CA2425366A1 - Method for the detection of cytosine methylations - Google Patents

Abstract

A method for the detection of cytosine methylations in genomic DNA is disclosed, whereby genomic DNA samples are firstly reacted with a chemical reagent, whereupon 5-methylcytosine and cytosine react differently and thus display a different base pair relationship in the DNA duplex after said reaction. The pre-treated DNA is then amplified by means of a polymerase and at least one primer oligonucleotide and the amplified material once again reacted with a chemical reagent as above. The cytosine bases and/or guanine bases remaining in the amplified material after the renewed chemical treatme nt are detected in the final step.

Description

Method for the detection of cytosine methylations The present invention describes a method for the detection of the degree of methylation of genomic DNA samples.
The levels of observation that have been well studied in molecular biology according to developments in methods in recent years include the genes themselves, the transcription of these genes into RNA and the translation to proteins therefrom. During the course of development of an individual, which gene is turned on and how the activation and inhibition of certain genes in certain cells and tissues are controlled can be correlated with the extent and nature of the methylation of the genes or of the genome. In 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, it plays 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. 5-Methylcytosine positions, however, cannot be identified by sequencing, since 5-methylcytosine has the same base-pairing behavior as cytosine. In addition, in the case of a PCR amplification, the epigenetic information which is borne by the 5-methylcytosines is completely lost.
A relatively new method that in the meantime has become the most widely used method for investigating DNA for 5-methylcytosine is based on the specific reaction of bisulfite with cytosine, which, after subsequent alkaline hydrolysis, is then converted to uracil, which corresponds in its base-pairing behavior to thymidine. In contrast, 5-methylcytosine is not modified under these conditions. Thus, the original DNA
is converted so that methylcytosine, which originally cannot be distinguished from cytosine by its hybridization behavior, can now be detected by "standard" molecular biology techniques as the only remaining cytosine, for example, by amplification and hybridization or sequencing. All of these techniques are based on base pairing, which is now fully utilized. The prior art, which concerns sensitivity, is defined by a method that incorporates the DNA to be investigated in an agarose matrix, so that the diffusion and renaturation of the DNA is prevented (bisulfite reacts only on single-stranded DNA) and all precipitation and purification steps are replaced by rapid dialysis. (Olek A, Oswald J, Walter J. A modified and improved method for bisulphite based cytosine methylation analysis. Nucleic Acids Res. 1996 Dec 15;24(24):5064-6). Individual cells can be investigated by this method, which illustrates the potential of the method. Of course, up until now, only individual regions of up to approximately 3000 base pairs long have been investigated; a global investigation of cells for thousands of possible methylation analyses is not possible. Of course, this method also cannot reliably analyze very small fragments of small quantities of sample. These are lost despite the protection from diffusion through the matrix.
An overview of other known possibilities for detecting 5-methylcytosines can be derived from the following review article: Rein T, DePamphilis ML, Zorbas H.
Identifying 5-methylcytosine and related modifications in DNA genomes. Nucleic Acids Res.

May 15;26(10):2255-64.
The bisulfite technique has been previously applied only in research, with a few exceptions (e.g., Zeschnigk M, Lich C, Buiting K, Doerfler W, Horsthemke B. A
single-tube PCR test for the diagnosis of Angelman and Prader-Willi syndrome based an allelic methylation differences at the SNRPN locus. Eur J Hum Genet 1997 Mar-Apr;S(2):94-8). However, short, specific segments of a known gene have always been amplified after a bisulfite treatment and either completely sequenced (Olek A, Walter J.
The pre-implantation ontogeny of the H19 methylation imprint. Nat Genet. 1997 Nov;17(3):275-6) or individual cytosine positions have been detected by a "primer extension reaction"
(Gonzalgo ML, Jones PA. Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE).
Nucleic Acids Res. 1997 Jun 15;25(12):2529-31, WO-Patent 95-00669) or an enzyme step (Xiong Z, Laird PW. COBRA: a sensitive and quantitative DNA methylation assay.
Nucleic Acids Res. 1997 Jun 15;25(12):2532-4). Detection by hybridization has also been described (Olek et al., WO-A 99-28498).
Other publications which are concerned with the application of the bisulfite technique for the detection of methylation in the case of individual genes are: Grigg G, Clark S.
Sequencing 5-methylcytosine residues in genomic DNA. Bioessays. 1994 Jun;16(6):431-6, 431; Zeschnigk M, Schmitz B, Dittrich B, Buiting K, Horsthemke B, Doerfler W. Imprinted segments in the human genome: different DNA methylation patterns in the Prader-WiIli/Angelman syndrome region as determined by the genomic sequencing method. Hum Mol Genet. 1997 Mar;6(3):387-95; Feil R, Charlton J, Bird AP, Walter J, Reik W. Methylation analysis on individual chromosomes: improved protocol for bisulphite genomic sequencing. Nucleic Acids Res. 1994 Feb 25;22(4):695-6; Martin V, Ribieras S, Song-Wang X, Rio MC, Dante R. Genomic sequencing indicates a correlation between DNA hypomethylation in the 5' region of the pS2 gene and its expression in human breast cancer cell lines. Gene. 1995 May 19;157(1-2):261-4; WO 97 46705, WO 95 15373 and WO 95 45560.
An overview of the state of the art in oligomer array production can be derived also from a special issue of Nature Genetics which appeared in January 1999 (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 in the case of reduced nonspecific background signal.
Probes with multiple fluorescent labels are used for scanning an immobilized DNA
array. Particularly suitable for fluorescent labels is the simple introduction of Cy3 and Cy5 dyes at the 5'-OH of the respective probe. The fluorescence of the hybridized probes is detected, for example, by means of a confocal microscope. The dyes Cy3 and CyS, among many others, are commercially available.
Matrix-assisted laser desarptions/ionization mass spectrometry (MALDI-TOF) is a very powerful development for the analysis of biomolecules (Karas M, Hillenkamp F.
Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem. 1988 Oct 15;60(20):2299-301 ). An analyte is embedded in a light-absorbing matrix. The matrix is vaporized by a short laser pulse and the analyte molecule is transported unfragmented into the gaseous phase. The analyte is ionized by collisions with matrix molecules. An applied voltage accelerates the ions in a field-free flight tube.
Ions are accelerated to varying degrees based on their different masses.
Smaller ions reach the detector sooner than large ions.
MALDI-TOF spectroscopy is excellently suitable for the analysis of peptides and proteins. The analysis of nucleic acids is somewhat more difficult (Gut, I. G.
and Beck, S. (1995), DNA and Matrix Assisted Laser Desorption Ionization Mass Spectrometry.
Molecular Biology: Current Innovations and Future Trends 1: 147-157.) For nucleic acids, the sensitivity is approximately 100 times poorer than for peptides and decreases overproportionally with increasing fragment size. For nucleic acids, which have a backbone with a multiple negative charge, the ionization process through the matrix is basically less efficient. In MALDI-TOF spectroscopy, the choice of matrix plays an imminently important role. Several very powerful matrices, which produce a very fine crystallization, have been found for the desorption of peptides. In the meantime, several effective matrices have been developed for DNA, but the difference in sensitivity was not reduced thereby. The difference in sensitivity can be reduced by modifying the DNA
chemically in such a way that it resembles a peptide. Phosphorothioate nucleic acids, in which the usual phosphates of the backbone are substituted by thiophosphates, can be converted by simple alkylation chemistry to a charge-neutral DNA (Gut, I. G.
and Beck, S. (1995), A procedure 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 in the presence of impurities, which make the detection of unmodified substrates very difficult.
Genomic DNA is obtained from DNA of cells, tissue or other test samples by standard methods. This standard methodology is found in references such as Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 1989.
Urea improves the efficiency of bisulfite treatment prior to the sequencing of methylcytosine in genomic DNA (Paulin R, Grigg GW, Davey MW, Piper AA. Urea improves efficiency of bisulphite-mediated sequencing of 5'-methylcytosine in genomic DNA. Nucleic Acids Res. 1998 Nov 1;26(21 ):5009-10).
Accordingly, there are methods that permit identification of cytosine methylation in that all unmethylated cytosines are converted to a specific base and only methylated cytosines are still present as cytosine after the amplification. However, it is not possible without anything further, on the other hand, to designate all of the cytosine bases that are still present after the amplification as formerly methylated cytosines, since in the amplification, cytosine is still incorporated in the counterstrand as the complementary base to the unconverted guanine. This, however, proves to be a problem, if one desires to utilize characteristic reactions for cytosine in a simple method, in order to determine methylated positions or the extent of methylation in the amplified segment.
This problem will be solved in the present invention.
The object of the invention is to provide a method, which permits labels to be incorporated in an amplified double-stranded DNA, which [labels] can be utilized for the clear identification of cytosine methylations in genomic DNA samples. These labels will permit the use of simple, established methods of molecular biology for the methylation analysis.
The object is solved according to the invention by a method for the detection of cytosine methylation in genomic DNA, whereby the following method steps are conducted:
a) the genomic DNA sample is chemically converted with a reagent, whereby 5-methylcytosine remains unchanged and cytosine is converted to uracil or another base similar to uracil in its base pairing behavior;
b) the pretreated DNA is amplified with the use of a polymerase and at least one primer oligonucleotide;

