EP1238112A2

EP1238112A2 - Method for the parallel detection of the degree of methylation of genomic dna

Info

Publication number: EP1238112A2
Application number: EP00989842A
Authority: EP
Inventors: Alexander Olek; Christian Piepenbrock
Original assignee: Epigenomics AG
Current assignee: Epigenomics AG
Priority date: 1999-12-06
Filing date: 2000-12-06
Publication date: 2002-09-11
Also published as: WO2001042493A2; DE10083729D2; AU778411B2; DE19959691A1; WO2001042493A3; AU2663201A; US20040248090A1; CA2395047A1

Abstract

The invention relates to a method for the parallel detection of the degree of methylation of genomic DNA wherein the following the steps are performed: (a) chemical treatment at the 5' position of non-methylated cytosine bases converts said bases into uracil, thymidine or another base which exhibits hybridization behavior different to that of cytosine in a genomic DNA sample; (b) more than ten different fragments, each having less than 2000 base pairs in said chemically treated genomic DNA sample, are amplified simultaneously using synthetic oligonucleotides as a primer, whereby said primers each contain genomic sequences which are involved in gene regulation and/or transcribed and/or translated, such as those sequences which should be obtained after execution of steps (a); (c) the sequence contexts of all or a portion of the CpG dinucleotides or CpNpG trinucleotides contained in the amplified fragments are determined.

Description

Method for the parallel detection of the methylation state of genomic DNA

The present invention relates to a method for the parallel detection of the methylation state of genomic DNA.

The observation levels that have been well studied in molecular biology according to the methodological developments of recent years are the genes themselves, the translation of these genes into RNA and the resulting proteins. When in the course of the development of an individual which gene is switched on and how activation and inhibition of certain genes in certain cells and tissues is controlled is highly likely to be correlated with the extent and character of the methylation of the genes or the genome. In this respect, it is reasonable to assume that pathogenic conditions are expressed in a changed methylation pattern of individual genes or of the genome.

State of the art are methods that allow the study of methylation patterns of individual genes. Recent developments in this method also allow the analysis of the smallest quantities of starting material. The present invention describes a method for parallel detection of the methylation state of genomic DNA samples, starting from a sample simultaneously amplifying numerous different fragments from sequences involved or / and transcribed and / or translated sequences and then the sequence context contained in the amplified fragments of CpG Dinucleotides is examined.

5-Methylcytosine is the most common covalently modified base in the DNA of eukaryotic cells. For example, it plays a role in the regulation of transcription, genomic imprinting and in tumorigenesis. The identification of 5-methylcytosine as a component of genetic information is therefore of considerable interest. However, 5-methylcytosine positions cannot be identified by sequencing since 5-methylcytosine has the same base pairing behavior like cytosine. In addition, in the case of PCR amplification, the epigenetic information which the 5-methylcytosines carry is completely lost. The modification of the genomic base cytosine to 5'-methylcytosine represents the most important and best-studied epigenetic parameter to date. Nevertheless, there are still methods to determine comprehensive genotypes of cells and individuals, but no comparable approaches to a large extent Generate and evaluate epigenotypic information.

In principle, three fundamentally different methods are known for determining the 5-methyl status of a cytosine in the sequence context.

The first principal method is based on the use of restriction endonucleases (RE), which are "methylation-sensitive". REs are characterized by the fact that they cut a DNA into a specific DNA sequence, usually between 4 and 8 bases long The position of such sections can then be verified by gel electrophoresis, transfer to a membrane and hybridization. Methylation-sensitive means that certain bases within the recognition sequence must be unmethylated in order for the section to be carried out according to the methylation pattern of the DNA However, the fewest methylable CpG are within the recognition sequences of REs and cannot be examined with this method.

The sensitivity of these methods is extremely low (Bird, AP, and Southern, EM, J. Mol. Biol. 118, 27-47). PCR combines a variant with this method; amplification by two primers located on both sides of the recognition sequence takes place after a cut only if the recognition sequence is methylated. In this case, the sensitivity increases theoretically to a single molecule of the target sequence, but only individual positions can be examined with great effort (Shemer, R. et al., PNAS 93, 6371-6376). Again, it is a prerequisite that the methylable position is within the recognition sequence of a RE. The second variant is based on partial chemical cleavage of total DNA, following the example of a Maxam-Gilbert sequencing reaction, ligation of adapters to the ends generated in this way, amplification with generic primers and separation on a gel electrophoresis. With this method, defined areas up to the size of less than a thousand base pairs can be examined. However, the process is so complicated and unreliable that it is practically no longer used (Ward, C. et al., J. Biol. Chem. 265, 3030-3033).

A relatively new and the most frequently used method for the investigation of DNA for 5-methylcytosine is based on the specific reaction of bisulfite with cytosine, which is converted into uracil after subsequent alkaline hydrolysis, which corresponds to the thymidine in its base-pairing behavior. However, 5-methylcytosine is not modified under these conditions. The original DNA is thus converted in such a way that methylcytosine, which originally cannot be distinguished from the cytosine by its hybridization behavior, can now be detected by "normal" molecular biological techniques as the only remaining cytosine, for example by amplification and hybridization or sequencing. All of these techniques are based on The state of the art in terms of sensitivity is defined by a method which includes the DNA to be examined in an agarose matrix, thereby the diffusion and renaturation of the DNA (bisulphite only reacts on single-stranded DNA) and all precipitation and purification steps are replaced by rapid dialysis (Olek, A. et al., Nucl. Acids. Res. 24, 5064-5066). Individual cells can be examined with this method, which illustrates the potential of the method However, so far only single region up to about 3000 base pairs in length, a global examination of cells for thousands of possible methylation events is not possible. However, this method, too, cannot reliably analyze very small fragments from small sample quantities. Despite the diffusion protection, these are lost through the matrix.

An overview of the other known options, 5-methylcytsosine can also be found in the following review article: Rein, T., DePamphilis, M. L, Zorbas, H., Nucleic Acids Res. 26, 2255 (1998).

The bisulphite technique has so far been used with a few exceptions (e.g. Zeschnigk, M. et al., Eur. J. Hum. Gen. 5, 94-98; Kubota T. et al., Nat. Genet. 16, 16-17 ) only used in research. However, short, specific pieces of a known gene are always amplified after bisulphite treatment and either completely sequenced (Olek, A. and Walter, J., Nat. Genet. 17, 275-276) or individual cytosine positions by a “primer Extension Reaction "(Gonzalgo, ML and Jones, PA, Nucl. Acids. Res. 25, 2529-2531) or Enzyme Cut (Xiong, Z. and Laird, PW, Nucl. Acids. Res. 25, 2532-2534) Detection by hybridization has also been described (Olek et al, WO9928498).

Similarities between promoters exist not only in the occurrence of TATA or GC boxes, but also in the transcription factors for which they have binding sites and the distance between them. The existing binding sites for a certain protein do not completely match in their sequence, but there are conserved sequences of at least 4 bases, which can be extended by inserting "wobbles", ie positions at which there are different bases Furthermore, these binding sites are at certain distances from one another.

The distribution of DNA in interphase chromatin, which takes up most of the nuclear volume, is subject to a very special order. The DNA is attached to the nuclear matrix in several places, a filamentous structure on the inside of the nuclear membrane. These regions are known as matrix attachment regions (MAR) or scaffold attachment regions (SAR). Attachment has a major impact on transcription or replication. These MAR fragments have no conservative sequences, but consist of 70% A and T, respectively, and are close to cis-acting regions that regulate transcription in general and topoisomerase II recognition sites. In addition to promoters and enhancers, there are other regulatory elements for various genes, so-called insulators. These insulators can, for example, inhibit the effect of the enhancer on the promoter if they are located between the enhancer and the promoter or, if they are located between heterochromatin and a gene, protect the active gene from the influence of the heterochromatin. Examples of such insulators are: 1. So-called LCR (locus control regions), which consists of several sites that are hypersensitive to DNAase I; 2. Certain sequences such as SCS (specialized chromatin structures) or SCS ', 350 or 200 bp long and highly resistant to degradation by DNAase I and flanked on both sides by hypersensitive sites (100 bp spacing). The protein BEAF-32 binds to scs'. These insulators can lie on both sides of the gene.

An overview of the state of the art in oligomer array production can also be found in a special edition of Nature Genetics published in January 1999 (Nature Genetics Supplement, Volume 21, January 1999) and the literature cited therein.

Patents related generally to the use of oligomer arrays and photolithographic mask design include e.g. B. US-A 5,837,832, US-A 5,856,174, WO-A 98/27430 and US-A 5,856,101. In addition, there are some substance and process patents that restrict the use of photolabile protective groups on nucleosides, such as. B. WO-A98 / 39348 and US-A 5,763,599.

Matrix-assisted laser desorption / ionization mass spectrometry (MALDI) is a new, very powerful development for the analysis of biomolecules (Karas, M. and Hillenkamp, F. 1988. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal. Chem 60: 2299-2301). An analyte molecule is embedded in a UV absorbing matrix. A short laser pulse evaporates the matrix into a vacuum, thus transporting the analyte unfragmented into the gas phase. An applied voltage accelerates the ions into a field-free flight tube. Due to their different masses, ions are accelerated to different extents. Smaller ions reach the detector earlier than larger ones and the flight time is in the mass of the Converted ions.

Fluorescent-labeled probes have been used in many cases for scanning an immobilized DNA array. The simple application of Cy3 and Cy5 dyes to the 5'OH of the respective probe is particularly suitable for fluorescence labeling. The fluorescence of the hybridized probes is detected, for example, using a confocal microscope. The dyes Cy3 and Cy5, among many others, are commercially available.

In order to calculate the expected number of amplified fragments on the basis of any template DNA and two primers that are not specific for a particular position, a statistical model of the structure of the genome must be used.

