Φ μ- rt -ι Φ -Q iQ Φ μ- d rt OJ μ- dd 3 EP φ P 3 •> CL co d CL ^ <! φ μ- H o H d -Q -¹ CL tr μ- CL Φ Φ Φ d he CΛ o Φ Φ he o

CQ Φ υq d Φ o Φ μ- Φ H CL QO co o 3 fr 1 CL co H he PJ 3 μ- d li H μ- JN ß P CL H among others CQ Φ α Φ • d - Φ o rt μ- πd μ- rt rt Φ μ- μ- d N

CL d rt s: fr N μ- Φ do <ld Φ Φ rt 1 μ- μ- Φ Φ li PJ Φ QN CQ Φ m CL d

Φ φ μ- Φ H φ ι VQ-iddoo H li NM d Φ o O d ι-i rt Ω d Φ μ- üa

H o μ- φ do iQ dd Φ μ- d Φ CQ fr it tr rt CQ Φ <1 tr μ- CQ rt

PJ <P rt o CL d iQ for μ- μ- Φ d Φ CL O ß: μ- Φ μ- rt tr O Φ Λ μ- φ

H φ φ co et φ Φ ß <! ιQ 3 HH d co Φ d <J Φ rt P PJ CQ Φ d rt H Ό d μ- • fr d O O d Φ CL CQ rt t? Φ CQ N co odo rt Ω H

H CL O 3 od fr rt PJ CL μ- H CL ß μ- ddo 3 N tr d

Φ P): o CΛ CL Φ 3 et ß 3 φ μ- d Φ d tr Φ Ω ΓΛ d - li d J

H d CL 13 CL do μ- μ- S fr Φ μ- d Q co ISI N ^ li ß tr it 13 CL μ- <1 μ- -QH

St. iQ ß φ Φ H et co φ CL o rt rt α φ s it P rt μ- Φ iQ do rt μ-

O φ XNH Ω μ- Ω et CQ φ et rt CΛ Φ tr φ li N rt CO N st od P rt PJ PJ

H rt μ- tr tr tr o μ- Φ 13 H * d μ- μ- Φ μ- μ- o rt P Φ dd iQ d Φ Φ φ Φ ^ CL t Φ he d rt CL Φ H Ω N he φ ß Φ o Holy rt rt d Φ co H CL d H Φ μ- Φ o NOH fr φ μ- μ- CQ tr ß o Φ x H CL Φ

Φ iQ H CQ μ- μ- μ- μ- li α P rt rt μ- PQ

*_* ß μ- μ- d Φ tr <! et Φ d α Φ co Φ φ φ Φ μ- φ Ω μ- 3 Φ> iQ li μ- CL

CL PJ: Φ Φ 3 ι-i N> <μ- μ- Q oo φ d tr tr o μ- CL od O Ω P Φ

CL Φ li d πd Q d • CL Φ d H "Q rt 13 H φ H rt rt o μ- P tr Φ CQ

PJ CQ he rt o φ 1 P φ H φ 3 φ μ- μ- Φ rt CΛ d φ Φ μ- dd he d • • d co d CΛ iQ "-d iQ CΛ li H μ- Φ CL N 13 H PJ μ- μ- fr <co O d 13 ^ φ ι-i co u PJ: rt CH φ μ- φ Q o tr tr φ o φ

CO CL φ 3 d O o Φ »P πd Φ rt CQ CQ φ N μ- S φ H ^ d CL Φ HH d μ- 0 φ φ N 3 He et N • iQ O he H φ oo μ- d φ d φ he ß 0 ß μ- Hi Hi ιQ P μ- li J Φ PJ μ- Φ Φ Φ PO CL rt ti 3 JP rt ιQ PJ