c) the amplified product is again chemically converted with a reagent, whereby methylcytosine remains unchanged and cytosine is converted to uracil or another base similar to uracil in its base pairing behavior;
d) the cytosine bases and/or guanine bases remaining in the amplified product after the repeated chemical treatment are detected.
The object is further solved by a method for the detection of cytosine methylation in genomic DNA, whereby the following method steps are conducted:
a) a genomic DNA sample is chemically converted with a reagent, whereby 5-methylcytosine remains unchanged and cytosine is converted to uracil or another base similar to uracil in its base pairing behavior;
b) the pretreated DNA is amplified with the use of a pofymerase and at least one primer oligonucleotide;
c) the amplified product is again chemically converted with a reagent, whereby methylcytosine remains unchanged and cytosine is converted to uracil or another base similar to uracil in its base pairing behavior;
d) the chemically treated amplified product from step c) is again amplified;
e) the cytosine bases and/or guanine bases remaining in the amplified product are detected. These method variants according to the invention comprise two amplification steps.
It is particularly preferred for the method according to the invention that the chemical treatment is conducted with sodium bisulfite (= hydrogen sulfite, disulfite).
It is also particularly preferred that the chemical treatment is conducted after embedding the DNA in agarose.
In the method according to the invention, it is preferred that in the chemical treatment, a reagent that denatures the DNA duplex and/or a radical trap is (are) present.
It is particularly preferred that the denaturing reagent is selected from one of the following substances:
polyethylene glycol dialkyl ether, dioxane and substituted derivatives, urea or derivatives, acetonitrile, primary alcohols, secondary alcohols, tertiary alcohols, diethylene glycol dialkyl ether, triethylene glycol dialkyl ether, tetraethylene glycol dialkyl ether, pentaethylene glycol dialkyl ether, hexaethylene glycol dialky! ether, DMSO or THF and/or that the radical trap is selected from one of the following substances:
di- and trihdroxybenzenes, green tea extract, pine bark extract (Pycnogenol), Ginkgo Biloba extract (EGb 761 ), a flavonoid blend of several fruit and vegetable extracts (GNLD), Bio-Normalizer (Sun-O Corp), DPPH (1,1-diphenyl-2-picrylhydrazyl), NDGA
(nordihydroguaiaretic acid), Trolox (6-hydroxy-2,5,7,8-tetramethylchromane 2-carboxylic acid), 2,6-di-tert-butylphenol, 4-methyl-di-tertbutylphenol, 4-methoxy-di-tert-butylphenol, 2,6-di-tert-butyl-p-cresol, 3,4-dihydroxybenzoic acid, vitamins C, E, Q, hydroquinone, ubiquinone, lignans, hydroxyterpenes, flavonoids, curcumin, tannins, retinoic acid compounds, Ge-132 bisbetacarboxyethy) germanium sesquioxides, superoxide dismutase (SOD), superoxide catalase, alpha-naphthoflavone, Ginkgo biloba extract (EGb 761 ), di-(2-methyl-5-chlorophenyl)dithionate and Cu(II) derivatives, mebendazole, CS (chloroform-soluble) afkafoidaf extract, 4-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-hydroxy-1,2-naphthoquinone, 4-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-methoxy-1,2-naphthoquinone, 4-(3,5-di-tert-butyl-4-hydroxyphenyl)-1,2-naphthoquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-bromo-1,4-naphthoquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-chloro-1,4-naphthoquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-methoxy-1,4-naphthoquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-hydroxy-1,4-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-1,2-anthraquinone, 4-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-methoxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,2-anthraquinone, 4-(3,5-di-tert-butyl-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,2-anthraquinone, 3-bromo-4-(3,5-di-tert-butyl-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,2-anthraquinone, 2-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidene)-indane-1,3-dione, 2-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidene)-3,4-epoxy-3-hydroxy-4-methoxy-3,4-dihydro-2H-naphthalene-1-one, 2-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidene)-3,4-epoxy-3,4-dimethoxy-3,4-dihydro-2H-naphthalene-1-one, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-indane-1-one, 3,3-bi-[2-(3,5-di-tert-butyl-4-hydroxyphenyl)-indene-1-onj-3-yl, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-bromo-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-chloro-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-methoxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone, 2-(3,5-di-tert-butyl-hydroxyphenyl)-3-hydroxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone, 2-bromo-3-(3-bromo-5-tert-butyl-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone, 2-bromo-3-(3,5-dibromo-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone, 2-bromo-3-(3-bromo-5-tert-butyl-4-hydroxyphenyl)-3-hydroxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone, 3-bromo-2-(3,5-di-tert-butyl-4-hydroxyphenyl)-1,4-anthraquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-methoxy-1,4-anthraquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-hydroxy-1,4-anthraquinone, 5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-1,3-diol, 3-methoxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-1-ol, 4-(3-chloro-5,5,8,8-tetramethyl-1,4-dioxo-1,4,5,6,7,8-hexahydroanthracen-2-yl) benzoic acid, methyl-4-(3-chloro-5,5,8,8-tetramethyl-1,4-dioxo-1,4,5,6,7,8-hexahydroanthracen-2-yl) benzoate, 4-(3-hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl) benzoic acid, methyl-(3-methoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl) benzoic acid, 4-(3-hydroxy-5,5,8,8-tetramethyl-1,4-dioxo-1,4,5,6,7,8-hexahydroanthracen-2-yl) benzoic acid, methyl-4-(3-hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl-azo) benzoate, 4-(3-hydroxy-5,5,8,8-tetramethyl-1,4-dioxo-1,4,5,6,7,8-hexahydroanthracen-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-tetrahydrocyclopenta[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-1,2,4-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-hydroxyphenyl)-2-methoxy-5,6-dimethyl-1,4-naphthoquinone, 2-(3,5-di-tert-butyl-hydroxyphenyl)-3-methoxy-5,7-dimethyl-1,4-naphthoquinone, 3-(3,5-di-tert-butyl-hydroxyphenyl)-2-methoxy-5,7-dimethyl-1,4-naphthoquinone, 2-(3,5-di-tert-butyl-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-hydroxyphenyl)-3-hydroxy-6,7-dimethyl-1,4-naphthoquinone, 2-(3,5-di-tert-butyl-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-hydroxyphenyl)-2-hydroxy-5,6-dimethyl-1,4-naphthoquinone, 2-(3,5-di-tert-butyl-hydroxyphenyl)-3-hydroxy-5,7-dimethyl-1,4-naphthoquinone, 3-(3,5-di-tert-butyl-hydroxyphenyl)-2-hydroxy-5,7-dimethyl-1,4-naphthoquinone.
It is also preferred according to the invention that the reagents which are used for the chemical treatment of DNA are completely or partially eliminated prior to the subsequent amplification. It is also preferred that the samples are diluted after the chemical treatment, prior to the amplification. In addition, it is preferred according to the invention that the amplification of several DNA segments is conducted simultaneously in one reaction vessel and/or that a heat-stable DNA polymerase is used for the amplification.
A method according to the invention is even more particularly preferred, however, in which a desulfonation of the DNA is conducted prior to the amplifications.
It is also preferred that the cytosine and/or guanine bases that remain after the second chemical treatment are detected by hybridization reactions and/or that the cytosine and/or guanine bases that remain after the second chemical treatment are detected and/or quantified by specific incorporation of detectable labels in the cytosine and/or guanine bases.
A method is also preferred according to the invention, in which in step d) according to the first method variant or step e) according to the second method variant for the detection of the pretreated DNA, the amplification products are hybridized to an oligonucleotide array and then the following substeps are conducted:
a) the amplified genomic DNA is hybridized to at least one oligonucleotide with the formation of a duplex, whereby said hybridized oligonucleotide lies directly adjacent at its 3'-end, or at a distance of up to 10 bases, to the positions which are to be investigated relative to their methylation in the genomic DNA sample;
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 the methylation state of the respective cytosine in the genomic DNA sample .
It is also preferred that the cytosine andlor guanine bases that remain after the second chemical treatment are detected by sequencing reactions.
It is also preferred that in step d) according to the first method variant or step e) according to the second method variant for the detection of the pretreated DNA, the amplification products are hybridized to an oligonucleotide array and then the following substeps are conducted:
(a) a set of oligonucleotides is hybridized to the amplified genomic DNA with the formation of a duplex, wherein this set of oligonucleotides is comprised of two different species and wherein the hybridized oligonucleotides of the first species lie directly adjacent at their 3'-end, or at a distance of up to 10 bases, to the positions which are to be investigated relative to their methylation in the genomic DNA sample and whereby 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 is separated by a gap of the magnitude of a single nucleotide or up to 10 nucleotides from the 3'-end of the hybridized oligonucleotide of the first species at the site of said selected position;
(b) the oligonucleotide of the first species is extended with a known sequence of n nucleotides by means of a polymerase by at most the number of nucleotides which lie between the 3'-end of the oligonucleotide of the first species and the 5'-end of the oligonucleotide of the second species, wherein the extension depends on the methylation state of the respective cytosine in the genomic DNA sample;
(c) the oligonucleotides are incubated in the presence of a ligase, whereby the oligonucleotide of the first species and the oligonucleotide of the second species that lie next to one another and that have been extended by the polymerase reaction are joined and a ligation product is obtained in this way, as long as an extension of the oligonucleotide of the first species has been produced in the preceding step, so that now the 3'-end with the present 3'-hydroxy function of the extended oligonucleotide is now directly adjacent to the 5'-end of the oligonucleotide of the second species.
Thus, in addition, it is particularly preferred that according to step d) in the first method variant for the detection of the pretreated DNA, the PCR products are hybridized to an oligonucleotide array and then the following substeps are conducted:
(a) the amplified genomic DNA is hybridized to at least one oligonucleotide with known sequence of n nucleotides with the formation of a duplex, whereby said hybridized oligonucleotides with their 3'-end hybridize partially or completely to the positions which are to be investigated with respect to their methylation in the genomic DNA
sample;
b) the oligonucleotide, insofar as it has previously hybridized by its 3'-terminus without erroneous base pairing to the position to be investigated, is extended by means of a polymerase by at least one nucleotide, whereby 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.