We give the calculation for three models here, but refer to the method described in Model 3 in this patent.

Model 1:

In the simplest case, it is assumed that a primary DNA strand is a random sequence of four bases that occur with the same frequency. This results in the probability that there is a perfect base pairing for any primer PrimA (length k) at a given location in the genome:

P _a {PrimA) = 0.25 ^k (model 1 for DNA)

(this probability is the same for the sense and anti-sense strand of the DNA)

In a bisulfite treatment of DNA, those cytosines are replaced by uracil that do not belong to a methylated CG. The base pairing behavior of the Uraciis corresponds to that of the thymine. Since CG is very rare in DNA (less than two percent), the statistical frequency of Cs after bisulfite treatment can be neglected. The probability that a primer PrimB (length k, thereof a As, t Ts, g Gs and c Cs) on bisulfite-treated DNA results in a perfect base pairing is different for a strand treated with bisulfite and the associated antisense strand:

P {PrimB) = 0.5 ^a * 0.25 '* 0.25 ^c * 0 ^g (model 1 for bisulfite DNA strand)

P _{! A} (PrimB) = 0.25 ^a * 0.5 '* 0 ^C * 0.25 ^g (model 1 for anti-sense strand to one

Bisulfite DNA strand)

(if the primer contains C or G, one of the probability values becomes 0).

Model 2:

DNA base frequency counts indicate that the four bases in the DNA are not equally distributed. Accordingly, the following frequencies (probabilities of occurrence) of the bases can be determined from DNA databases.

_DAM U) = 0.281 1 _ßAM (7 0.2784 _DAM (C) = 0.2206 P _DNA (G) = 0.2199

6% of the genome from Homo Sapiens from high throughput sequencing projects (database "htgs" from NIH / NCBI from 6.9.1999) serve as the basis for these statistics (and the following for models 2 and 3). The total amount of data is more than 1.5x10 ⁸ base pairs, which corresponds to an estimation error for the individual probabilities less than 10 ^{~ 5} .

Model 1 can be improved with the help of these values.

This means that the probability that a primer PrimC (length k, of which a

As, t Ts, g Gs and c Cs) results in a perfect base pairing: P ₂ (PrimC) = P _DNA (τy * P _DNA (A) '* P _DNA (C) ^g * P _DNA (G) ^c (Model 3 for DNA)

The following probabilities result for the strand treated with bisulfite, assuming that all CpG positions are methylated (the same statistics are obtained for the bisulfite treatment of the DNA sense and DNA antisense strands):

P _bDNA P _bDNA (C) = 0.0lA0 P _bυNA (G) = .2l99 P _büN4 (T) = 0ΛS50

This results in the probability that there is a perfect base pairing for a primer PritnD (length k, thereof a As, Ts, g Gs and c Cs):

P _2s (PrimD) = P _bυNA (τγ * P _bDNA (A) '* P _bDNA (CY * P _büNA (G) ^c (model 3 for bisulfite DNA strand)

P _2a (PrimD) = P _bϋNA (A r * P _bDNA (T) '* P _bDNA (G) ^s * P _bυNA (cγ (model 3 for anti-sense strand to a bisulfite DNA strand)

Model 3:

Significant estimation errors in model 2, especially in the case of the bisulfite-treated DNA, result from the fact that C can only occur in the context of CG. Model 3 takes this property into account and assumes that the primary DNA is a random sequence with dependence on directly adjacent bases (Markov chain of the first order). The pairwise base probabilities determined empirically from the database (completely methylated; treated with bisulphite) result for both DNA strands as P _bDNA (from; to) from the following table:

R ^ UHO.2811 P _bDNA (C) = .0 \ A0 P _bDNA (G) = 0.2l99

P _bDNA (T) = 0.A $ 50

and for the reverse complementary strand (by correspondingly exchanging the entries) R _rADΛM ( ^from • ^' n ch)

rbDNA (.4) = 0.4850 * ™ (00.2199 rbDNA (G) = 0.0140 rbDNA

The probability that a primer PrimE (with the base sequence BB ₂ B ₃ B ₄ ...; e.g. ATTG ...) results in a perfect base pairing depends on the exact sequence of the bases and results as the product:

P _3s (PrimE) = P _rbDNA (B) P rbDNA ( ^B \ • ^B l) P rbDNA ( ^B 2>^'ß l) ^P ' rbDNA ( ^B 3>^'B Λ)

(Model 3 for rbDNA w rbDNA (B ₂ ) rbDNA (B ₃ ) Bisulfite DNA strand)

_■ , „_ _■ .. - (Model 3 for anti-sense strand to a bisulfite DNA strand)

Calculation of the number of amplified fragments to be expected

The bisulfite treated DNA is amplified using a number of primers. From the point of view of the model, the DNA consists of one sense and one anti-sense strand with a length of N bases (all chromosomes are summarized here). A primer can be expected to be on the sense strand

N * P XPrim)

results in perfect base pairings - the functions P _1s , P _2s or P _3s of model 1, 2 or 3 can be used for this calculation, depending on the desired estimation quality. If several primers (PrimU, PrimV, PrimW, PrimX, etc.) are used at the same time, the probability of a perfect base pairing on the sense strand at a given position is as follows:

i ^> ₄ (primers) = P, {prim U)

+ {l- P PrimU)) P {PrimV)

+ {l-P {PrimU)) (l-P {PrimV)) P PrimW)

+ (l-P {PrimU)) {l-P {PrimV)) {l-P ^ PrimW)) P PrimX)

+ ...

And thus as the number of perfect base savings to be expected with any of the primers

N * P ⁽ primers) The analog equations are used to determine P _a (primers) on the anti-sense strand. An amplificate is created if, when there is a perfect base pairing on the sense strand within the maximum fragment length / W, a primer on the opposite strand forms a perfect base pairing. The probability of this is

, M - (l - P _a (primers))

For large M and small P _a (primers) this can be calculated using the following expression:

-PΛ primers) _{r /} ,,, _u

^■ {(1 -P „{Printer s)) ^M - l log (1 —P _a {Primers))

For the total number F of fragments that can be expected from the amplification of both strands, the result is:

(1 - P (primers))

F = N * P, (primers) _λ . , ". _. ^ [(1 - P _a (Primers))" - l log (1 - P _a (Primers))

(1 - P (primers))

^{+ N * P} - ^lta, Ϊ ^ ^ » ^{lπ" fJf} ™ ^{m) l} ''

This method provides a precise expectation for predicting the number of binding sites of certain sequences to any genomic DNA fragment previously treated with bisulfite. It serves as the basis for the calculation of the statistically expected number of amplificates in a PCR reaction based on two primer sequences and a DNA of length N, whereby only the amplificates that do not exceed a number of M nucleotides are taken into account. This patent assumes that M is 2000. In principle, the known methods for the detection of cytosine methylations in genomic DNA are not designed in such a way that a large number of target regions in the genome to be examined are detected simultaneously. The object of the present invention is to create a method with which a sample of genomic DNA can be examined simultaneously at several positions for cytosine methylation.

The object is achieved by the characterizing features of claim 1. Advantageous further developments of the feature are characterized in the dependent claims.

In contrast to other methods, after chemical pretreatment of the DNA, many target regions can be amplified simultaneously using appropriately adapted primer pairs. It is not absolutely necessary to know the sequence context of all these target regions in advance, since in many cases, as also exemplified below, consensus sequences from the sequencing of related target regions are known, which, as described below, are used for the design of specific target regions of specific or selective primer pairs can be. The method is successfully applied when the amplification of the chemically pretreated genomic DNA yields more fragments up to a maximum of 2000 base pairs in length than can be statistically expected from the target regions to be examined in each case.

The statistical expected value for the number of these fragments is calculated using the formulas listed in the prior art. The number of fragments produced in the amplification step, however, can be detected by any molecular biological, chemical or physical method.

The following values are assumed for carrying out the required statistical considerations, which are also relevant for the claims listed below: The human haploid genome contains 3 billion base pairs and 100,000 genes, which in turn code an average of 2,000 base pairs long mRNA, the genes including the introns are on average 15,000 base pairs long. Promoters cover an average of 1000 base pairs per gene. If the statistical expected value for the number of amplified products that are based on two primers in transcribed sequences must therefore be calculated, the expected value for the entire genome must first be calculated using the above formula (method 3) and with the proportion of the transcribed sequences in the total genome to calculate. The same procedure is used for parts of any genome as well as for promoters and translated sequences (coding for mRNA).

The present invention thus describes a method for the parallel detection of the methylation state of genomic DNA. Several cytosine methylations in a DNA sample are to be analyzed simultaneously. The following process steps are carried out one after the other:

First, a genomic DNA sample is chemically treated in such a way that at the 5'-position unmethylated cytosine bases are converted into uracil, thymine or another base which is unlike the cytosine in hybridization behavior. The treatment of genomic DNA with bisulfite (hydrogen sulfite, disulfite) and subsequent alkaline hydrolysis, which leads to a conversion of unmethylated cytosine nucleobases into uracil, is preferably used for this purpose.

In a second process step, more than ten different fragments are simultaneously amplified from the pretreated genomic DNA by using synthetic oligonucleotides as primers, whereby more than twice as many fragments as statistically expected come from sequences involved in transcription and / or translation that are involved in gene regulation. This can be achieved using various methods.

In a preferred variant of the method, at least one of the oligonucleotides used for the amplification contains fewer nucleobases than statistically a sequence-specific hybridization to the chemically treated genomic DNA sample would be required, which can lead to the amplification of several fragments at the same time. The total number of nucleobases contained in this oligonucleotide is less than 17. In a particularly preferred variant of the method, the number of nucleobases contained in this oligonucleotide is less than 14.

