EP1261740A1

EP1261740A1 - Ligase/polymerase method for detecting cytosine methylation in dna samples

Info

Publication number: EP1261740A1
Application number: EP01915053A
Authority: EP
Inventors: Alexander Olek; Kurt Berlin
Original assignee: Epigenomics AG
Current assignee: Epigenomics AG
Priority date: 2000-02-25
Filing date: 2001-02-23
Publication date: 2002-12-04
Also published as: US20030119025A1; DE10010281B4; AU2001242279B2; AU2006236059A1; JP2003523752A; DE10010281A1; CA2401198A1; US7405040B2; WO2001062961A1; AU4227901A

Abstract

The invention relates to a method for detecting 5-methylcytosine in genomic DNA samples. Firstly, a genomic DNA from a DNA sample is chemically reacted with a reagent, whereby 5-methylcytosine and cytosine react differently. Afterwards, the pretreated DNA is amplified while using a polymerase and at least one primer. In the next step, the amplified genomic DNA is hybridized to at least two oligonucleotides, whereby the latter are assembled by inserting at least one oligonucleotide. In the case of the ligation product, a nucleotide carries a detectable tagging, and the lengthening is subject to the methylation status of the respective cytosine in the genomic DNA sample. In the following step, the lengthened oligonucleotides are examined for the presence of the tagging.

Description

Ligase / polymerase method for the detection of cytosine methylation in DNA samples

The invention relates to a method for the detection of 5-methylcytosine in genomic DNA samples.

The levels of observation 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 can 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.

The present invention describes a method for the detection of the methylation state of genomic DNA samples. The method can also be used to detect point mutations and single nucleotide polymorphisms (SNPs).

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, in genetic 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 because 5-methylcytosine has the same base pairing behavior as cytosine. In addition, a PCR Amplification completely lost the epigenetic information that the 5-methylcytosines carry.

A relatively new and now the most widely used method for examining DNA for 5-

Methylcytosine is based on the specific reaction of bisulfite with cytosine, which is converted into üracil after subsequent alkaline hydrolysis, which corresponds to the thymidine in its base-pairing behavior. 5-Methylcytosine, however, is not modified under these conditions. Thus the original DNA is punched so umgewan ^¬ that methylcytosine, which can be originally not distinguished by its hybridization behavior from cytosine, now normal through ",, molecular biology techniques can be detected 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 being fully exploited. 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 preventing the diffusion and renaturation of the DNA (bisulfite only reacts on single-stranded DNA) and all precipitation and purification steps replaced by rapid dialysis (Olek, A. et al., Nucl. Acids. Res. 1996, 24,

5064-5066). With this method, individual cells can be examined, which illustrates the potential of the method. However, only individual regions up to about 3000 base pairs in length have so far been investigated; a global examination of cells for thousands of possible methylation analyzes 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 possibilities for detecting 5-methylcytosine can be found in the following overview article: Rein, T., DePamphilis, ML, Zorbas, H., Nucleic Acids Res. 1998, 26, 2255.

The bisulfite technique has so far been used only in research with a few exceptions (e.g. Zechnigk, M. et al., Eur. J. Hum. Gen. 1997, 5, 94-98). However, short, specific pieces of a known gene are always amplified after bisulfite treatment and either completely sequenced (Olek, A. and Walter, J., Nat. Genet. 1997, 17, 275-276) or individual cytosine positions by a "Primer extension reaction" (Gonzalgo, ML and Jones, PA, Nucl. Acids Res. 1997, 25, 2529-2531, WO patent 9500669) or an enzyme cut (Xiong, Z. and Laird, PW, Nucl. Acids Res 1997, 25, 2532-2534). Detection by hybridization has also been described (Olek et al., WO 99 28498).