A method is also preferred according to the invention, in which the PCR
products and or extension products and/or ligation products are provided with a detectable label for detection. It is preferred that the labels are fluorescent labels or that the labels are radionuclides or that the labels are removable mass labels, which are detected in a mass spectrometer.
It is also preferred that in one of the amplifications, one of the primers is bound to a solid phase.
It is particularly preferred that the PCR products andlor extension products and/or ligation products overall are detected in the mass spectrometer and thus are clearly characterized by their mass. It is also preferred that a fragment of the PCR
products and/or extension products and/or ligation products are detected in the mass spectrometer. It is particularly preferred here that the fragment of the PCR
product and/or extension product and/or ligation product is produced by digestion with one or more exo- or endonucleases. It is more particularly preferred according to the invention that the produced fragments have a single positive or negative net charge for better detectability in the mass spectrometer.
1t is also preferred according to the invention that the PCR products and/or extension products andlor ligation products are detected and visualized by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF) or by means of electrospray mass spectrometry (ES/).
A method according to the invention is particularly preferred wherein the genomic DNA
has been obtained from a DNA sample, whereby sources for DNA include, e.g., cell lines, blood, sputum, stool, urine, cerebrospinal fluid, tissue embedded in paraffin, for example tissue from eyes, intestine, kidney, brain, heart, prostate, lungs, breast or liver, histological slides and all possible combinations thereof.
Another subject of the present invention is a use of a method according to the invention 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; cancer diseases; CNS malfunctions, damage or disease; symptoms of aggression or behavioral disturbances; clinical, psychological and social consequences of brain damage; 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; lesion, inflammation, infection, immunity and/or convalescence;
malfunction, damage or disease of the body as a consequence of an abnormality in the development process; malfunction, damage or disorder of the skin, the muscles, the connective tissue or the bones; endocrine and metabolic malfunction; headaches or sexual malfunction.
The use of a method according to the invention is preferred for distinguishing cell types or tissues or for investigating cell differentiation.
The subject of the present invention is also a kit, comprising a reagent containing bisulfite, denaturing reagents or solvents, as well as radical traps and optional primers for the production of amplified products, as well as, optionally, instructions for conducting an assay according to one of claims 1-28.
The present invention describes a method for the production of double-stranded DNA
from a genomic DNA sample, which, after the method has been conducted, has the property that CG base pairs are still present only at the positions at which methylcytosine bases were previously found in the genomic DNA. Subsequently any method can be used, which utilizes the chemical, biological or physical properties of CG
base pairs, in order to identify the positions at which methylcytosine bases were found in the genomic DNA sample. For this purpose, many preferred embodiment variants are given below, but is is assumed that the person of average skill in the art could depart from these or could find other obvious alternative possibilities for detecting CG base pairings and to utilize such for the objective given here.
The genomic DNA used in the method is preferably obtained from a DNA sample, whereby sources for DNA include, e.g., cell lines, blood, sputum, stool, urine, cerebrospinal fluid, tissue embedded in paraffin, for example tissue from eyes, intestine, kidney, brain, heart, prostate, lungs, breast or liver, histological slides and all possible combinations thereof.
The method for the detection of cytosine methylation in genomic DNA is characterized by the following steps: First, a genomic DNA sample is chemically converted with a reagent, whereby 5-methylcytosine remains unchanged and cytosine is converted to uracil or another base similar to uracil in its base pairing behavior. Then, in the second step, the pretreated DNA is amplified with the use of a polymerase and at least one primer oligonucfeotide. In the third step, the amplified product is again chemically converted with a reagent, whereby 5-methylcytosine remains unchanged and cytosine is converted to uracil or another base similar to uracil in its base pairing behavior. In the last step, the cytosine bases and/or guanine bases remaining in the amplified product after the repeated chemical treatment are detected.
In a particularly preferred variant, the method is characterized by the following steps:
The method for the detection of cytosine methylation in genomic DNA is characterized by the following steps: First, a genomic DNA sample is chemically converted with a reagent, whereby 5-methylcytosine remains unchanged and cytosine is converted to uracil or another base similar to uracil in its base pairing behavior. Then, in the second step, the pretreated DNA is amplified with the use of a polymerase and at least one primer oligonucleotide. In the third step, the amplified product is again chemically converted with a reagent, whereby 5-methylcytosine remains unchanged and cytosine is converted to uracil or another base similar to uracil in its base pairing behavior. In the fourth step, the chemically treated amplified products from the third step are again amplified. In the last step, the cytosine bases and/or guanine bases remaining in the amplified product after the repeated chemical treatment are detected.
The chemical treatment is particularly preferably conducted with a solution of a hydrogen sulfite (= disulfite, bisulfite). Under suitable conditions, as is known to the person of average skill in the art, this treatment leads to an addition of hydrogen sulfite to unmethylated cytosine bases. A practically quantitative conversion of the amino function of the cytosine into a keto function follows this reaction, so that a sulfonated uracil is formed, which is converted to uracil by alkaline hydrolysis. A
selective conversion of the unmethylated cytosine bases to uracil thus results in this way, whereas the 5-methylcytosines remain unchanged. This reaction occurs only if the DNA
is present in single-strand form. In a particularly preferred variant of the method, therefore, the chemical treatment of the DNA is conducted in a way that permits the DNA present to be embedded in agarose. The formation of duplexes is prevented in this way.
In another particularly preferred variant of the method, during the chemical treatment, a solvent or a reagent is added, which denatures the DNA duplex. These solvents or reagents preferably involve: polyethylene glycol dialkyl ether, dioxane and substituted derivatives, urea or derivatives, acetonitrile, primary alcohols, secondary alcohols, tertiary alcohols, diethylene glycol dialkyl ether, triethylene glycol dialkyl ether, tetraethylene glycol dialkyl ether, pentaethylene glycol dialkyl ether, hexaethylene glycol dialkyl ether, DMSO or THF.
In addition, a radical trap is preferably added, which prevents the decomposition of the DNA under the reaction conditions. These radical traps preferably involve:
di- and trihdroxybenzenes, green tea extract, pine bark extract (Pycnogenol), Ginkgo Biloba extract (EGb 761 ), a flavonoid blend of several fruit and vegetable extracts (GNLD), Bio-Normalizer (Sun-O Corp), DPPH (1,1-diphenyl-2-picrylhydrazyl), NDGA
(nordihydroguaiaretic acid), Trolox (6-hydroxy-2,5,7,8-tetramethylchromane 2-carboxylic acid), 2,6-di-tert-butylphenol, 4-methyl-di-tertbutylphenol, 4-methoxy-di-tert-butylphenol, 2,6-di-tert-butyl-p-cresol, 3,4-dihydroxybenzoic acid, vitamins C, E, Q, hydroquinone, ubiquinone, lignans, hydroxyterpenes, flavonoids, curcumin, tannins, retinoic acid compounds, Ge-132 bisbetacarboxyethyl germanium sesquioxides, superoxide dismutase (SOD), superoxide catalase, alpha-naphthoflavone, Ginkgo biioba extract (EGb 761 ), di-(2-methyl-5-chlorophenyl)dithionate and Cull) derivatives, mebendazole, CS (chloroform-soluble) alkaloidal extract, 4-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-hydroxy-1,2-naphthoquinone, 4-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-methoxy-1,2-naphthoquinone, 4-(3,5-di-tert-butyl-4-hydroxyphenyl)-1,2-naphthoquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-bromo-1,4-naphthoquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-chloro-1,4-naphthoquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-methoxy-1,4-naphthoquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-hydroxy-1,4-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-1,2-anthraquinone, 4-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-methoxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,2-anthraquinone, 4-(3,5-di-tert-butyl-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,2-anthraquinone, 3-bromo-4-(3,5-di-tert-butyl-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,2-anthraquinone, 2-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidene)-indane-1,3-dione, 2-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidene)-3,4-epoxy-3-hydroxy-4-methoxy-3,4-dihydro-2H-naphthalene-1-one, 2-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidene)-3,4-epoxy-3,4-dimethoxy-3,4-dihydro-2H-naphthalene-1-one, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-indane-1-one, 3,3-bi-[2-(3,5-di-tert-butyl-4-hydroxyphenyl)-indene-1-on]-3-yl, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-bromo-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-chloro-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-methoxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone, 2-(3,5-di-tert-butyl-hydroxyphenyl)-3-hydroxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone, 2-bromo-3-(3-bromo-5-tert-butyl-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone, 2-bromo-3-(3,5-dibromo-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone, 2-bromo-3-(3-bromo-5-tert-butyl-4-hydroxyphenyl)-3-hydroxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone, 3-bromo-2-(3,5-di-tert-butyl-4-hydroxyphenyl)-1,4-anthraquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-methoxy-1,4-anthraquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-hydroxy-1,4-anthraquinone, 5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-1,3-diol, 3-methoxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-1-ol, 4-(3-chloro-5,5,8,8-tetramethyl-1,4-dioxo-1,4,5,6,7,8-hexahydroanthracen-2-yl) benzoic acid, methyl-4-(3-chloro-5,5,8,8-tetramethyl-1,4-dioxo-1,4,5,6,7,8-hexahydroanthracen-2-yl) benzoate, 4-(3-hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl) benzoic acid, methyl-(3-methoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl) benzoic acid, 4-(3-hydroxy-5,5,8,8-tetramethyl-1,4-dioxo-1,4,5,6,7,8-hexahydroanthracen-2-yl) benzoic acid, methyl-4-(3-hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl-azo) benzoate, 4-(3-hydroxy-5,5,8,8-tetramethyl-1,4-dioxo-1,4,5,6,7,8-hexahydroanthracen-2-yl-azo) benzoic acid, 3-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidene)-5,5,8,8-tetramethyf-5,6,7,8-tetrahydrocycfopenta[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-1,2,4-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-hydroxyphenyl)-2-methoxy-5,6-dimethyl-1,4-naphthoquinone, 2-(3,5-di-tert-butyl-hydroxyphenyl)-3-methoxy-5,7-dimethyl-1,4-naphthoquinone, 3-(3,5-di-tert-butyl-hydroxyphenyl)-2-methoxy-5,7-dimethyl-1,4-naphthoquinone, 2-(3,5-di-tert-butyl-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-hydroxyphenyl)-3-hydroxy-6,7-dimethyl-1,4-naphthoquinone, 2-(3,5-di-tert-butyl-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-hydroxyphenyl)-2-hydroxy-5,6-dimethyl-1,4-naphthoquinone, 2-(3,5-di-tert-butyl-hydroxyphenyl)-3-hydroxy-5,7-dimethyl-1,4-naphthoquinone, 3-(3,5-di-tert-butyl-hydroxyphenyl)-2-hydroxy-5,7-dimethyl-1,4-naphthoquinone.