In a further preferred variant of the method, more than 4 oligonucleotides with different sequences are used simultaneously in one reaction vessel for the amplification. In a particularly preferred variant, more than 26 different oligonucleotides are used simultaneously to produce a complex amplificate. In a particularly preferred variant of the method, more than twice the number that is statistically to be expected comes from genome sections involved in the regulation of genes, e.g. Promoters and enhancers, comes as would be expected with a purely random choice of the oligonucleotide sequences. In a further particularly preferred variant of the method, more than twice the number of the amplified fragments originates from genome sections which are transcribed in mRNA in at least one cell of the respective organism, or from genome sections (exons) spliced into mRNA after transcription than would be expected if the oligonucleotide sequences were chosen at random.

In a further particularly preferred variant of the method, more than twice the number of the amplified fragments comes from genome sections which code for parts of one or more gene families, or else they come from genome sections which are used for so-called “matrix attachment sites” (MARs). contain characteristic sequences than would be expected with a purely random selection of the oligonucleotide sequences.

In a further particularly preferred variant of the method, more than twice as many of the amplified fragments come from genome sections which organize the packing density of the chromatin as so-called “boundary elements”, or else they come from multiple drug resistance genes (MDR) - Promoters or coding regions than would be expected with a purely random choice of the oligonucleotide sequences.

In a further, particularly preferred variant of the method, two oligonucleotides or two classes of oligonucleotides are used to amplify the fragments described, one or a class of which, except in the context of CpG or CpNpG, may contain base C but not base G and which the other or the other class may contain the base G, but not the base C, except in the context of CpG or CpNpG.

In a further preferred variant of the method, the amplification is carried out by means of two oligonucleotides, one of which contains a four to sixteen base long sequence which is complementary to or corresponds to such a DNA as it would arise if an equally long DNA fragment, which one of the factors

AhR / Arnt aryl hydrocarbon receptor / aryl hydrocarbon receptor nuclear translocator

Amt aryl hydrocarbon receptor nuclear translocator AML-1a CBFA2; core binding factor, runt domain, alpha subunit 2

(acute myeloid leukemia 1; aml1 oncogene)

AP-1 activator protein-1 (AP-1); Synonyms: c-Jun

C / EBP CCAAT / enhancer binding protein

C / EBPalpha CCAAT / enhancer binding protein (C / EBP), alpha

C / EBPbeta CCAAT / enhancer binding protein (C / EBP), beta

CDP CUTL1; cut (Drosophila) -Iike 1 (CCAAT displacement protein)

CDP CR1 complement component (3b / 4b) receptor 1 CDP CR3 complement component (3b / 4b) receptor 3 CHOP-C / EBPalpha DDIT; DNA-damage-inducible transcript 3 / CCAAT / enhancer binding protein (C / EBP), alpha c-Myc / Max avian myelocytomatosis viral oncogene / MYC-ASSOCIATED

FACTOR X

CREB cAMP responsive element binding protein CRE-BP1 CYCLIC AMP RESPONSE ELEMENT-BINDING PROTEIN

2, CREB2, CREBP1; now ATF2; activating transcription factor 2

CRE-BP1 / c-Jun activator protein-1 (AP-1); Synonyms: c-Jun CREB MP responsive element binding protein E2F E2F transcription factor (originally identified as a DNA- binding protein essential E1A-dependent activation of the adenovirus E2 promoter)

E47 transcription factor 3 (E2A immunoglobulin enhancer binding factors E12 / E47)

Egr-1 early growth response 1 Egr-2 early growth response 2 (Krox-20 (Drosophila) homolog) ELK-1 ELK1, member of ETS (environmental tobacco smoke) oncogene family

Freac-2 FKHL6; forkhead (Drosophila) -Iike 6; FORKHEAD-RELATED ACTIVATOR 2; FREAC2

Freac-3 FKHL7; forkhead (Drosophila) -Iike 7; FORKHEAD-RELATED ACTIVATOR 3; FREAC3

Freac-4 FKHL8; forkhead (Drosophila) -Iike 8; FORKHEAD-RELATED ACTIVATOR 4; FREAC4

Freac-7 FKHL11; forkhead (Drosophila) -Iike 9; FORKHEAD-RELATED ACTIVATOR 7; FREAC7

GATA-1 GATA-binding protein 1 / enhancer-binding protein GATA1 GATA-1 GATA-binding protein 1 / enhancer-binding protein GATA1 GATA-1 GATA-binding protein 1 / enhancer-binding protein GATA1 GATA-2 GATA-binding protein 2 / Enhancer-binding protein GATA2 GATA-3 GATA-binding protein 3 / Enhancer-binding protein GATA3 GATA-X HFH-3 FKHL10; forkhead (Drosophila) -Iike 10; FORKHEAD-RELATED ACTIVATOR 6; FREAC6

HNF-1 TCF1; transcription factor 1, hepatic; LF-B1, hepatic nuclear factor (HNF1), albumin proximal factor

HNF-4 hepatocyte nuclear factor 4

IRF-1 interferon regulatory factor 1

ISRE interferon-stimulated response element

Lmo2 complex LIM domain only 2 (rhombotin-like 1)

MEF-2 MADS box transcription enhancer factor 2, polypeptide A (myocyte enhancer factor 2A)

MEF-2 MADS box transcription enhancer factor 2, polypeptide A (myocyte enhancer factor 2A) myogenin / NF-1 myogenin (myogenic factor 4) / neurofibromin 1; NEUROFIBROMATOSIS, TYPE I

MZF1 ZNF42; zinc finger protein 42 (myeloid-specific retinoic acid-responsive)

NF-E2 NFE2; nuclear factor (erythroid-derived 2), 45kD NF-kappaB (p50) nuclear factor of kappa light polypeptide gene enhancer in B-cells p50 subunit

NF-kappaB (p65) nuclear factor of kappa light polypeptide gene enhancer in B- cells p65 subunit

NF-kappaB nuclear factor of kappa light polypeptide gene enhancer in B cells

NRSF NEURON RESTRICTIVE SILENCER FACTOR; REST; RE1- silencing transcription factor

Oct-1 OCTAMER-BINDING TRANSCRIPTION FACTOR 1;

POU2F1; POU domain, class 2, transcription factor 1

Oct-1 OCTAMER-BINDING TRANSCRIPTION FACTOR 1;

POU2F1; POU domain, class 2, transcription factor 1

Oct-1 OCTAMER-BINDING TRANSCRIPTION FACTOR 1;

POU2F1; POU domain, class 2, transcription factor 1

Oct-1 OCTAMER-BINDING TRANSCRIPTION FACTOR 1;

POU2F1; POU domain, class 2, transcription factor 1

Oct-1 OCTAMER-BINDING TRANSCRIPTION FACTOR 1;

POU2F1; POU domain, class 2, transcription factor 1

P300 E1A (adenovirus E1A oncoprotein) BINDING PROTEIN,

300-KD

P53 tumor protein p53 (Li-Fraumeni syndrome); TP53

Pax-1 paired box gene 1

Pax-3 paired box gene 3 (Waardenburg syndrome 1)

Pax-6 paired box gene 6 (aniridia, keratitis)

Pbx lb pre-B-cell leukemia transcription factor

Pbx-1 pre-B-cell leukemia transcription factor 1

RORalpha2 RAR-RELATED ORPHAN RECEPTOR ALPHA; retinoic

ACID-BINDING RECEPTOR ALPHA

RREB-1 ras responsive element binding protein 1

SP1 simian virus 40 protein 1

SREBP-1 sterol regulatory element binding transcription factor 1

SRF serum response factor (c-fos serum response element-binding transcription factor)

SRY sex determining region Y

STAT3 signal transducer and activator of transcription 1, 91 kD

Tal-1alpha / E47 T-cell acute lymphocytic leukemia 1 / transcription factor 3

(E2A immunoglobulin enhancer binding factors E12 / E47)

TATA cellular and viral TATA box elements Tax / CREB Transiently-expressed axonal glycoprotein / cAMP responsive element binding protein

Tax / CREB Transiently-expressed axonal glycoprotein / cAMP responsive element binding protein

TCF11 / MafG v-maf musculoaponeurotic fibrosarcoma (avian) oncogene family, protein G

TCF11 Transcription Factor 11; TCF11; NFE2L1; nuclear factor

(erythroid-derived 2) -like 1

USF upstream stimulating factor Whn winged-helix nude X-BP-1 X-box binding protein 1 or

YY1 ubiquitously distributed transcription factor belonging to theGLI-Kruppel class of zinc finger proteins

binds, would be treated chemically in such a way that at the 5'-position unmethylated cytosine bases are converted into uracil, thymidine or another base which is unlike the cytosine in terms of hybridization behavior.

In a further preferred variant of the method, the amplification is carried out by means of two oligonucleotides or two classes of oligonucleotides, of which one or the one class contains the four to sixteen base long sequence which is complementary to or corresponds to such a DNA as it arises would, if an equally long DNA fragment, which can bring about the specific localization of genome / chromatin sections within the cell nucleus via its sequence or secondary structure, were treated in such a way that unmethylated cytosine bases in the 5'-position in uracil, thymidine or another of the Hybridization behavior from the base which is dissimilar to the cytosine can be transformed.