P o Φ φ J li et Φ co fi ⁽ J fr it CL CL CL CQ tr PJ μ- d inter alia μ- O tr

PH s: o CQ et PJ ß CQ Ό rt ^ PJ φ o Φ ß ß PJ • <P co o CL PH

CL d P Φ he φ φ Φ Q do H "li li o PJ d \ μ- Φ Φ d φ

PJ fr H d H Φ tr NH d PJ μ- Ω CQ d CL μ- 1 co ISI μ- d fr P

CO μ- μ- d φ μ- J: CL μ- μ- CL PJ rt VQ iQ he tr φ μ- μ- M s: d H CQ

Φ 3 Φ CL PJ dd Φ φ φ μ- CQ Φ rt o μ- CL Φ 3 Φ li d Φ φ Φ J Φ co op rr Φ iQ o 1 o φ Φ H Φ 1 Φ Q J μ- O ß CL H μ- H d O

\ 1 1 li et d μ- CQ d 1 d φ 1 1

1> ■ 1 φ

End with existing 3 adjacent '-Hydroxyfunktion the extended oligonucleotide immediately adjacent to the 5' end of the oligonucleotide of the second species, which is present preferably phosphorylated.

The oligonucleotides used to the first species and / or the oligonucleotides of the second species used particularly preferably contain either only the bases T, A and C or the bases T, A and G.

In another preferred variant of the method is carried out after the hybridization to an oligonucleotide array by the following sub steps:

(A) hybridizing the amplified genomic DNA of at least one oligonucleotide with known sequence of n nucleotides forming a duplex, said hybridized oligonucleotides with their 3 'end partially or completely hybridize to the positions relative to their methylation in the genomic DNA - sample should be examined;

(B) the oligonucleotide is, if it with its

3 'terminus hybridized previously with no mismatches to the to be examined position, extended by means of a polymerase by at least one nucleotide, at least one nucleotide bears a detectable label and the extension depends on the methylation state of the respective cytosine in the genomic DNA sample.

The PCR products and / or extension products and / or ligation products are particularly preferably provided for detection with a detectable label.

Preferably, the markings of the PCR products and / or extension products and / or ligation products are Fluorescent labels, radionuclides or removable mass labels, which are detected in a mass spectrometer.

The PCR products and / or extension products and / or ligation products can preferably be detected as a whole in the mass spectrometer and are thus clearly characterized by their mass.

Particular preference is given in each case has a fragment of

PCR products and / or extension products and / or ligation products in the mass spectrometer after.

The fragment of the PCR product and / or Verlängerungspro- domestic product and / or ligation product generated is preferably by digestion with one or more exo- or endonucleases.

For better detectability in the mass spectrometer further the produced fragments particularly preferably a single positive or negative net charge on sen.

The PCR products and / or extension products and / or ligation products are detected and visualized it preferably means of matrix assisted laser desorption ons / ionization mass spectrometry (MALDI-TOF) or by means of electrospray mass spectrometry (ESI).

The present method is preferably used for the diagnosis and / or prognosis of adverse events for patients or individuals, whereby these adverse events belong to at least one of the following categories: undesired drug interactions; Cancers; CNS malfunctions, damage or disease; Aggression or behavioral disturbances; clinical, psychological and social consequences of Gehirnschädi- conditions; psychotic disturbances and personality disorders; Dementia and / or associated syndromes; cardiovascular disease, malfunction and damage; Malfunction, damage or disease of the gastrointestinal tract; Malfunction, damage or disease of the respiratory system; Injury, inflammation, infection, immunity and / or convalescence; Malfunction, damage or disease of the body as an abnormality in the development process; Malfunction, damage or disease of the skin, muscles, connective tissue or the bones; endocrine and metabolic malfunction, damage or disease; Headaches or sexual malfunction.

The new method also serves especially preferred for distinguishing cell types, tissues or for investigating cell differentiation.

The present invention further provides a kit comprising a bisulfite contained reagent denaturing reagents or solvents, as well as radical scavengers according

List 1 contains primers for producing the amplified and instructions for performing an assay.