A variant of the method is also preferred in which the reagents, which are utilized for the chemical treatment of DNA, are wholly or partially separated prior to the subsequent amplification. For example, this can be conducted by binding the DNA to a solid phase and removing the reagents by washing steps. In a particularly preferred variant of the method, the sample is diluted after the chemical treatment, but only so that the reagents are present in such a low concentration that they do not disrupt the amplification that immediately follows. In a particularly preferred variant of the method, the amplification of several DNA segments is conducted simultaneously in one reaction vessel. It is also particularly preferred that a heat-stable DNA polymerase is used and that PCR
is employed for the amplification, as it is known in principle to the person of average skill in the art.
It is also preferred to conduct a desulfonation prior to the amplifications, since sulfonic acid groups may be present due to the chemical pretreatment. It is particularly preferred that this desulfonation be conducted in a basic buffer and ideally this basic buffer is the same buffer that is also used for the amplification reaction.
Several methods will now be described below, which are familiar in principle to the person of average skill in the art, since the cytosine or guanine bases that remain after the method has been conducted, which now serve as indicators for the presence of cytosine methylation in the initial genomic DNA sample, can be detected.
The remaining cytosine andlor guanine bases or cytosine-guanine base pairs are detected by hybridization reactions. This detection can preferably be conducted by hybridizing the amplified products to an oligonucleotide array and then conducting the following substeps: In the first step, the amplified DNA is hybridized to at least one oligonucleotide with the formation of a duplex, whereby said hybridized oligonucleotide lies directly adjacent at its 3'-end, or at a distance of up to 10 bases, to the positions which are to be investigated relative to their methylation in the genomic DNA
sample.
The oligonucieotide or oligonucleotides with known sequence of n nucleotides is (are) now extended in the next step by means of a polymerase by at least one nucleotide, whereby the nucleotide bears a detectable label and the extension depends on the methylation state of the respective cytosine in the original genomic DNA
sample. Thus, it is possible, for example, that an extension reaction occurs only if a guanine lies adjacent to the 3'-end of the oligonucleotide on the template DNA, which permits the incorporation of a labeled cytosine. The coupling of the labeled cytosine to the oligonucleotide then finally serves as the detection reaction for the presence of a methylated cytosine in the initial genomic sample.
It is also possible that the cytosine and/or guanine bases remaining after the second chemical treatment (and optional second amplification) are detected by sequencing reactions The person of average skill in the art is particularly familiar with sequencing according to Sanger and sequencing according to Maxam-Gilbert. The latter is particularly appropriate, since only those CG base pairs are still present in the duplex at sites where a methylcytosine was present in the initial genomic DNA sample. It is thus possible to generate fragments by C and/or G-specific cleavage reactions, and the size of the fragment directly depends on the methylation state of the sample. The size of these fragments, for example, can then be determined on sequencing gels or by capillary electrophoresis. In comparison to only a simple bisulfite treatment, as is described in the prior art, the method described here in this variant also displays the particular advantage that the cleavage of the respective counterstrand cannot occur, since this strand does not contain either C or G. The number of fragments that are formed is clearly reduced in this way and also, if several amplified products are investigated simultaneously, a very clear and comprehensive evaluation is possible.
A variant of the method is also preferred, in which the amplification products are hybridized to an oligonucleotide array and then the following substeps are conducted: In the first step, a set of oligonucleotides is hybridized to the amplified genomic DNA with the formation of a duplex, wherein this set of oligonucleotides is comprised of two different species and wherein the hybridized oligonucleotides of the first species lie directly adjacent at their 3'-end, or at a distance of up to 10 bases, to the positions which are to be investigated relative to their methylation in the genomic DNA
sample and whereby 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 is separated by a gap of the magnitude of a single nucleotide or up to nucleotides from the 3'-end of the hybridized oligonucleotide of the first species at the site of said selected position. In the second step, the oligonucleotide of the first species with known sequence of n nucleotides is extended by means of a polymerase by at most the number of nucleotides which lie between the 3'-end of the oligonucleotide of the first species and the 5'-end of the oligonucleotide of the second species, wherein the extension depends on the methylation state of the respective cytosine in the genomic DNA sample. For example, it is possible that an extension of the 3'-end only occurs if a thymine is present instead of a cytosine in the chemically pretreated DNA, since, for example, in the nucleotide mix used, guanine is present only as a terminator, and thus the extension is interrupted if a cytosine is present. In turn, a cytosine is present after the chemical treatment, only if a 5-methylcytosine was present at the respective position in the original DNA sample.
In the last step, the oligonucleotide is incubated in the presence of a ligase, whereby the oligonucleotide of the first species and the oligonucleotide of the second species that lie next to one another and that have been extended by the polymerase reaction are joined and a ligation product is obtained in this way, as long as an extension of the oligonucleotide of the first species has been produced in the preceding step, such that now the 3'-end with the present 3'-hydroxy function of the extended oligonucleotide is now directly adjacent to the 5'-end of the oligonucleotide of the second species.
A method is also preferred, in which the PCR products are hybridized to an oligonucleotide array for the detection of the pretreated DNAand then the following substeps are conducted: In the first step, the amplified genomic DNA is hybridized to at least one oligonucleotide with known sequence of n nucleotides with the formation of a duplex, whereby said hybridized oligonucleotides with their 3'-end hybridize partially or completely to the positions which are to be investigated with respect to their methylation in the genomic DNA sample. In the second step, the oligonucleotide, insofar as it has previously hybridized by its 3'-terminus without erroneous base pairing to the position to be investigated, is extended by means of a polymerase by at least one nucleotide, whereby 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.
Thus, for example, an extension can occur only if the genomic DNA was originally unmethylated at the position at which the above-named oligonucleotide binds by its 3'-end, since if this were not so, after the chemical pretreatment, a cytosine would remain at this position, which causes a mismatch at the 3'-end after the binding of the oligonucleotide, which interrupts the extension. If a methylation is not present, then in contrast, the primer can bind by its 3'-end to the template DNA without mismatch.
The analysis of DNA oligomers which are formed and which serve for the detection of cytosine methylation can be conducted, for example, by means of fluorescent labels, which are incorporated into the DNA by means of [using] labeled nucleotides.
The person of average skill in the art is also familiar with how most of the described detection reactions can be conducted so that the participating oligonucleotides are bound to a solid phase.
Thus, for example, it is possible and particularly preferred to conduct the PCR after the second chemical treatment so that radioactively or fluorescently labeled C
andlor G
nucleotides are incorporated and also one of the PCR primers is bound to a solid phase. After the removal of all educts and products not bound to the solid phase, a PCR
product bound to the solid phase remains, in which the number of incorporated labels is proportional to the number of the guanine and/or cytosine bases remaining after the second chemical treatment, and thus directly depends on the number of cytosine methylations in the genomic sample. Thus, a direct quantification of the methylated cytosines is possible in this PCR product.
A method is also particularly preferred, in which the PCR products and/or extension products and/or ligation products are provided with a detectable label for the detection.
The labels are particularly preferably fluorescent labels, radionuclides, or removable mass labels, which are detected in a mass spectrometer, or combinations thereof.
In another preferred variant of the method, the PCR products and/or extension products and/or ligation products overall are detected in the mass spectrometer and thus are clearly characterized by their mass. In another particularly preferred variant of the method, a fragment of the PCR products and/or extension products and/or ligation products is detected in the mass spectrometer. It is particularly preferred that these fragments of the PCR products and/or extension products and/or ligation products are produced by digestion with one or more exo- or endonucleases. In another particularly preferred embodiment of the method, the generated fragments have a single positive or negative net charge for better detectability in the mass spectrometer.
It is particularly preferred that the PCR products and/or extension products and/or ligation products are detected and visualized by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF) or by means of electrospray mass spectrometry (ES/).
The subject of the present invention is also the use of the described method 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; cancer diseases; CNS malfunctions, damage or disease; symptoms of aggression or behavioral disturbances; clinical, psychological and social consequences of brain damage; psychotic disturbances and personality disorders; dementia andlor associated syndromes; cardiovascular disease, malfunction and damage;