In a further preferred variant of the method, the amplification is carried out by means of two oligonucleotides or two classes of oligonucleotides, one or one of which is one of the sequences

TCGCGTGTA, TACACGCGA, TGTACGCGA, TCGCGTACA, TTGCGTGTT, AACACGCAA, GGTACGTAA, TTACGTACC, TCGCGTGTT, AACACGCGA, GGTACGCGA, TCGCGTACG, TTGTACGCTA, TCGCGTACG

ATTGCGTGT, ACACGCAAT, GTACGTAAT, ATTACGTAC, ATTGCGTGA, TCACGCAAT, TTACGTAAT, ATTACGTAA, ATCGCGTGA, TCACGCGAT, TTACGCGAT, ATCGCGATA, ATCGCGCTGAT, GTACTGGT

TGAGTTAG, CTAACTCA, TTGATTTA, TAAATCAA, TGATTTAG, CTAAATCA, TTGAGTTA, TAACTCAA, TTTGGT, ACCAAA, ATTAAA, TTTAAT, TGTGGA, TCCACA, TTTATA, TATAAA, TTTGGA, TCCAAA, TTTAAA, TTTAAA, TGTGGT, ACCACA, ATTATA, TATAAT,

ATTAT, ATAAT, GTAAT, ATTAC, ATTGT, ACAAT, GTAAT, ATTAC,

GAAAG, CTTTC, TTTTT, AAAAA,

GTAAT, ATTAC, ATTGT, ACAAT,

GAAAT, ATTTC, ATTTT, AAAAT,

GTAAG, CTTAC, TTTGT, ACAAA,

TTAATAATCGAT, ATCGATTATTAA, ATCGATTATTGG, CCAATAATCGAT,

ATCGATTA, TAATCGAT, TAATCGAT, ATCGATTA,

ATCGATCGG, CCGATCGAT, TCGATCGAT, ATCGATCGA, ATCGATCGT, ACGATCGAT, GCGATCGAT, ATCGATCGC,

TATCGATA, TATCGATA, TATCGGTG, CACCGATA, TATTAATA, TATTAATA, TATTGGTG, CACCAATA,

GTGTAATATTT, AAATATTACAC, GGGTATTGTAT, ATACAATACCC, GTGTAATTTTT, AAAAATTACAC, GGGGATTGTAT, ATACAATCCCC, ATGTAATTTTT, AAAAATTACAT, GGGGATTGTAT, ATACAATCCCC, ATGTAATATTT, AAATATTACAT, GGGTATTGTAT, ATACAATACCC, ATTACGTGGT, ACCACGTAAT, ATTACGTGGT, ACCACGTAAT, TGACGTAA, TTACGTCA, TTACGTTA, TAACGTAA, TGACGTTA, TAACGTCA, TGACGTTA, TAACGTCA, TTACGTAA, TTACGTAA, TTACGTAA, TTACGTAA, TGACGTTA, TAACGTCA, TAACGTTA, TAACGTTA,

TGACGT, ACGTCA, GCGTTA, TAACGC, TGACGT, ACGTCA, ACGTTA, TAACGT, TTTCGCGT, ACGCGAAA, GCGCGAAA, TTTCGCGC, TTTGGCGT, ACGCCAAA, GCGTTAAA, TTTAACGC,

TAGGTGTTA, TAACACCTA, TAATATTTG, CAAATATTA, TAGGTGTTT, AAACACCTA, GAATATTTG, CAAATATTC,

GTAGGTGG, CCACCTAC, TTATTTGT, ACAAATAA, GTAGGTGT, ACACCTAC, ATATTTGT, ACAAATAT,

TGCGTGGGCGG, CCGCCCACGCA, TCGTTTACGTA, TACGTAAACGA, TGCGTGGGCGT, ACGCCCACGCA, ACGTTTACGTA, TACGTAAACGT,

TGCGTAGGCGT, ACGCCTACGCA, ACGTTTACGTA, TACGTAAACGT, TGCGTAGGCGG, CCGCCTACGCA, TCGTTTACGTA, TACGTAAACGA, ATAGGAAGT, ACTTCCTAT, ATTTTTTGT, ACAAAAAAT TCGGAAGT, ACTTCCGA, ATTTTCGG, CCGAAAAT, TCGGAAGT, ACTTCCGA, GTTTTCGG, CCGAAAAC, TCGGAAAT, ATTTCCGA, ATTTTCGG, CCGAAAAT, TCGGAAAT, ATTTCCGA, GTTTTCGG, CCGAAAACGTTAATTAAT

AAAGTAAATA, TATTTACTTT, TGTTTATTTT, AAAATAAACA, AATGTAAATA, TATTTACATT, TGTTTATATT, AATATAAACA, TAAGTAAATA, TATTTACTTA, TGTTTATTTA, TAAATAAACA, TATGTAAATA, TATTTATATA, TGTTAATA

ATAAATA, TATTTAT, TGTTTAT, ATAAACA, ATAAATA, TATTTAT, TATTTAT, ATAAATA, GATA, TATC, TATT, AATA,

TAGATAA, TTATCTA, TTATTTG, CAAATAA, TTGATAA, TTATCAA, TTATTAG, CTAATAA, GATAA, TTATC, TTATT, AATAA,

GATG, CATC, TATT, AATA,

GATAG, CTATC, TTATT, AATAA, GATAAG, CTTATC, TTTATT, AATAAA,

TGTTTATTTA, TAAATAAACA, TAAATAAATA, TATTTATTTA, TGTTTGTTTA, TAAACAAACA, TAAATAAATA, TATTTATTTA, TATTTATTTA, TAAATAAATA, TAAATAAATA, TATTTATTTA, TATTTGTTTA, TAAACAAATA, TAAATAATA

GTTAATGATT, AATCATTAAC, AATTATTAAT, ATTAATAATT, GTTAATTATT, AATAATTAAC, AATAATTAAT, ATTAATTATT, GTTAATTAAT, ATTAATTAAC, ATTAATTAAT, ATTAATTAAT, GTTAATGAAT, ATTCATTAAC, ATTTATTAAT, ATTAATAAAT,

TAAAGTTTA, TAAACTTTA, TGAATTTTG, CAAAATTCA, TAAAGGTTA, TAACCTTTA, TGATTTTTG, CAAAAATCA,

AAAGTGAAATT, AATTTCACTTT, GGTTTTATTTT, AAAATAAAACC, AAAGCGAAATT, AATTTCGCTTT, GGTTTCGTTTT, AAAACGAAACC,

TAGTTTTATTTTTTT, AAAAAAATAAAACTA, GGGAAAGTGAAATTG,

CAATTTCACTTTCCC,

TAGTTTTATTTTTTT, AAAAAAATAAAACTA, GGAAAAGTGAAATTG,

CAATTTCACTTTTCC,

TAGTTTTTTTTTTTT, AAAAAAAAAAAACTA, GGAAAAGAGAAATTG,

CAATTTCTCTTTTCC, TAGTTTTTTTTTTTT, AAAAAAAAAAAACTA, GGGAAAGAGAAATTG,

CAATTTCTCTTTCCC,

TAGGTG, CACCTA, TATTTG, CAAATA,

TTTTAAAAATAATTTT, AAAATTATTTTTAAAA, AGGGTTATTTTTAGAG,

CTCTAAAAATAACCCT,

TTTTAAAAATAATTTT, AAAATTATTTTTAAAA, GGAGTTATTTTTAGAG,

CTCTAAAAATAACTCC,

TTTTAAAAATAATTTT, AAAATTATTTTTAAAA, AGAGTTATTTTTAGAG,

CTCTAAAAATAACTCT,

TTTTAAAAATAATTTT, AAAATTATTTTTAAAA, GGGGTTATTTTTAGAG,

CTCTAAAAATAACCCC,

TGTTATTAAAAATAGAAA, TTTCTATTTTTAATAACA, TTTTTATTTTTAGTAATA, TATTACTAAAAATAAAAA, TGTTATTAAAAATAGAAT, ATTCTATTTTTAATAACA, GTTTTATTTTTAGTAATA, TATTACTAAAAATAAAAC, TTACTACAAA, TTACTAGCACCA

TAGGGG, CCCCTA, TTTTTA, TAAAAA, GAGGGG, CCCCTC, TTTTTT, AAAAAA,

TGTTGAGTTAT, ATAACTCAACA, ATGATTTAGTA, TACTAAATCAT, TGTTGATTTAT, ATAAATCAACA, GTGAGTTAGTA, TACTAACTCAC, TGTTGAGTTAT, ATAACTCAACA, ATGATTTAGTA, TACTAAATCAT, TGTTGATTTAT, ATAAATCAACA, GTGAGTTAGTA, TACTAACTCAC, GGGGATTTTT, AAAAATCCCC, GGGAATTTTT, AAAAATTCCC, GGGGATTTTT, AAAAATCCCC, GGGGATTTTT, AAAAATCCCC, GGGGATTTTT, AAAAATCCCC, GGAAATTTTT, AAAAATTTCC, GGGAATTTTT, AAAAATTCCC, GGAAATTTTT, AAAAATTTCC, GGGAATTTTT, AAAAATTCCC, GGAAATTTTT, AAAAATTTCC, GGGATTTTTT, AAAAAATCCC, GGAAAGTTTT, AAAACTTTCC, GGGAATTTTT, AAAAATTCCC, GGGAATTTTT, AAAAATTCCC, GGGATTTTTT, AAAAAATCCC, GGGAAGTTTT, AAAACTTCCC, GGGATTTTTTA, TAAAAAATCCC, TGGAAAGTTTT, AAAACTTTCCA, TTTAGTATTACGGATAGAGGT, ACCTCTATCCGTAATACTAAA, GTTTTTGTTCGTGGTGTTGAA, TTCAACACCACGAACAAAAAC, TTTAGTATTACGGATAGAGTT, AACTCTATCCGTAATACTAAA, GGTTTTGTTCGTGGTGTTGAA, TTCAACACCACGAACAAAACC, TTTAGTATTACGGATAGCGTT, AACGCTATCCGTAATACTAAA, GGCGTTGTTCGTGGTGTTGAA, TTCAACACCACGAACAACGCC, TTTAGTATTACGGATAGCGGT, ACCGCTATCCGTAATACTAAA, GTCGTTGTTCGTGGTGTTGAA, TTCAACACCA CGAACAACGAC,