List 1:

Di-, trihydroxybenzenes, green tea extract (green tea extract), Pycnogenol (pine bark extract), Ginkgo Biloba

Extract (EGb 761), flavonoid mixture of different fruit and vegetable extracts (GNLD), Bio-Normalizer (Sun-

0 Corp)

DPPH (1, l-diphenyl-2-picrylhydrazyl), NDGA (Nordihydrogu- ajaret acid),

Trolox (6-hydroxy-2, 5,7, 8-tetramethylchroman-2-carboxylic acid), 2, 6-di-tert-butylphenol, 4-methyl-di-tert-butylphenol, 4-methoxy-di-tert-butylphenol, 2, 6-di-tert-butyl-p-cresol, 3, 4-dihydroxybenzoic acid, vitamin C, vitamin e, vitamin Q, hydroquinone, ubiquinone, lignans, Hydroxyterpene, flavonoids, curcumin, tannins, Retinsäureverbindungen, Ge 132 Bisbetacarboxyethyl germanium sesquioxide, superoxide dismutase (SOD), superoxide catalase, alpha naphthoflavone , Di (2-methyl-5-chlorophenyl) dithionate and Cu (II) - derivatives, mebendazole, CS (Chloroformlöslicher) alkaloid extract,

4- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-hydroxy-l, 2-naphthoquinone,

4- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-methoxy-l, 2-naphthoquinone, 4- (3, 5-di-tert-butyl-4-hydroxyphenyl) -1,2- naphthoquinone, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-bromo-l, 4- naphthoquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-chloro-l, 4- naphthoquinone, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-methoxy- l, 4-naphthoquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-hydroxy-l, 4- naphthoquinone, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -1, 4- naphthoquinone, 4- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-hydroxy-5, 5,8,8- tetramethyl-5, 6, 7, 8-tetrahydro-l, 2-anthraquinone, 4- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-methoxy-5, 5,8,8- tetramethyl-5, 6, 7, 8-tetrahydro-l, 2-anthraquinone, 4- (3, 5-di-tert-butyl-4-hydroxyphenyl) -5, 5, 8, 8- tetramethyl-5, 6, 7, 8-tetrahydro-l, 2-anthraquinone,

3-bromo-4- (3, 5-di-tert-butyl-4-hydroxyphenyl) -5,5,8,8- tetramethyl-5, 6, 7, 8-tetrahydro-l, 2-anthraquinone, 2- (3, 5-di-tert-butyl-4-oxocyclohexa-2, 5-dienylidene) - indan-1, 3-dione, 2- (3, 5-di-tert-butyl-4-oxocyclohexa-2, 5 -dienyliden) -3, 4-epoxy-3-hydroxy-4-methoxy-3, 4-dihydro-2H-naphthalen-l-one, 2- (3, 5-di-tert-butyl-4-oxocyclohexa-2, 5-dienylidene) -3, 4-epoxy-3, 4-dimethoxy-3, 4-dihydro-2H-naphthalen-l-one, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -indan-1-one, 3, 3-bi- [2- (3, 5-di-tert-butyl-4-hydroxyphenyl) indene -1-one] -3-yl, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-bromo-5, 5,8,8- tetramethyl-5, 6, 7, 8- tetrahydro-l, 4-anthraquinone, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-chloro-5, 5,8,8- tetramethyl-5, 6,7, 8-tetrahydro- l, 4-anthraquinone, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-methoxy-5, 5, 8, 8- tetramethyl-5, 6, 7, 8-tetrahydro-l, 4-anthraquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-hydroxy-5, 5, 8, 8- tetramethyl-5, 6, 7, 8-tetrahydro-l, 4-anthraquinone, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -5,5,8,8- tetramethyl-5, 6, 7, 8-tetrahydro-l, 4-anthraquinone, 2-bromo-3- (3 -bromo-5-tert-butyl-4-hydroxyphenyl) -5,5,8,8- tetramethyl-5, 6, 7, 8-tetrahydro-l, 4-anthraquinone, 2-bromo-3- (3, 5 -dibromo-4-hydroxyphenyl) -5, 5,8,8- tetramethyl-5, 6, 7, 8-tetrahydro-l, 4-anthraquinone, 2-bromo-3- (3-bromo-5-tert-butyl -4-hydroxyphenyl) -3-hydroxy- 5,5,8, 8-tetramethyl-5, 6, 7, 8-tetrahydro-l, 4-anthraquinone, 3-bromo-2- (3, 5-di-tert -butyl-4-hydroxyphenyl) -1, 4-anthraquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-methoxy-l, 4-anthraquinone, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-hydroxy- l, 4-anthraquinone,