malfunction, damage or disease of the gastrointestinal tract; malfunction, damage or disease of the respiratory system; lesion, inflammation, infection, immunity and/or convalescence;
malfunction, damage or disease of the body as a consequence of an abnormality in the development process; malfunction, damage or disorder of the skin, the muscles, the connective tissue or the bones; endocrine and metabolic malfunction; headaches or sexual malfunction.
The use of the method is also particularly preferred for distinguishing cell types or tissues or for investigating cell differentiation.
The subject of the present invention is also a kit, comprising a reagent containing bisulfate, denaturing reagents or solvents, as well as radical traps andlor primers for the production of amplified products, as well as, optionally, instructions for conducting the method according to the invention. The following examples explain the invention, but it is not limited to these.
Example 1: Modification of a genomic sequence by two treatments with bisulfate The following double-stranded DNA sequence, which contains a potentially methylated GG dinucleotide, will serve as an example for explaining the method:
A 3'-TGATCTAGCATGACTAC-5' B 5'-ACTAGATCGTACTGATG-3' The double-stranded sequence can be used in order to investigate adverse events of a specific category for patients or individuals. A genomic, unknown methylated DNA
sample (20 ng), which was digested with a restriction enzyme, here Mss1, is used. The digested DNA is chemically converted with hydrogen sulfite, (bisulfate, disulfite) and a radical trap at elevated temperature. A reagent or solvent, which supports denaturing, is added. The DNA is first thermally denatured and then reacted with bisulfate, the radical trap and the denaturing reagent, and is incubated for a long time at elevated temperature. The bisulfate reaction leads to the conversion of all unmethylated cytosine bases to uracil. In order to purify the bisulfite-treated DNA, it is bound to a reversed-phase C18 solid phase and is freed of chemicals by washing with a suitable buffer solution. Then the DNA is eluted with a polar solvent, such as, e.g., acetonitrile or water and concentrated to a smaller volume. The bisulfite-treated fragments are subjected to alkaline hydrolysis directly prior to specific amplification under basic conditions. After this, the sample is amplified with the use of 25 pmol of specific primer oligonucleotides (containing no CG dinucleotides). After the first treatment with a solution of a hydrogen sulfite, in which the unmethylated cytosine bases are converted to uracil, and subsequent PCR, which amplifies the strands that are no longer complementary after the chemical treatment with hydrogen sulfite and at the same time uracil is also sustantially replaced by thymine, the following double-stranded DNA sequences result:
A) for the case in which CG (+) was methylated:
from the lower strand B:
C (+) 3'-TAATCTAGCATAACTAC-5' B' (+) 5'-ATTAGATCGTATTGATG-3' Sequence L (up) from the upper strand A:
A' (+) 3 TGATTTAGCATGATTAT-5' D (+) 5'-ACTAAATCGTACTAATA-3' Sequence U (up) A) for the case in which CG (-) was unmethylated:
from the lower strand B:
C (-) 3'-TAATCTAACATAACTAC-5' B' (-) 5'-ATTAGATTGTATTGATG-3' Sequence L (down) from the upper strand A:
A' (-) 3'-TGATTTAGTATGATTAT-5' D (-) 5'-ACTAAATCATACTAATA-3' Sequence U (down) The second treatment with hydrogen sulfite is conducted analogously to the first. The amplified products are first thermally denatured and this is supported by the addition of a reagent or solvent, and then they are chemically converted, and subsequently amplified, whereby alkaline hydrolysis is conducted again just prior to the amplification.
The second specific amplification can be conducted with a part of the bisulfite-treated sample. The reaction solution can be diluted until the chemicals required for the hydrogen sulfite reaction no longer disturb the PCR. On the other hand, the sample can again be purified by means of a reversed-phase C18 solid phase. The DNA that has been treated twice with hydrogen sulfite is amplified with the use of 25 pmol of specific primer oligonucleotides (containing no CG dinucleotides). Thus, the sequences of the primer oligonucleotides of the first and second amplifications need not correspond.
After the second treatment with a solution of a hydrogen sulfite, in which again the unmethylated cytosine bases are converted to uracil, and subsequent PCR, which amplifies the strands that are no longer complementary after the chemical treatment with hydrogen sulfite and at the same time uracil is also substantially replaced by thymine, the following double-stranded DNA sequences now result:
(A) for the case in which CG (+) was methylated:
from the lower strand B' and C:
E (+) 3'-TAATCTAACATAACTAC-5' Sequence LL (up) B " (+) 5'-ATTAGATTGTATTGATG-3' C' (+) 3'-TAATTTAGTATAATTAT-5' F (+) 5'-ATTAAATCATATTAATA-3' Sequence LU (up) from the upper strand D and A':
G (+) 3'-TAATTTAACATAATTAT-5' D' (+) 5'-ATTAAATTGTATTAATA-3' Sequence UL (up) H (+) 3'-TGATTTAGTATGATTAT-5' A" (+) 5'-ACTAAATCATACTAATA-3' Sequence UU (up) B) for the case in which CG (-) was unmethylated:
from the lower strand C and B':
E (-) 3'-TAATCTAACATAACTAC-5' B" (-) 5'-ATTAGATTGTATTGATG-3' Sequence LL (down) C' (-) 3'-TAATTTAATATAATTAT-5' F (-) 5'-ATTAAATTATATTAATA-3' Sequence LU (down) from the upper strand D and A':
G (-) 3'-TAATTTAATATAATTAT-5' D' (-) 5'-ATTAAATTATATTAATA-3' Sequence UL (down) H (-) 3'-TGATTTAGTATGATTAT-5' A " (-) 5'-ACTAAATCATACTAATA-3' Sequence UU (down) The sequences UU are identical, independent of the methylation state of the sample;
the same also applies to the sequences LL. Thus, both sequences cannot be used for determining the methylation state. LU and UL, however, differ at the former CG
positions depending on the methylation state of the DNA sample (emphasis provided in the sequences). It is worth noting and also the actual advantage of the method is particularly that in the respective strands, cytosine and guanine are only formed if a methylation was present previously. Practically, therefore, there exists a conversion of the methylcytosine bases in the genomic sample into a base that is also characteristic for the double strand, while in the case of simple bisulfite treatment, the base is characteristic for methylcytosine only in one of the two strands. This now permits application to practically all of the methods for sequencing known to the person of average skill in the art (such as, for example, Maxam-Gilbert sequencing or for genotyping, without needing to take into consideration the counterstrand. If, for example, a sequence-specific cleavage is conducted at cytosine bases of the DNA
amplified after the second bisulfate treatment, then only one cleavage of one strand results, while the other remains unchanged.
Example 2: Modification of a genomic sequence by two treatments with bisulfate The gene HSMDR1 of the human genome was selected as an example. In the experiment, after a first bisulfate treatment, DNA fragments of this gene with a length of 2010 by were produced and simultaneously gc-rich domains were generated at the 5'-and 3'-ends. Since these "artificial" ends at the 5'- and 3'-ends of the PCR
products were produced after the first bisulfate treatment, they are subject to a simple conversion in the second bisulfate treatment. This leads to a higher specificity of these domains, since they still contain C's and G's (in contrast to the other sequence). Thus they are particularly suitable for the design of primers for an amplification after the second bisulfate treatment. The amplification of a short piece of the target DNA
takes place with primers that exclusively consist of A's and T's. The sequences of the genomic DNA
with a single bisulfate treatment and a double bisulfate treatment are shown at the end.
Description of the Figures:
Figure 1:
Diagram for the production of the PCR product with gc-domains of DNA from a single bisulfate treatment:
a) 5'gtgatcccgggcgagctcccTAAGTATGTTGAAGAAAGATTATTG;
b) 3'gcttgggctgcaggtcgaccTTTTAACCTTCTATCTCATCAAC;
c) bisulfate-treated DNA, single strand Figure 2:
Genomic DNA of the neighborhood of the HSMDR1 gene: LOCUS HSMDR1A2932 by DNA human MDR1 (multidrug resistance) gene for the P-glycoprotein Figure 3:
Sequence of the upper strand after the first bisulfite treatment for the case of complete methylation of the CG's (PCR product with primers 1 and 2) Figure 4:
Sequences of the lower strand after the second bisulfite treatment for the case of complete methyfation of all CG's a) PCR product with primers 3 and 4 (154 bp) b) PCR product with primers 5 and 6 (2010 bp) Procedure:
a) Restriction of human DNA of unknown methylation state with Mss1 b) Bisulfite treatment with the agarose method c) Bisulfite-specific PCR for the production of MDR1 fragments with a length of 1936 bp.
"Artificial" gc-rich ends are simultaneously generated on the PCR products.
d) Second bisulfite treatment without agarose with subsequent ZipTip purification e) Amplification of DNA with two bisulfite treatments by means of primers for the gc-domains Primers used:
Bisulfite primer, product size: 2070 by 1 MDR1-B-U-gc gtgatcccgggcgagctcccTAAGTATGTTGAAGAAAGATTATTG
2 MDR1-B-L-gc gcttgggctgcaggtcgacCTTTTAACCTTCTATCTCATCAAC
<--gc-domain---------><-gene-specific sequence-->
Primer for DNA with two bisulfite treatments without gc-domains, product size:
754 by 3 MDR1-2B-U3 tttttttttatttttttattat 4 MDR1-2B-L3 atttttttttattattttttaat Primer for DNA with two bisulfate treatments with gc-domains; product size:
2090 by MDR1-2B-L-gc GTTTGGGTTGTAGGTTGAT
6 MDR1-2B-U-gc ataatcccaaacaaactccc Test conditions:
The restriction of human genomic DNA (Promega) was conducted with Mss1 (Fermentas) according to the manufacturer's instructions.
The first bisulfate treatment of the digested DNA was conducted with the agarose method, as it is described in the prior art.
The amplification of DNA was conducted under the following PCR conditions (Qiagen):
--1 p.1 DNA (10 ng of bisulfate-treated DNA) --0.2 p1 Taq (1 Unit) --0.2 p,1 dNTP (25 mM each) 0.25 mM final --1 p1 primer 1 (12.5 pmol/p,l) 0.5 pmol/p.l final --1 w1 primer 2 (12.5 pmol/~l) 0.5 pmol/p.l final --2.5 p1 10X PCR buffer --19.1 H20 (molecular grade) The following cycler programs were used:
--PCR with domain primers (primers 1 and 2) after the first bisulfate treatment:
95°C/20:00; 95°C/1:00; 56 °C/0:45; 72°C/2:00;
cycles: 40; 72°C/10:00;
4°C/end --PCR after the second bisulfate treatment (primers 3 and 4):
95°C/20:00;
95°C/1:00; 40 °C/0:45; 72°C/1:00; cycles: 40;
72°C/10:00; 4°C/end --PCR after the second bisulfite treatment (primers 5 and 5 [sic]):
95°C/20:00; 95°C/1:00; 56 °C/0:45; 72°C/2:00;
cycles: 40; 72°C/10:00;
4°C/end The second bisulfite treatment of the produced PCR product was conducted without agarose in solution. The DNA with two bisulfite treatments was purified by means of the ZipTip~ method (Millipore) for the removal of salts and chemicals. The procedure was as follows:
--new tips rinsed 3x with 10 p,1 of 2M TEAR buffer --35 ~.I of PCR product loaded --1x rinse with 10 ~I of 2M TEAR
--3x rinse with 10 ~I of 0.1 M TEAA
--3x rinse with 10 ~I of water (2x distilled) --Elution of the product with 100 ~I of MeCN (10x10 ~I of fresh MeCN), loaded and eluted in a tube --100 u1 of eluate were dried under vacuum, resuspended in 30 ~I of water (2x distilled) and immediately used in the PCR.