ATATGTAAAT, ATTTACATAT, ATTTGTATAT, ATATACAAAT, TTATGTAAAT, ATTTACATAA, ATTTGTATAA, TTATACAAAT,

GAATATTTA, TAAATATTC, TGAATATTT, AAATATTCA, GAATATGTA, TACATATTC, TGTATATTT, AAATATACA,

ATAAT, ATTAT, ATTAT, ATAAT, GTAAT, ATTAC, ATTAT, ATAAT,

AATGTAAAT, ATTTACATT, ATTTGTATT, AATACAAAT,

ATTTGTATATT, AATATACAAAT, GGTATGTAAAT, ATTTACATACC, ATTTGTATATT, AATATACAAAT, AATATGTAAAT, ATTTACATATT, ATTTGTATATT, AATATACAAAT, AGTATGTATAT, ATTTACATACT, ATTTATATATAT, ATTTGATATAT

AGGAGT, ACTCCT, ATTTTT, AAAAAT, GGGAGT, ACTCCC, ATTTTT, AAAAAT, GGATATGTTCGGGTATGTTT, AAACATACCCGAACATATCC, GGATATGTTCGGGTATGTTT, AAACATACCCGAACATATCC, GGATATGTTCGGGTATGTTT, AAACATACCCGAACATATCC, AGATATGTTCGGGTATGTTT, AAACATACCCGAACATATCT, TCGTTTCGTTTTAGATAT, ATATCTAAAACGAAACGA, ATATTTAGAGCGGAACGG, CCGTTCCGCTCTAAATAT,

CGTTACGGTT, AACCGTAACG, AATCGTGACG, CGTCACGATT, CGTTACGGTT, AACCGTAACG, GATCGTGACG, CGTCACGATC, CGTTACGTTT, AAACGTAACG, AAGCGTGACG, CGTCACGACGTTCGGACGTC

TTTACGTATGA, TCATACGTAAA, TTATGCGTGAA, TTCACGCATAA, TTTACGTTTGA, TCAAACGTAAA, TTAAGCGTGAA, TTCACGCTTAA, TTTACGTTTTA, TAAAACGTAAA, TGAAGCGTGAA, TTCACGGGTACA

AATTAATTAA, TTAATTAATT, TTGATTGATT, AATCAATCAA, TATTAATTAA, TTAATTAATA, TTGATTGATG, CATCAATCAA,

TAATTAT, ATAATTA, ATGATTG, CAATCAT,

TAGGTTA, TAACCTA, TGATTTA, TAAATCA,

TTTTAAATATTTTT, AAAAATATTTAAAA, GGGGGTGTTTGGGG,

CCCCAAACACCCCC,

TTTTAAATTATTTT, AAAATAATTTAAAA, GGGGTGGTTTGGGG,

CCCCAAACCACCCC,

TTTTAAATTTTTTT, AAAAAAATTTAAAA, GGGGGGGTTTGGGG,

CCCCAAACCCCCCC,

TTTTAAATAATTTT, AAAATTATTTAAAA, GGGGTTGTTTGGGG,

CCCCAAACAACCCC,

GAGGCGGGG, CCCCGCCTC, TTTCGTTTT, AAAACGAAA, GAGGTAGGG, CCCTACCTC, TTTTGTTTT, AAAACAAAA, AAGGCGGGG, CCCCGCCTT, TTTCGTTTT, AAAACGAAA, AAGGTAGGG, CCCTACCTT, TTTTGTTTT, AAAACAAAA,

GGGGGCGGGGT, ACCCCGCCCCC, ATTTCGTTTTT, AAAAACGAAAT, GGGGGCGGGGT, ACCCCGCCCCC, GTTTCGTTTTT, AAAAACGAAAC, TATTATTTTAT, ATAAAATAATA, GTGGGGTGATAT, TATCAAGATAT, TATCAAGATAT, TATCAAGATAT

ATTACGTGAT, ATCACGTAAT, ATTACGTGAT, ATCACGTAAT, ATTACGTGAT, ATCACGTAAT, GTTACGTGAT, ATCACGTAAC,

TTTTATATGG, CCATATAAAA, TTATATAAGG, CCTTATATAA, TTATATATGG, CCATATATAA, TTATATATGG, CCATATATAA, AAATAAT, ATTATTT, GTTGTTT, AAACAAC, AAATTAA, TTAATTT, TTAGTTT, AAACTAATTATT, AAACTAATATT, AAAT

ATTTTTCGGAAATG, CATTTC CG AAAAAT, TATTTTCGGGAAAT,

ATTTCCCGAAAATA,

ATTTTTCGGAAATG, CATTTC CG AAAAAT, TATTTTCGGGAAAT,

ATTTCCCGAAAATA,

ATTTTCGGGAAATG, CATTTCCCGAAAAT, TATTTTTCGGAAAT,

ATTTCCGAAAAATA,

ATTTTCGGGAAGTG, CACTTCCCGAAAAT, TATTTTTCGGAAAT,

ATTTCCGAAAAATA,

AATAGATGTT, AACATCTATT, AATATTTGTT, AACAAATATT, AATAGATGGT, ACCATCTATT, ATTATTTGTT, AACAAATAAT,

GTATAAATA, TATTTATAC, TATTTATAT, ATATAAATA, GTATAAATG, CATTTATAC, TATTTATAT, ATATAAATA, GTATAAAAA, TTTTTATAT, TTTTTATAT, ATATAAAAA, GTATAAAAG, CTTTTATAT, TTTTTATAT, ATATAAAAA, TTATAAATATA TTTTTATAA, TTTTTATAG, CTATAAAAA, TTATAAAAG, CTTTTATAA, TTTTTATAG, CTATAAAAA, GGGGGTTGACGTA, TACGTCAACCCCC, TGCGTTAATTTTT, AAAAATTAACGCA,

GGGGGTTGACGTA, TACGTCAACCCCC, TACGTTAATTTTT, AAAAATTAACGTA,

TGACGTATATTTTT, AAAAATATACGTCA, GGGGATATGCGTTA,

TAACGCATATCCCC,

TGACGTATATTTTT, AAAAATATACGTCA, GGGGGTATGCGTTA,

TAACGCATACCCCC, ATGATTTAGTA, TACTAAATCAT, TGTTGAGTTAT, ATAACTCAACA, GTTAT, ATAAC, ATGAT, ATCAT,

TTACGTGA, TCACGTAA, TTACGTGG, CCACGTAA, TTACGTGG, CCACGTAA, TTACGTGG, CCACGTAA, TTACGTGG, CCACGTAA, TTACGTGA, TCACGTAA, TTACGTGA, TCACGTAA, TTACTTA, GACGTACA, TCACGTACA

TGACGTGT, ACACGTCA, ATACGTTA, TAACGTAT, TGACGTGG, CCACGTCA, TTACGTTA, TAACGTAA, CGGTTATTTTG, CAAAATAACCG, TAAGATGGTCG or CGACCATCTTA

contains which is complementary to or corresponds to such DNA as it would arise if an equally long DNA fragment, which via its sequence or secondary structure can bring about the specific localization of genome / chromatin sections within the cell nucleus, were treated chemically in such a way that at the 5'-position unmethylated cytosine bases are converted into uracil, thymidine or another base which is unlike the cytosine in terms of hybridization behavior.

In a particularly preferred variant of the method, the oligonucleotides used for the amplification contain, in addition to the consensus sequences defined above, several positions at which either one of the three bases G, A and T or any of the bases C, A and T can be present.

In a particularly preferred variant of the method, the oligonucleotides used for the amplification contain, apart from one of the consensus sequences described above, a maximum of as many additional bases as are required for the simultaneous amplification of more than one hundred different fragments per reaction from the DNA treated chemically as above.

In a third method step, the sequence context of all or part of the CpG dinucleotides or CpNpG trinucleotides contained in the amplified fragments is examined. In a particularly preferred variant of the method, the analysis is carried out by hybridizing the fragments already provided with a fluorescence marker in the amplification to an oligonucleotide array (DNA chip). The fluorescent marker can be introduced either via the primers used or through a fluorescence-labeled nucleotide (eg Cy5-dCTP, commercially available from Amersham-Pharmacia).

Complementary fragments hybridize to the respective oligomers immobilized on the chip surface, non-complementary fragments are removed in one or more washing steps. The fluorescence at the respective hybridization sites on the chip then allows conclusions to be drawn about the sequence context of the CpG dinucleotides or CpNpG trinucleotides contained in the amplified fragments.

In a further preferred variant of the method, the amplified fragments are immobilized on a surface and then hybridization is carried out with a combinatorial library of distinguishable oligonucleotide or PNA oligomer probes. Again, non-complementary probes are removed by one or more washing steps. The hybridized probes are either detected via their fluorescent markers or, in a further particularly preferred variant of the method, are detected using matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) on the basis of their unique mass. The probe libraries are synthesized in such a way that the mass of each component can be clearly assigned to its sequence.

In a further preferred variant of the method, the amplification products can also be influenced in terms of their average size by changing the chain extension times in the amplification step. Since mainly smaller fragments (approx. 200-500 base pairs) are examined here, a shortening of the chain extension steps is e.g. B. a PCR useful.

In a further preferred variant of the method, the amplified products are Gel electrophoresis is separated and the fragments in the desired size range are cut out before your analysis. In a further particularly preferred variant, the amplificates cut out of the gel are amplified again using the same set of primers. Then only fragments of the desired size can be created, since others are no longer available as templates.

Another object of the present invention is a kit containing at least two primer pairs, reagents and auxiliary substances for amplification and / or reagents and auxiliary substances for chemical treatment and / or a combinatorial probe library and / or an oligonucleotide array (DNA chip), insofar as they are necessary or useful for carrying out the method according to the invention.

The following examples illustrate the invention.