5,5,8, 8-tetramethyl-5, 6, 7, 8-tetrahydronaphthalene-l, 3- diol, 3-methoxy-5, 5, 8, 8-tetramethyl-5, 6, 7, 8- tetrahydronaphthalene 1-ol,

4- (3-chloro-5, 5, 8, 8-tetramethyl-l, 4-dioxo-l, 4, 5, 6, 7, 8-hexahydroanthracene-2-yl) benzoic acid,

Methyl-4- (3-chloro-5, 5, 8, 8-tetramethyl-l, 4-dioxo-

1, 4, 5, 6, 7, 8-hexahydroanthracene-2-yl) benzoate, 4- (3-hydroxy-l, 4-dioxo-l, 4-dihydronaphthalene-2-yl) - benzoic acid, Methyl (3-methoxy-l, 4-dioxo-l, 4-dihydronaphthalene-2-yl) - benzoic acid,

4- (3-hydroxy-5, 5, 8, 8-tetramethyl-l, 4-dioxo-l, 4,5,6,7,8- hexahydroanthracene-2-yl) benzoic acid, methyl-4- (3 -hydroxy-l, 4-dioxo-l, 4-dihydronaphthalene-2-yl-azo) benzoate,

4- (3-hydroxy-5, 5, 8, 8-tetramethyl-l, 4-dioxo-l, 4,5, 6,7,8-hexahydroanthracene-2-yl-azo) benzoic acid,

3- (3, 5-di-tert-butyl-4-oxocyclohexa-2, 5-dienylidene) - 5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-cyclopenta [b] naphthalene-1, 2-dione,

3- (3, 5-di-tert-butyl-4-oxocyclohexa-2, 5-dienylidene) -

5,5,8, 8-tetramethyl-5, - 6, 7, 8-tetrahydroanthracene-3H-l, 2, 4- trione, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3 -methoxy-5, 8- dimethyl-1, 4-naphthoquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-methoxy-6, 7-dimethyl-1, 4-naphthoquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-methoxy-5-methyl-1, 4-naphthoquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -2-methoxy-5-methyl-

1, 4-naphthoquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-methoxy-6-methyl-

1, 4-naphthoquinone, 3- (3, 5-di-tert-butyl-4-hydroxyphenyl) -2-methoxy-6-methyl-

1, 4-naphthoquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-methoxy-5, 6- dimethyl-1, 4-naphthoquinone,

3- (3, 5-di-tert-butyl-4-hydroxyphenyl) -2-methoxy-5, 6- dimethyl-1, 4-naphthoquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-methoxy-5, 7- dimethyl-1, 4-naphthoquinone,

3- (3, 5-di-tert-butyl-4-hydroxyphenyl) -2-methoxy-5, 7- dimethyl-1, 4-naphthoquinone, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl ) -3-ethylthio-5-methyl-1, 4-naphthoquinone, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-ethylthio-6-methyl-1, 4-naphthoquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-hydroxy-5, 8-dimethyl-1, 4-naphthoquinone, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl ) -3-hydroxy-6, 7-dimethyl-1, 4-naphthoquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-hydroxy-5-methyl-