SEQUENCE LISTING
<110> Epigenomics AG
<120> Method for Detection of Cytosine Methylations <130> E01/1241/WO
<140> PCT/DE 01103901 <141> 2001-10-10 <160> 24 <170> PatentIn version 3.1 <210> 1 <211> 17 <212> DNA
<213> Artificial <900> 1 catcagtacg atctagt 17 <210> 2 <211> 17 <212> DNA
<213> Artificial <400> 2 actagatcgt actgatg 17 <210> 3 <211> 17 <212> DNA
<213> Artificial <400> 3 catcaatacg atctaat 17 <210> 4 <211> 17 <212> DNA
<213> Artificial <400> 4 attagatcgt attgatg 17 <210> 5 <211> 17 <212> DNA
<213> Artificial <400> 5 tattagtacg atttagt 17 <210> 6 <211> 17 <212> DNA
<213> Artificial <400> 6 actaaatcgt actaata 17 <210> 7 <211> 17 <212> DNA
<213> Artificial <400> 7 catcaataca atctaat 17 <210> 8 <211> 17 <212> DNA
<213> Artificial <400> 8 attagattgt attgatg 17 <210> 9 <211> 17 <212> DNA
<213> Artificial <400> 9 tattagtatg atttagt 17 <210> 10 <211> 17 <212> DNA
<213> Artificial <400> 10 actaaatcat actaata 17 <210> 11 <211> 17 <212> DNA
<213> Artificial <400> 11 catcaataca atctaat 17 <210> 12 <211> 17 <212> DNA
<213> Artificial <400> 12 tattaatatg atttaat 17 <210> 13 <211> 17 <212> DNA
<213> Artificial <400> 13 attaaatcat attaata 17 <210> 14 <211> 17 <212> DNA
<213> Artificial <400> 14 tattaataca atttaat 17 <210> 15 <211> 17 <212> DNA
<213> Artificial <400> 15 attaaattgt attaata 17 <210> 16 <211> 17 <212> DNA
<213> Artificial <900> 16 tattaatata atttaat 17 <210> 17 <211> 17 <212> DNA
<213> Artificial <400> 17 attaaattat attaata 17 <210> 18 <211> 45 <212> DNA
<213> Artificial <400> is gtgatcccgg gcgagctccc taagtatgtt gaagaaagat tattg 95 <210> 19 <211> 93 <212> DNA
<213> Artificial <400> 19 caactactct atcttccaat tttccagctg gacgtcgggt tcg 43 <210> 20 <211> 43 <212> DNA
<213> Artificial <400> 20 gcttgggctg caggtcgacc ttttaacctt ctatctcatc aac 43 <210> 21 <211> 22 <212> DNA
<213> Artificial <400> 21 ttttttttta tttttttatt at 22 <210> 22 <211> 23 <212> DNA
<213> Artificial <400> 22 attttttttt attatttttt aat 23 <210> 23 <211> 19 <212> DNA
<213> Artificial <400> 23 gtttgggttg taggttgat 19 <210> 24 <211> 20 <212> DNA
<213> Artificial <900> 24 ataatcccaa acaaactccc 20