Examples:

Example 1 :

Primers for preferential amplification of CG rich regions in the human genome

The CG-rich regions in the human genome are so-called CpG islands, which have a regulatory function. We define CpG Islands in such a way that they have at least 500 bp and a GC content of> 50%, and the ratio CG / GC is> 0.6. Under these conditions, 16 Mb are CpG Islands. This means that about 0.5% of the genome sequence lies in these CpG islands, if one also considers a region up to 1000 bp downstream. This consideration is based on data from the Ensembl Database from 10/31/00, source Sanger Center. The sequence available there was approx. 3.5 GB, and the repeats were masked for the calculations.

Statistically, it would be expected for 12meres that they hybridize only 0.005 times as often to one of the CG-rich regions as to any other region in the Genome. Primers have now been found which bind 1.8 times more often to a region rich in CG. In addition, the reverse primer found gives almost a specificity for these CpG islands.

In this example the primers are AGTAGTAGTAGT (Seq. ID 1) AAAACAAAAACC (Seq. ID 2) and alternatively AGTAGTAGTAGT (Seq. ID 19) and ACAAAAACTAAA (seq. ID 20). The first pair of primers leads at least to the amplificates Seq. ID 3 to 18, the second pair of primers for the amplicons of Seq. ID 21 to 31.

Example 2:

Calculation of the prediction of the number of amplicons in genomic regions.

According to claim 8 in the patent, it is shown that more than twice as many amplificates can be produced than would be statistically expected according to Formula 1.

(P „(Primers)) _r ,,, _U

F specifies the number of predicted amplicons that can be expected if one considers N bases as a database from the genome. P is the respective probability of hybridization of a primer oligonucleotide, separated after hybridization in the sense and antisense strand. M is the maximum permissible length of the amplicons to be expected.

The probability P is determined by a first order Markov chain. The assumption is made that the DNA is a random sequence depending on neighboring bases. The transition probabilities of neighboring bases are necessary for the calculation of a Markov chain. These were determined empirically from 12% of the assembled human genome, which was completely treated with bisulfite, and summarized in Table 1. Table 2 shows the transition probabilities for the corresponding complementary reverse Strand specified. These result from simply swapping the entries in Table 1.

Table 1

with P bDNA U) = 0.2811

J \ α «(00.2199

/ W _Λ (00.4850 and for the reverse complementary strand (by exchanging the entries accordingly) P ^ _DNA ( ^from >^' to)

Table 2

R O0.4850 (00.2199 * ™ (00.0140 O0.2811

This depends on the probability that a PrimE (with the Base sequence Bi B ₂ B ₃ B ₄ ...; eg ATTG ...) results in a perfect base pairing, based on the exact sequence of the bases and results in the product: r _b DNA _\ ^B _\ , ^' B ₂ ) P _rbDNA (B ₂ ; B ₃ ) P _rbDNA B ₃ ; B

P _3s (PrimE) = P _rbDNA (BA!

"rbDNA ^B \) rbDNA \ 2) P rbDNA \ 31

(Bisulfite DNA strand)

(anti-sense strand to a bisulfite DNA strand); for a primer prim on the sense strand result

N * PχPrim) perfect base pairings - If several primers (PrimU, PrimV, PrimW, PrimX, etc.) are used at the same time, the probability for a perfect base pairing on the sense strand at a given position is: P _s (primers) = P _S {Prim U)

+ (l -P _i (PrimU)) P _i (PrimV) + (l -P (PrimU)) (l -P PrimV)) P \ PrimW) + (l -PXPrimU)) (l -PχPrimV)) (l -P (PrimW)) P (PrimX) + ... (PrimU, PrimV, PrimW ... are different primers with different base pairings) and thus as the number of perfect base pairings to be expected with any of the primers

N * P (primers). The analog equations are used to determine P _a (primers) on the anti-sense strand.

For the example with two primers (one sense primer and one antisense primer), the following probabilities result: P (AGTAGTAGTAGT) = 0.000000860027 P (AACAAAAACTAA) = 0.000030005828

On the CpG Islands, which contain a total of approximately 30,000,000 bases, a frequency of hybridizations is expected for: AGTAGTAGTAGT: 25.80 on the sense strand

AACAAAAACTAA: 900.17 on the complementary reverse strand.

The primers cannot hybridize on the other strands, since no bis occur in the sense strand due to the bisulfite treatment outside the context CG and accordingly complementarily on the antisense strand.

An amplificate is created if, when there is a perfect base pairing on the sense strand within the maximum fragment length M, a primer on the opposite strand forms a perfect base pairing, which is the probability

P _a (primers) 2_, (l - P _a (primers)) '' for large M and small P _a (primers) this is calculated by the following expression:

P _a (primers) _{. M} (l -P _a (primers)) ^M - l] log (1-P _a (primers)) for the total number F of the amplificates that are to be expected from the amplification of both strands is obtained

F = N * P _s (primers) ^? * ^ (\ -P _a (primers)) ^M - l] g (l -P _a (primers)) formula l

+ N * P (primers) - - ,, ^nmers > ⁾ (ι _ (primers)) ^M -] ^{a K} '\ og (l -P _s (primers) y ^{κ Λ} " ^J

For the example given above, there are 3.0498 amplificates for the CpG islands with 30 mega bases. However, we can show (see Example 1) that primers that are specific for certain regions can produce more than statistically predicted amplicons.

Claims

claims

I. Method for the parallel detection of the methylation state of genomic DNA, characterized in that the following steps are carried out:

a) in a genomic DNA sample, chemical treatment at the 5 'position converts unmethylated cytosine bases to uracil, thymidine or another base which is unlike the cytosine in terms of hybridization behavior;

b) from this chemically treated genomic DNA, more than ten different fragments, each of which are less than 2000 base pairs long, are amplified simultaneously using synthetic oligonucleotides as primers, these primers in each case sequences from gene regulation involved and / or transcribed and / or or contain translated genomic sequences as would be present after treatment according to step a);

c) the sequence context of all or part of the CpG dinucleotides or CpNpG trinucleotides contained in the amplified fragments is determined.

2. The method according to claim 1, characterized in that one carries out the chemical treatment by means of a solution of a bisulfite, bisulfite or disulfite.

3. The method according to claim 1 or 2, characterized in that at least one of the oligonucleotides used in step b) contains fewer nucleobases than would be statistically required for a sequence-specific hybridization to the chemically treated genomic DNA sample.

4. The method according to any one of claims 1 to 3, characterized in that at least one of the oligonucleotides used in step b) of claim 1 is shorter than 18 nucleobases.

5. The method according to any one of claims 1 to 3, characterized in that at least one of the oligonucleotides used in step b) of claim 1 is shorter than 15 nucleobases.

6. The method according to claim 1 or 2, characterized in that in step b) of claim 1 more than 4 different oligonucleotides are used simultaneously for the amplification.

7. The method according to claim 1 or 2, characterized in that in step b) of claim 1 more than 26 different oligonucleotides are used simultaneously for the amplification.

8. The method according to any one of the preceding claims, characterized in that in step b) of claim 1 more than twice as many amplified fragments as calculated according to formula 1 from genome sections involved in the regulation of genes, such as promoters and enhancers, comes as with a purely random choice of the oligonucleotide sequences would be expected, or else their share in the total detectable fragments is more than twice as high as calculated according to formula 1,

(P (primers))

^ ' ^(M »" _lφ ( ₁ - _f , ( _WM ,)) ^l "- ^f - ^lw *'^>)'' - ¹¹ _{Formula 1}

[P (primers))

doing the calculation as follows:

in the case of DNA treated with bisulfite, C can only occur in the context of CG, so it is assumed that the primary DNA is a random sequence with a dependence on directly neighboring bases (first-order Markov chain); which were empirically determined from the database (completely methylated; treated with bisullfit) Base probabilities in pairs result for both DNA strands as P _bDNA (from; to) from the following table:

With

J O0.2811

^{/ 0} . ⁰¹⁴⁰

^/ O ⁰ .4 ⁸⁵⁰

and for the reverse complementary strand (by corresponding exchange of the entries) P _rbDNA (vo; nach)

rbDNA (Λ) = 0.4850 rbDNA (C) = 0.2199 rbDNA (G) = 0.0140 rbDNA (7 ^, ) = 0.2811

; thus the probability that a primer PrimE (with the base sequence Bi B ₂ B ₃ B ₄ ...; e.g. ATTG ...) results in a perfect base pairing depends on the exact sequence of the bases and results as that Product:

(Bisulfite DNA strand)

P _D3a t (Pτ, n ■ m -

N * P (prim); perfect base pairings - If several primers (PrimU, PrimV, PrimW, PrimX, etc.) are used at the same time, the probability of a perfect base pairing on the sense strand at a given position results: P _s (Primers) = P (Prim U)

+ (l -P (PrimU)) P _s (PrimV)

+ (l -P SPrimU)) (l -P (PrimV)) P _s (PrimW)

+ {\ -P SPrimU)) (l -P (PrimV)) (l ~ P (PrimW)) P _s (PrimX)

+ ... and thus as the number of perfect base savings to be expected with any of the primers

N * P primers); the analogous equations are used for the determination of P _a (primers) on the anti-sense strand; An amplificate is produced if and when there is a perfect base pairing on the sense strand within the maximum fragment length M, a primer on the opposite strand forms a perfect base pairing, which is the probability

(l —P _a (primers)) '' for large M and small P _a (primers) this is calculated by the following expression:

for the total number F of the amplificates that are to be expected from the amplification of both strands F = N * P, (primers) ^ "^ ™ ^, Λ (l -P _a (primers)) ^M - 1} log (l -P _a (primers)) formula l

(P i PviϊϊiQTS))

₊ ^ ^ „(^ _Gra ) _{log (1 (frimera))} [(. - (Η, _era ))" - i]

9. The method according to any one of claims 1 to 7, characterized in that in step b) of claim 1 more than twice as many amplified fragments as calculated according to claim 8 comes from genome sections which are transcribed in at least one cell of the respective organism in mRNA than would be expected with a purely random selection of the oligonucleotide sequences, or else their proportion in the total detectable fragments is more than twice as high as calculated according to claim 8.