1, 4-naphthoquinone,

3- (3, 5-di-tert-butyl-4-hydroxyphenyl) -2-hydroxy-5-methyl-1, 4-naphthoquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-hydroxy-6-methyl-

1, 4-naphthoquinone,

3- (3, 5-di-tert-butyl-4-hydroxyphenyl) -2-hydroxy-6-methyl-

1, 4-naphthoquinone, 2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-hydroxy-5, 6- dimethyl-1, 4-naphthoquinone,

2- (3-bromo-5-tert-butyl-4-hydroxyphenyl) -3-hydroxy-5, 6- dimethyl-1, 4-naphthoquinone,

3- (3, 5-di-tert-butyl-4-hydroxyphenyl) -2-hydroxy-5, 6- dimethyl-1, 4-naphthoquinone,

2- (3, 5-di-tert-butyl-4-hydroxyphenyl) -3-hydroxy-5, 7- dimethyl-1, 4-naphthoquinone,

3- (3, 5-di-tert-butyl-4-hydroxyphenyl) -2-hydroxy-5, 7- dimethyl-1, 4-naphthoquinone.

The following example illustrates the invention.

Example:

Automated execution of the bisulfite reaction.

In the present example, the application of the method for detecting the methylation status of cytosines in the factor VIII gene of a genomic DNA sample with a restriction endonuclease as specified by the manufacturer is the coupler was treated as described. The method relies on the use of an automatic pipetting system (MWG RoboSeq 4204) with four separate vertically movable adapters for exchangeable pipetting tips that exclude cross-contamination. The pipetting system allows pipetting lOOμl with an error of less than ± 2μl. The worktop of the automatic pipetting system is equipped with six racks for pipetting tips and eight pipetting positions, two of which can be cooled, a coolable reagents frame, a stacking system for 10 microtiter plates, a Pipettierspitzen- wash station and a device for separating the pipette tips from the adapter. The automatic pipetting system is connected via a serial interface with a computer and a software program that allows the free programming of all pipetting steps necessary for the application of the process is controlled.

In the first process step, an aliquot of the DNA sample is pipetted into a 96 freely selectable positions of a microtiter plate by hand. The microtiter plate is then heated using an Eppendorf Mastercyc- coupler for denaturing the pretreated DNA sample to 96 ° C. The microtiter plate is then transferred to the automatic pipetting system. In all positions, which contain DNA, are programmatically sequentially from the reagent rack aliquots of a denaturing agent (dioxane), a 3.3M sodium hydrogen sulfite solution, and a solution of a radical scavenger used in the denaturing agent added with a pipette. Subsequently, the microtiter plate in Eppendorf Master cycler is incubated that in the DNA sample under the influence of sodium hydrosulfite all unmethylated cytosine residues are converted into a bisulfite adduct. CO co M IV) ¹ cπ o o Cπ cπ o Cπ

d I-¹ he Ω CL LJ. tr πd N rt rt CL ≤ rt H CΛ s; O CL σ rt QN he tc rt H3 Hi CL 3

Φ φ Φ tr Φ Φ PJ: dd Φ Φ μ- μ- Φ ^ rt 3 oy μ- 3 Φ H O dt μ- o φ μ- • ß: Φ PJ r "H <Φ ii CL Hi Φ li! H rt H CQ M li d HH d Φ d CQ φ co H "od tr rt Hi 3 Ω h φ od Φ ι-i 13 Φ Φ L rt μ- PJ H CL μ- μ- iQ d rt POH tr o fr ß: li li CL 0 "" • Φ co l-¹ P ι-i Φ φ P μ->> Φ öd iQ d rt rt πd Φ H3

3 he rt Φ Φ ii rt PJ d μ- n iQ Hi O φ φ Φ li H s: Φ Hi ß J <! tr CL μ- H Φ CL H πd PJ_^ rt PJ: 3 ß rt O μ-> μ- μ- Hi N φ Φ d μ- Hi D ß O φ φ