Claims (32)

Claims

1. A method for the detection of cytosine methylation in genomic DNA, hereby characterized in that the following method steps are conducted:
a) a genomic DNA sample is chemically converted with a reagent, whereby 5-methylcytosine remains unchanged and cytosine is converted to uracil or another base similar to uracil in its base pairing behavior;
b) the pretreated DNA is amplified with the use of a polymerase and at least one primer oligonucleotide;
c) the amplified product is again chemically converted with a reagent, whereby methylcytosine remains unchanged and cytosine is converted to uracil or another base similar to uracil in its base pairing behavior;
d) the cytosine bases and/or guanine bases remaining in the amplified product after the repeated chemical treatment are detected.

2. The method for the detection of cytosine methylation in genomic DNA
according to claim 1, further characterized in that the following method steps are conducted:
a) a genomic DNA sample is chemically converted with a reagent, whereby 5-methylcytosine remains unchanged and cytosine is converted to uracil or another base similar to uracil in its base pairing behavior;
b) the pretreated DNA is amplified with the use of a polymerase and at least one primer oligonucleotide;
c) the amplified product is again chemically converted with a reagent, whereby methylcytosine remains unchanged and cytosine is converted to uracil or another base similar to uracil in its base pairing behavior;
d) the chemically treated amplified product from step c) is again amplified;
e) the cytosine bases and/or guanine bases remaining in the amplified product are detected.

3. The method according to claim 1 or 2, further characterized in that the chemical treatment is conducted with sodium bisulfite (= hydrogen sulfite, disulfite).

4. The method according to claim 3, further characterized in that the chemical treatment is conducted after embedding the DNA in agarose.

5. The method according to one of the preceding claims, further characterized in that a reagent that denatures the DNA duplex and/or a radical trap is (are) present in the chemical treatment.

6. The method according to claim 5, further characterized in that the denaturing reagent is selected from one of the following substances: polyethylene glycol dialkyl ether, dioxane and substituted derivatives, urea or derivatives, acetonitrile, primary alcohols, secondary alcohols, tertiary alcohols, diethylene glycol dialkyl ether, triethylene glycol dialkyl ether, tetraethylene glycol dialkyl ether, pentaethylene glycol dialkyl ether, hexaethylene glycol dialkyl ether, DMSO or THF.

7. The method according to claim 5 or 6, further characterized in that the radical trap is selected from one of the following substances:
di- and trihdroxybenzenes, green tea extract, pine bark extract (Pycnogenol), Ginkgo Biloba extract (EGb 761 ), a flavonoid blend of several fruit and vegetable extracts (GNLD), Bio-Normalizer (Sun-O Corp), DPPH (1,1-diphenyl-2-picrylhydrazyl), NDGA
(nordihydroguaiaretic acid), Trolox (6-hydroxy-2,5,7,8-tetramethylchromane 2-carboxylic acid), 2,6-di-tert-butylphenol, 4-methyl-di-tertbutylphenol, 4-methoxy-di-tert-butylphenol, 2,6-di-tert-butyl-p-cresol, 3,4-dihydroxybenzoic acid, vitamins C, E, Q, hydroquinone, ubiquinone, lignans, hydroxyterpenes, flavonoids, curcumin, tannins, retinoic acid compounds, Ge-132 bisbetacarboxyethyl germanium sesquioxides, superoxide dismutase (SOD), superoxide catalase, alpha-naphthoflavone, Ginkgo biloba extract (EGb 761), di-(2-methyl-5-chlorophenyl)dithionate and Cu(II) derivatives, mebendazole, CS (chloroform-soluble) alkaloidal extract, 4-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-hydroxy-1,2-naphthoquinone, 4-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-methoxy-1,2-naphthoquinone, 4-(3,5-di-tert-butyl-4-hydroxyphenyl)-1,2-naphthoquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-bromo-1,4-naphthoquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-chloro-1,4-naphthoquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-methoxy-1,4-naphthoquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-hydroxy-1,4-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-1,2-anthraquinone, 4-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-methoxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,2-anthraquinone, 4-(3,5-di-tert-butyl-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,2-anthraquinone, 3-bromo-4-(3,5-di-tert-butyl-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,2-anthraquinone, 2-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidene)-indane-1,3-dione, 2-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidene)-3,4-epoxy-3-hydroxy-4-methoxy-3,4-dihydro-2H-naphthalene-1-one, 2-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidene)-3,4-epoxy-3,4-dimethoxy-3,4-dihydro-2H-naphthalene-1-one, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-indane-1-one, 3,3-bi-[2-(3,5-di-tert-butyl-4-hydroxyphenyl)-indene-1-on]-3-yl, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-bromo-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-chloro-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-methoxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone, 2-(3,5-di-tert-butyl-hydroxyphenyl)-3-hydroxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone, 2-bromo-3-(3-bromo-5-tert-butyl-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone, 2-bromo-3-(3,5-dibromo-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone, 2-bromo-3-(3-bromo-5-tert-butyl-4-hydroxyphenyl)-3-hydroxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone, 3-bromo-2-(3,5-di-tert-butyl-4-hydroxyphenyl)-1,4-anthraquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-methoxy-1,4-anthraquinone, 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-hydroxy-1,4-anthraquinone, 5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-1,3-diol, 3-methoxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-1-ol, 4-(3-chloro-5,5,8,8-tetramethyl-1,4-dioxo-1,4,5,6,7,8-hexahydroanthracen-2-yl) benzoic acid, methyl-4-(3-chloro-5,5,8,8-tetramethyl-1,4-dioxo-1,4,5,6,7,8-hexahydroanthracen-2-yl) benzoate, 4-(3-hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl) benzoic acid, methyl-(3-methoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl) benzoic acid, 4-(3-hydroxy-5,5,8,8-tetramethyl-1,4-dioxo-1,4,5,6,7,8-hexahydroanthracen-2-yl) benzoic acid, methyl-4-(3-hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl-azo) benzoate, 4-(3-hydroxy-5,5,8,8-tetramethyl-1,4-dioxo-1,4,5,6,7,8-hexahydroanthracen-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-tetrahydrocyclopenta[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-1,2,4-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-hydroxyphenyl)-2-methoxy-5,6-dimethyl-1,4-naphthoquinone, 2-(3,5-di-tert-butyl-hydroxyphenyl)-3-methoxy-5,7-dimethyl-1,4-naphthoquinone, 3-(3,5-di-tert-butyl-hydroxyphenyl)-2-methoxy-5,7-dimethyl-1,4-naphthoquinone, 2-(3,5-di-tert-butyl-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-hydroxyphenyl)-3-hydroxy-6,7-dimethyl-1,4-naphthoquinone, 2-(3,5-di-tert-butyl-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-hydroxyphenyl)-2-hydroxy-5,6-dimethyl-1,4-naphthoquinone, 2-(3,5-di-tert-butyl-hydroxyphenyl)-3-hydroxy-5,7-dimethyl-1,4-naphthoquinone, 3-(3,5-di-tert-butyl-hydroxyphenyl)-2-hydroxy-5,7-dimethyl-1,4-naphthoquinone.