10.The method according to any one of claims 1 to 7, characterized in that in step b) of claim 1 more than twice as many amplified fragments as calculated according to claim 8 comes from genome sections spliced after transcription in mRNA than exons a purely random choice of the oligonucleotide sequences would be expected, or else their proportion in the total detectable fragments is more than twice as high as calculated according to claim 8.

11. The method according to any one of claims 1 to 7, characterized in that in step b) of claim 1 more than twice as many amplified fragments as calculated according to claim 8 come from genome sections which code for parts of one or more gene families than in one a purely random choice of the oligonucleotide sequences would be expected, or else their proportion in the total detectable fragments is more than twice as high as calculated according to claim 8.

12.The method according to any one of claims 1 to 7, characterized in that in step b) of claim 1 more than twice as many amplified fragments than calculated according to claim 8 come from genome sections which are used for so-called "matrix attachment sites" (MARs). - characteristic sequences contained than would be expected with a purely random selection of the oligonucleotide sequences, or else their share in the total detectable fragments is more than twice as high as calculated according to claim 8.

13.The method according to any one of claims 1 to 7, characterized in that in step b) of claim 1 more than twice as many amplified fragments as calculated according to claim 8 come from genome sections which organize the packing density of the chromatin as so-called "boundary elements" than would be expected with a purely random selection of the oligonucleotide sequences, or else their proportion in the total detectable fragments is more than twice as high as calculated according to claim 8.

14.The method according to any one of claims 1 to 7, characterized in that in step b) of claim 1 more than twice as many amplified fragments as calculated according to claim 8 come from "multiple drug resistance gene" (MDR) promoters or coding regions than would be expected with a purely random selection of the oligonucleotide sequences, or their share in the total detectable fragments is more than twice as high as calculated according to claim 8.

15.The method according to any one of the preceding claims, characterized in that two oligonucleotides or two classes of oligonucleotides are used to amplify the fragments described in claim 1, of which one or a class may contain the base C except in the context of CpG or CpNpG, but not the base G and of which the other or the other class may contain the base G, but not the base C, except in the context of CpG or CpNpG.

16. The method according to any one of claims 1 to 4, characterized in that the amplification described in claim 1 is carried out by means of two oligonucleotides, one of which contains a four to sixteen base long sequence which is complementary to or corresponds to such DNA, how they arise if an equally long DNA fragment to which one of the transcription factors

Amt aryl hydrocarbon receptor nuclear translocator AML-1a CBFA2; core-binding factor, runt domain, alpha subunit 2 (acute myeloid leukemia 1; aml1 oncogene)

AP-1 activator protein-1 (AP-1); Synonyms: c-Jun

C / EBP CCAAT / enhancer binding protein

C / EBPalpha CCAAT / enhancer binding protein (C / EBP), alpha

C / EBPbeta CCAAT / enhancer binding protein (C / EBP), beta

CDP CUTL1; cut (Drosophila) -Iike 1 (CCAAT displacement protein)

CDP CR1 complement component (3b / 4b) receptor 1 CDP CR3 complement component (3b / 4b) receptor 3 CHOP-C / EBPalpha DDIT; DNA-damage-inducible transcript 3 / CCAAT / enhancer binding protein (C / EBP), alpha c-Myc / Max avian myelocytomatosis viral oncogene / MYC-ASSOCIATED FACTOR X

CREB cAMP responsive element binding protein CRE-BP1 CYCLIC AMP RESPONSE ELEMENT-BINDING PROTEIN 2, CREB2, CREBP1; now ATF2; activating transcription factor 2

CRE-BP1 / c-Jun activator protein-1 (AP-1); Synonyms: c-Jun

CREB MP responsive element binding protein

E2F E2F transcription factor (originally identified as a DNA- binding protein essential E1A-dependent activation of the adenovirus E2 promoter)

Freac-7 FKHL11; forkhead (Drosophila) -Iike 9; FORKHEAD-RELATED ACTIVATOR 7; FREAC7 GATA-1 GATA-binding protein 1 / enhancer binding protein GATA1

GATA-1 GATA-binding protein 1 / enhancer binding protein GATA1

GATA-2 GATA-binding protein 2 / enhancer binding protein GATA2

GATA-3 GATA-binding protein 3 / enhancer binding protein GATA3

GATA-X

HFH-3 FKHL10; forkhead (Drosophila) -Iike 10; FORKHEAD-RELATED ACTIVATOR 6; FREAC6

HNF-4 hepatocyte nuclear factor 4

IRF-1 interferon regulatory factor 1

ISRE interferon-stimulated response element

Lmo2 complex LIM domain only 2 (rhombotin-like 1)

MZF1 ZNF42; zinc finger protein 42 (myeloid-specific retinoic acid-responsive)

NF-kappaB (p65) nuclear factor of kappa light polypeptide gene enhancer in B-cells p65 subunit

NF-kappaB nuclear factor of kappa light polypeptide gene enhancer in B cells

Oct-1 OCTAMER-BINDING TRANSCRIPTION FACTOR 1; POU2F1; POU domain, class 2, transcription factor 1

P300 E1A (adenovirus E1A oncoprotein) BINDING PROTEIN, 300-KD

P53 tumor protein p53 (Li-Fraumeni syndrome); TP53 Pax-1 paired box gene 1

Pax-3 paired box gene 3 (Waardenburg syndrome 1)

Pax-6 paired box gene 6 (aniridia, keratitis)

Pbx l b pre-B-cell leukemia transcription factor

Pbx-1 pre-B-cell leukemia transcription factor 1

RORalpha2 RAR-RELATED ORPHAN RECEPTOR ALPHA; retinoic

ACID-BINDING RECEPTOR ALPHA

RREB-1 ras responsive element binding protein 1

SP1 simian virus 40 protein 1

SREBP-1 sterol regulatory element binding transcription factor 1

SRY sex determining region Y

STAT3 signal transducer and activator of transcription 1, 91 kD

Tal-1alpha / E47 T-cell acute lymphocytic leukemia 1 / transcription factor 3

(E2A immunoglobulin enhancer binding factors E12 / E47)

TCF11 Transcription Factor 11; TCF11; NFE2L1; nuclear factor

(erythroid-derived 2) -like 1

USF upstream stimulating factor Whn winged-helix nude X-BP-1 X-box binding protein 1 or YY1 ubiquitously distributed transcription factor belonging to theGLI-Kruppel class of zinc finger proteins

binds, would undergo a chemical treatment according to claim 1.

17. The method according to any one of claims 1 to 4, characterized in that one carries out the amplification described in claim 1 by means of two oligonucleotides, one of which contains a four to sixteen base long sequence which is complementary to or corresponds to such a DNA how it would arise if a DNA fragment of the same length, which, via its sequence or secondary structure, can bring about the specific localization of genome / chromatin sections within the cell nucleus, were subjected to a chemical treatment according to claim 1. I δ.Method according to one of claims 1 to 4, characterized in that the amplification described in claim 1 is carried out by means of two oligonucleotides, of which at least one of the sequences (from 5 ^' to 3 ^' )

TGAGTTAG, CTAACTCA, TTGATTTA, TAAATCAA, TGATTTAG, CTAAATCA, TTGAGTTA, TAACTCAA,

TTTGGT, ACCAAA, ATTAAA, TTTAAT, TGTGGA, TCCACA, TTTATA, TATAAA, TTTGGA, TCCAAA, TTTAAA, TTTAAA, TGTGGT, ACCACA, ATTATA, TATAAT,

ATTAT, ATAAT, GTAAT, ATTAC, ATTGT, ACAAT, GTAAT, ATTAC,

GAAAG, CTTTC, TTTTT, AAAAA,

GTAAT, ATTAC, ATTGT, ACAAT,

GAAAT, ATTTC, ATTTT, AAAAT,

GTAAG, CTTAC, TTTGT, ACAAA,

TTAATAATCGAT, ATCGATTATTAA, ATCGATTATTGG, CCAATAATCGAT,

ATCGATTA, TAATCGAT, TAATCGAT, ATCGATTA,

TATCGATA, TATCGATA, TATCGGTG, CACCGATA, TATTAATA, TATTAATA, TATTGGTG, CACCAATA,

GTGTAATATTT, AAATATTACAC, GGGTATTGTAT, ATACAATACCC, GTGTAATTTTT, AAAAATTACAC, GGGGATTGTAT, ATACAATCCCC, ATGTAATTTTT, AAAAATTACAT, GGGGATTGTAT, ATACAATCCCC, ATGTAATATTTTTATTATTATTAT, ACC TGACGTAA, TTACGTCA, TTACGTTA, TAACGTAA, TGACGTTA, TAACGTCA, TGACGTTA, TAACGTCA, TTACGTAA, TTACGTAA, TTACGTAA, TTACGTAA, TGACGTTA, TAACGTCA, TAACGTTA, TAACGTTA,

GTAGGTGG, CCACCTAC, TTATTTGT, ACAAATAA, GTAGGTGT, ACACCTAC, ATATTTGT, ACAAATAT,

TGCGTAGGCGT, ACGCCTACGCA, ACGTTTACGTA, TACGTAAACGT, TGCGTAGGCGG, CCGCCTACGCA, TCGTTTACGTA, TACGTAAACGA, ATAGGAAGT, ACTTCCTAT, ATTTTTTGT, ACAAAAAAT