CL o H μ- πd CL O d Φ Λ rt PJ 3 φ fr d O co μ- CQ H rt Hi Hi 3 h M is H

13 Hi Φ o ß: CO rt Φ μ- Φ d rt PJ PJ -Q

CO! QM 13 Φ rt IS he Φ td CO 3 μ- tr ß: CO P o μ- μ- μ- d rt d Φ o P rt Q πd iQ μ- φ Φ μ- Φ li 1 Φ o Dd

CL o tr Φ μ- d rt 3 d Φ <1 Φ rt st ^ μ- μ- _^^ Q li rt rt Φ d fr tr πd Φ t Ω μ- φ H rt rt μ- PJ φ μ- PJ O PJ rt Ω o CΛ μ- φ NH PJ _* - HH Φ μ- • <: Q d Φ rt N μ- Φ O rt H d l-¹ CL 3 PO tr d H> 3 CL ß odd O iQ H Ω ß ß rt O odd μ- σ μ- Φ φ Φ J CL CQ rt O φ ds μ- rt CL tc he Φ rt CL μ- φ = s H d Q 3 Λ d co rt Φ μ- Φ 1 _^^ ι-s ii Φ α Φ μ- Φ Φ H • φ Hi

Hl he μ- CL Ω> ß he rt o μ- d ι_ι. μ- H CΛ d Ω li d H rt υD CQ φ li μ- μ- d li σ CL μ- φ tr 1 o μ- Φ Φ co rt u Φ P α M PJ PJ r tu Φ rt rt • Φ .mu.J μ - rt

N d CL ß Φ CQ H πd rt o Ω Φ Φ CL lO PJ CQ μ- Ω μ- H Φ cπ d Φ dd μ- he μ- CL HH d 13 O d he πd tr t co O co IV) 1 n φ φ Hι μ- o d 13 - rt P φ d φ φ Ω N μ- φ M φ φ o d rt Ω - H μ- μ- d d H tr tr PJ

H PJ σ tr CL Hi st 13 t <H Hi co d P li Φ tr φ • - 'σ CL P CQ f li H PJ α ß PJ: SN CL PJ et d DJ Hi li μ- φ φ gd μ- μ- CL li • • φ φ φ φ PJ Holy rt 3 d - μ- ≤ PJ d

CQ o ß: μ- μ- rt rt rt rt Φ Φ ι-3 πd φ d Φ Hi <! μ »O 1 et co μ- rt rt CL fr Φ Φ o CL

- _^ Ω tr rt rt rt he HH σ μ- • μ- μ- μ- d o Φ - πd CΛ μ- rt rt Φ _^ H μ-

^ Tr πd H rt Φ Φ μ- PJ Φ o 13 13 •_^ Ω d H ii ι-3 O rt O Φ J μ- PJ o "rt PJ ß μ- -> o ß Φ tr d φ O CΛ tr d CQ φ tr Φ VQ s; ß - <HP d CL d: dd st rt PJ φ dd ιQ μ- »dd JH Φ rt rt PJ Φ φ rt co H Φ P 13 * PJ σ CO μ- μ- g rt iQ

• Φ 1 iQ ds rt J dd <! rt li Φ do 3 d φ Φ CL dd Ω 1-5 rt 3 S o

EP πd 2 CL μ- • ß 3 CL O μ- I-¹ o σ μ- CL φ iQ μ- 3 fr ι-s μ- tr Φ φ μ- 3 s:

Φ li CL μ- Φ for d 3 φ J li Φ PJ CL 2 Ω Φ ι-3> li - d VQ rt rt Φ HH fr PJ μ-

- PO Φ for σ H CL H d li iQ ß φ> tr rt ϊ PJ Φ Φ φ

• li μ- N 13 d lJ rt li

CL CL H li et O P> O: rt Hl Φ CQ he H 1 _"« _^^ CQ CL os; J Φ Φ d H "•_^ o μ-

Φ Φ ß o rt d πd CQ Φ H¹ • rt CΛ he> Φ Φ PJ iQ d P Φ PJ rt o

^ Fr πd rt d μ- PJ H ß CL Φ CQ • d rt μ- CL ι-3 Q fr li CΛ d Φ Φ Φ rt rt H CL μ- Ω μ-> rt O rt μ- Ω μ- P σ Φ -Q rt d ι-i CO PJ OO Φ et H d Q d CQ rt rt tr Φ μ- ιQ d * ι rt σ Φ tr 3 iQ 3 li rt Φ ^ <! J d co o rt he ii Φ. 13 L rt Φ Φ φ Φ li d hi ​​s: 1 Φ 2 li φ • 3 ß: O d¹ O rt μ- pι d H Φ ι-i

H PJ: μ- PO li li t 13 1 Φ H iQ Hl o Φ CQ d rt μ- H Φ st φ Φ d 13 πd S

HH φ 13 μ^j μ- πd HH φ ω μ- μ- fe Ώ d ß iQ • Ω d Ω μ- μ- μ- -¹ H- he μ- μ- CL J PJ li CL μ- H CO d μ- CQ •• »rt φ ι-i PP pj PJ 13 d fr fr PJ d rt L φ Φ μ- he rt 5 ^ ι- i μ- Φ tr tr

O rt P ι-3 Φ H, O rt CL CL Φ Φ iQ rt h Φ

N d CL et rt rt CO he Φ J Φ φ PJ: CL Φ Q s ι-3 PJ μ- 3 Φ P he ß rt rt o φ i ß μ- rt CL Φ PJ φ Φ Φ tr dd 13 ι-3 rt φ H α φ PJ he μ- φ H rt μ- H o φ μ- ß ι-3 μ- CL CL πd CQ PJ φ o rt he μ- rt φ μ- co μ- <! Φ μ- CQ 3 Ω d Φ Φ rt ^ d Φ S Φ li o μ- CO d N t-3 μ- Φ 3 he tr d μ- Q PJ CL ii rt rt tr μ- ß: μ- O 13 Φ d μ- μ- t iQ Φ α μ- o Holy O^{^} ; PJ πd co ß Φ o rt φ φ μ- he d 3 co d rt rt fr ϊ 1 d Φ Hi H3 ι-3 d PJ <! co o

Φ> Ω H φ CQ ^ H

CL> P d J tr li PJ rt PO μ- μ- μ- μ- μ- odo co H tr Hl d o CQ 13

DJ M iQ li et rt πd PJ: o 3 Φ Φ PP rt O HI. _^ LH Φ co μ- μ- Φ H rt rt Φ

CQ rt rt J CL CQ I-¹ μ- O rt 3 PJ μ- Φ Ω ι-3 Hl Φ πd h-¹ H Ω rt rt ii Λ Φ Φ P⁾ tr H Φ li 13 μ- CQ φ Ud rt μ- Φ S fr α Ω d tr OO μ- rt tr μ- d er er rt 3

Q μ- O d PJ li Λ Ω d 1 1 ι-i μ- rt H tr φ o rt 2 φ Φ φ oo ι-3 Φ Φ πd rt

Φ O iQ Φ ß tr rt d fr μ- PJ rt α CD Φ iQ ddd rt H tr O d B: Φ μ- Φ o Φ 1 O 1 s: H o Ω 3 3> Ώ • d φ μ- PJ CO he

1 1 ^ iQ "_* P rt PJ μ- od μ- Φ o cn do t-¹ CL CL co d μ- Φ PJ

Φ Ω d ii 1 H 1 1 ι-3 1 O: Φ Φ 1 1 1 1-5 1 1 ß CQ CQ CL 1 1 1 1 li CO

picture of a PCR-amplified DNA strand bisulfitbehandelten (left: molecular weight marker, right: PCR product)