8. The method according to one of the preceding claims, further characterized in that the reagents which are used for the chemical treatment of DNA are completely or partially eliminated prior to the subsequent amplification.

9. The method according to one of the preceding claims, further characterized in that the samples are diluted after the chemical treatment, prior to the amplification.

10. The method according to one of the preceding claims, further characterized in that the amplification of several DNA segments is conducted simultaneously in one reaction vessel.

11. The method according to one of the preceding claims, further characterized in that a heat-stable DNA polymerase is used for the amplification.

12. The method according to one of the preceding claims, further characterized in that a desulfonation of the DNA is conducted prior to the amplifications in claims 1 or 2.

13. The method according to one of claims 1 to 12, further characterized in that the cytosine and/or guanine bases that remain after the second chemical treatment are detected by hybridization reactions.

14. The method according to one of claims 1 to 12, further characterized in that the cytosine and/or guanine bases that remain after the second chemical treatment are detected and/or quantified by specific incorporation of detectable labels in the cytosine and/or guanine bases.

15. The method according to one of claims 1 to 12, further characterized in that in step d) according to claim 1 or step e) according to claim 2 for the detection of the pretreated DNA, the amplification products are hybridized to an oligonucleotide array and then the following substeps are conducted: a) the amplified genomic DNA is hybridized to at least one oligonucleotide with the formation of a duplex, whereby said hybridized oligonucleotide lies directly adjacent at its 3'-end, or at a distance of up to 10 bases, to the positions which are to be investigated relative to their methylation in the genomic DNA sample;
b) the oligonucleotide with known sequence of n nucleotides is extended by means of a polymerise by at least one nucleotide, whereby the nucleotide bears a detectable label and the extension of the respective cytosine in the genomic DNA sample depends on the methylation state.

16. The method according to one of claims 1 to 12, further characterized in that the cytosine and/or guanine bases that remain after the second chemical treatment are detected by sequencing reactions.

17. The method according to one of claims 1 to 12, further characterized in that in step d) according to claim 1 or step e) according to claim 2 for the detection of the pretreated DNA, the amplification products are hybridized to an oligonucleotide array and then the following substeps are conducted:
(a) a set of oligonucleotides is hybridized to the amplified genomic DNA with the formation of a duplex, wherein this set of oligonucleotides is comprised of two different species and wherein the hybridized oligonucleotides of the first species lie directly adjacent at their 3'-end, or at a distance of up to 10 bases, to the positions which are to be investigated relative to their methylation in the genomic DNA sample and whereby 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 is separated by a gap of the magnitude of a single nucleotide or up to 10 nucleotides from the 3'-end of the hybridized oligonucleotide of the first species at the site of said selected position;
(b) the oligonucleotide of the first species with known sequence of n nucleotides is extended by means of a polymerise by at most the number of nucleotides which lie between the 3'-end of the oligonucleotide of the first species and the 5'-end of the oligonucleotide of the second species, wherein the extension depends on the methylation state of the respective cytosine in the genomic DNA sample;
(c) the oligonucleotide is incubated in the presence of a ligase, whereby the oligonucleotide of the first species and the oligonucleotide of the second species that lie next to one another and that have been extended by the polymerase reaction are joined and a ligation product is obtained in this way, as long as an extension of the oligonucleotide of the first species has been produced in the preceding step, so that now the 3'-end with the present 3'-hydroxy function of the extended oligonucleotide is directly adjacent to the 5'-end of the oligonucleotide of the second species.

18. The method according to one of claims 1 to 12, further characterized in that according to step d) in claim 1, for the detection of the pretreated DNA, the PCR
products are hybridized to an oligonucleotide array and then the following substeps are conducted:
(a) the amplified genomic DNA is hybridized to at least one oligonucleotide with known sequence of n nucleotides with the formation of a duplex, whereby said hybridized oligonucleotides with their 3'-end hybridize partially or completely to the positions which are to be investigated with respect to their methylation in the genomic DNA
sample;
b) the oligonucleotide, insofar as it has previously hybridized by its 3'-terminus without erroneous base pairing to the position to be investigated, is extended by means of a polymerase by at least one nucleotide, whereby at least one nucleotide bears a detectable label and the extension of the respective cytosine in the genomic DNA
sample depends on the methylation state.

19. The method according to one of the preceding claims, further characterized in that the PCR products and/or extension products and/or ligation products are provided with a detectable label for the detection.

20. The method according to one of the preceding claims, further characterized in that the labels are fluorescent labels.

21. The method according to one of the preceding claims, further characterized in that the labels are radionuclides.

22. The method according to one of claims 1 to 19, further characterized in that the labels are removable mass labels which are detected in a mass spectrometer.

23. The method according to one of the preceding claims, further characterized in that in one of the amplifications, one of the primers is bound to a solid phase.

24. The method according to one of claims 1 to 19, further characterized in that the PCR products and/or extension products and/or ligation products overall are detected in the mass spectrometer and thus are clearly characterized by their mass.

25. The method according to one of claims 1 to 19, further characterized in that a fragment of the PCR products and/or extension products and/or ligation products is detected each time in the mass spectrometer.

26. The method according to claim 25, further characterized in that the fragment of the PCR product and/or extension product and/or ligation product is produced by digestion with one or more exo- or endonucleases.

27. The method according to claim 25 or 26, further characterized in that the generated fragments have a single positive or negative net charge for better detectability in the mass spectrometer.

28. The method according to one of claims 1 to 19, further characterized in that the PCR products and/or extension products and/or ligation products are detected and visualized by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF) or by means of electrospray mass spectrometry (ESI).

29. The method according to one of the preceding claims, wherein the genomic DNA
has been obtained from a DNA sample, whereby sources for DNA include, e.g., cell lines, blood, sputum, stool, urine, cerebrospinal fluid, tissue embedded in paraffin, for example tissue from eyes, intestine, kidney, brain, heart, prostate, lungs, breast or liver, histological slides and all possible combinations thereof.

30. Use of a method according to one of the preceding claims 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; cancer diseases; CNS malfunctions, damage or disease; symptoms of aggression or behavioral disturbances; clinical, psychological and social consequences of brain damage; 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; lesion, inflammation, infection, immunity and/or convalescence;
malfunction, damage or disease of the body as a consequence of an abnormality in the development process; malfunction, damage or disorder of the skin, the muscles, the connective tissue or the bones; endocrine and metabolic malfunction; headaches or sexual malfunctions.

31. Use of the method according to one of claims 1 to 29 for distinguishing cell types or tissues or for investigating cell differentiation.

32. A kit, comprising a reagent containing bisulfite, denaturing reagents or solvents, as well as radical traps and optional primers for the production of amplified products, as well as, optionally, instructions for conducting an assay according to one of claims 1-29.