TCGGAAGT, ACTTCCGA, ATTTTCGG, CCGAAAAT, TCGGAAGT, ACTTCCGA, GTTTTCGG, CCGAAAAC, TCGGAAAT, ATTTCCGA, ATTTTCGG, CCGAAAAT, TCGGAAAT, ATTTCCGA, GTTTTCGG, CCGAAAACGTTAATTAAT

GATG, CATC, TATT, AATA,

GATAG, CTATC, TTATT, AATAA, GATAAG, CTTATC, TTTATT, AATAAA, TGTTTATTTA, TAAATAAACA, TAAATAAATA, TATTTATTTA, TGTTTGTTTA, TAAACAAACA, TAAATAAATA, TATTTATTTA, TATTTATTTA, TAAATAAATA, TAAATAAATA, TATTTATTTA, TATTTGTTTA, TAAACAAATA, TAAATAATTA

TAGTTTTATTTTTTT, AAAAAAATAAAACTA, GGGAAAGTGAAATTG,

CAATTTCACTTTCCC,

TAGTTTTATTTTTTT, AAAAAAATAAAACTA, GGAAAAGTGAAATTG,

CAATTTCACTTTTCC,

TAGTTTTTTTTTTTT, AAAAAAAAAAAACTA, GGAAAAGAGAAATTG,

CAATTTCTCTTTTCC,

TAGTTTTTTTTTTTT, AAAAAAAAAAAACTA, GGGAAAGAGAAATTG,

CAATTTCTCTTTCCC,

TAGGTG, CACCTA, TATTTG, CAAATA,

TTTTAAAAATAATTTT, AAAATTATTTTTAAAA, AGGGTTATTTTTAGAG,

CTCTAAAAATAACCCT,

TTTTAAAAATAATTTT, AAAATTATTTTTAAAA, GGAGTTATTTTTAGAG,

CTCTAAAAATAACTCC,

TTTTAAAAATAATTTT, AAAATTATTTTTAAAA, AGAGTTATTTTTAGAG,

CTCTAAAAATAACTCT,

TTTTAAAAATAATTTT, AAAATTATTTTTAAAA, GGGGTTATTTTTAGAG,

CTCTAAAAATAACCCC,

TAGGGG, CCCCTA, TTTTTA, TAAAAA,

GAGGGG, CCCCTC, TTTTTT, AAAAAA,

TGTTGAGTTAT, ATAACTCAACA, ATGATTTAGTA, TACTAAATCAT,

TGTTGATTTAT, ATAAATCAACA, GTGAGTTAGTA, TACTAACTCAC,

TGTTGAGTTAT, ATAACTCAACA, ATGATTTAGTA, TACTAAATCAT,

TGTTGATTTAT, ATAAATCAACA, GTGAGTTAGTA, TACTAACTCAC, GGGGATTTTT, AAAAATCCCC, GGGAATTTTT, AAAAATTCCC, GGGGATTTTT, AAAAATCCCC, GGGGATTTTT, AAAAATCCCC, GGGGATTTTT, AAAAATCCCC, GGAAATTTTT, AAAAATTTCC, GGGAATTTTT, AAAAATTCCC, GGAAATTTTT, AAAAATTTCC, GGGAATTTTT, AAAAATTCCC, GGAAATTTTT, AAAAATTTCC, GGGATTTTTT, AAAAAATCCC, GGAAAGTTTT, AAAACTTTCC, GGGAATTTTT, AAAAATTCCC, GGGAATTTTT, AAAAATTCCC, GGGATTTTTT, AAAAAATCCC, GGGAAGTTTT, AAAACTTCCC, GGGATTTTTTA, TAAAAAATCCC, TGGAAAGTTTT, AAAACTTTCCA, TTTAGTATTACGGATAGAGGT, ACCTCTATCCGTAATACTAAA, GTTTTTGTTCGTGGTGTTGAA, TTCAACACCACGAACAAAAAC, TTTAGTATTACGGATAGAGTT, AACTCTATCCGTAATACTAAA, GGTTTTGTTCGTGGTGTTGAA, TTCAACACCACGAACAAAACC, TTTAGTATTACGGATAGCGTT, AACGCTATCCGTAATACTAAA, GGCGTTGTTCGTGGTGTTGAA, TTCAACACCACGAACAACGCC, TTTAGTATTACGGATAGCGGT, ACCGCTATCCGTAATACTAAA, GTCGTTGTTCGTGGTGTTGAA, TTCAACACCACGAACAACGAC,

ATAAT, ATTAT, ATTAT, ATAAT, GTAAT, ATTAC, ATTAT, ATAAT,

AATGTAAAT, ATTTACATT, ATTTGTATT, AATACAAAT,

CGTTACGGTT, AACCGTAACG, AATCGTGACG, CGTCACGATT, CGTTACGGTT, AACCGTAACG, GATCGTGACG, CGTCACGATC, CGTTACGTTT, AAACGTAACG, AAGCGTGACG, CGTCACGACGTTCGGACGTC TTTACGTATGA, TCATACGTAAA, TTATGCGTGAA, TTCACGCATAA, TTTACGTTTGA, TCAAACGTAAA, TTAAGCGTGAA, TTCACGCTTAA, TTTACGTTTTA, TAAAACGTAAA, TGAAGCGTGAA, TTCACGGGTACA

TAATTAT, ATAATTA, ATGATTG, CAATCAT,

TAGGTTA, TAACCTA, TGATTTA, TAAATCA,

TTTTAAATATTTTT, AAAAATATTTAAAA, GGGGGTGTTTGGGG,

CCCCAAACACCCCC,

TTTTAAATTATTTT, AAAATAATTTAAAA, GGGGTGGTTTGGGG,

CCCCAAACCACCCC,

TTTTAAATTTTTTT, AAAAAAATTTAAAA, GGGGGGGTTTGGGG,

CCCCAAACCCCCCC,

TTTTAAATAATTTT, AAAATTATTTAAAA, GGGGTTGTTTGGGG,

CCCCAAACAACCCC,

GAGGCGGGG, CCCCGCCTC, TTTCGTTTT, AAAACGAAA, GAGGTAGGG, CCCTACCTC, TTTTGTTTT, AAAACAAAA, AAGGCGGGG, CCCCGCCTT, TTTCGTTTT, AAAACGAAA, AAGTTCTGTAA, CCC

ATTTTTCGGAAATG, CATTTC CG AAAAAT, TATTTTCGGGAAAT,

ATTTCCCGAAAATA,

ATTTTTCGGAAATG, CATTTC CG AAAAAT, TATTTTCGGGAAAT,

ATTTCCCGAAAATA,

ATTTTCGGGAAATG, CATTTCCCGAAAAT, TATTTTTCGGAAAT,

ATTTCCGAAAAATA,

ATTTTCGGGAAGTG, CACTTCCCGAAAAT, TATTTTTCGGAAAT, ATTTCCGAAAAATA,

GGGGGTTGACGTA, TACGTCAACCCCC, TACGTTAATTTTT, AAAAATTAACGTA,

TGACGTATATTTTT, AAAAATATACGTCA, GGGGATATGCGTTA,

TAACGCATATCCCC,

TGACGTATATTTTT, AAAAATATACGTCA, GGGGGTATGCGTTA,

TAACGCATACCCCC,

ATGATTTAGTA, TACTAAATCAT, TGTTGAGTTAT, ATAACTCAACA,

GTTAT, ATAAC, ATGAT, ATCAT,

contains, which is complementary to such a DNA or corresponds to it as it would arise if an equally long DNA fragment, which can bring about the specific localization of genome / chromatin sections within the cell nucleus via its sequence or secondary structure, a chemical treatment according to claim 1 would undergo.

19. The method according to any one of claims 16 to 18, characterized in that the oligonucleotides used for the amplification contain several positions in addition to the consensus sequences defined in claims 16 to 18, at which either any of the three bases G, A and T or any of bases C, A and T may be present.

20. The method according to claim 19, characterized in that the oligonucleotides used for the amplification, apart from one of the consensus sequences described in claim 18, contain a maximum of as many additional bases as are required for the simultaneous amplification of more than one hundred different fragments per reaction from the chemical treated DNA, calculated according to claim 8, is required.

21. The method according to any one of the preceding claims, characterized in that the examination of the sequence context of all or part of the CpG dinucleotides or CpNpG trinucleotides contained in the amplified fragments according to claim 1 c) by hybridization of the fragments already provided in the amplification with a fluorescent marker to one Oligonucleotide array (DNA chip) takes place.

22. The method according to any one of claims 1 to 20, characterized in that the amplified fragments are immobilized on a surface and then a hybridization is carried out with a combinatorial library of distinguishable oligonucleotide or PNA oligomer probes.

23. The method according to claim 22, characterized in that the probes are detected by means of matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) on the basis of their unique mass and thus the sequence context of all or part of the CpG dinucleotides contained in the amplified fragments or CpNpG trinucleotides is decrypted.

24. The method according to any one of the preceding claims, characterized in that the amplification as described in step b) of claim 1 is carried out by a polymerase chain reaction in which the size of the amplified fragments by means of chain extension steps shortened to less than 30 s is limited.

25.A method according to one of the preceding claims, characterized in that after the amplification according to step b) of claim 1, the products are separated by gel electrophoresis and the fragments which are smaller than 2000 base pairs or smaller than any limit value below 2000 base pairs, by cutting out the other amplification products before the evaluation according to step c) of claim 1.

26. The method according to claim 25, characterized in that after the separation of amplificates of a certain size, these are amplified again before carrying out step c) of claim 1.

27. Kit containing at least two primer pairs, reagents and auxiliary substances for the amplification and / or reagents and auxiliary substances for the chemical treatment according to claim 1 a) and / or a combinatorial probe library and / or an oligonucleotide array (DNA chip), insofar as they are necessary or useful for carrying out the method according to the invention.