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

An overview of the state of the art in oligomer array production can 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. Various methods exist to DNA Sieren to immobili ^¬. The best known method is the binding of a DNA, which is functionalized with biotin, to a streptavidin-coated surface (Uhlen, M. et al. 1988, Nucleic Acids Res. 16, 3025-3038). The bond strength of this system corresponds to that of a covalent chemical bond without being one. In order to be able to covalently bind a target DNA to a chemically prepared surface, the target DNA must have a corresponding functionality. DNA itself has no functionalization that is suitable. There are different ways of introducing a suitable functionalization into a target DNA: two easy-to-use functionalizations are primary, aliphatic amines and thiols. Such amines are reacted quantitatively with N-hydroxysuccinimide esters, and thiols react quantitatively with alkyl iodides under suitable conditions. One difficulty is in introducing such functionalization into DNA. The simplest variant is the introduction by means of a PCR primer. Variants shown use 5'-modified primers (NH ₂ and SH) and a bifunctional linker.

An essential part of immobilization on a surface is its condition. Systems described so far are mainly made of silicon or metal. Another method for binding a target DNA is based on using a short recognition sequence (for example 20 bases) in the target DNA for hybridization to a surface-immobilized oligonucleotide. Enzymatic variants for introducing chemically activated positions on a target DNA have also been described. Here, a 5'-NH ₂ functionalization is carried out enzymatically on a target DNA. Fluorescent-labeled probes have been used in many cases for scanning an immobilized DNA array. The simple attachment of Cy3 and Cy5 dyes to the 5 'OH of the respective probe is particularly suitable for fluorescent labels. 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.

An overview of the prior art in oligomer array production can be found in a special edition of Nature Genetics published in January 1999 (Nature Genetics Supplement, Volume 21, January 1999), the literature cited therein and US patent 5994065 on methods for production from solid supports for target molecules such as oligonucleotides with a reduced non-specific background signal.

More recent methods for detecting mutations are listed below:

As a special case of sequencing, the single-base primer extension (Genetic Bit Analysis) is worth mentioning (Head, SR., Rogers, YH., Parikh K., Lan, G., Anderson, S., Goelet, P., Boycejacino MT ., Nucleic Acids Research. 25 (24): 5065-5071, 1997; Picoult-Newberg, L., Genome Res. 9 (2): 167-174, 1999). Combined amplification and sequencing is described in US Pat. No. 5,928,906, where base-specific termination on matrix molecules is used. Another method uses a ligase / polymerase reaction for the identification of nucleotides (US Pat. No. 5,952,174).

Matrix-assisted laser desorption / ionization mass spectrometry (MALDI) is a very powerful development for the analysis of biomolecules (Karas, M. and Hillenkamp, F. (1988), Laser desorption ionization of proteins with molecular masses exeeding 10000 daltons. Anal. Chem. 60: 2299-2301). An analyte is embedded in a light-absorbing matrix. The matrix is evaporated by a short laser pulse and the analyte molecule is thus transported unfragmented into the gas phase. The ionization of the analyte is achieved by collisions with matrix molecules. 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.

Maldi is ideal for the analysis of peptides and proteins. The analysis of nucleic acids is somewhat more difficult (Gut, IG and Beck, S. (1995), DNA and Matrix Assisted Laser Desorption Ionization Mass Spectrometry. Molecular Biology: Current Innovations and Future Trends 1: 147-157.) Für The sensitivity of nucleic acids is about 100 times worse than for peptides and decreases disproportionately with increasing fragment size. For nucleic acids that have a multiple negative charge

Backbone, the ionization process through the matrix is much more inefficient. For MALDI, the choice of the matrix plays an eminently important role. Some very powerful matrices have been found for the desorption of peptides, which result in a very fine crystallization. There are now some attractive matrices for DNA, but this did not reduce the difference in sensitivity. The difference in sensitivity can be reduced by chemically modifying the DNA to make it more similar to a peptide. Phosphorothioate nucleic acids, in which the usual phosphates of the backbone are substituted by thiophosphates, can be converted into a charge-neutral DNA by simple alkylation chemistry (Gut, IG 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 "batch tagging" is the increased stability of the analysis against contamination, which makes the detection of unmodified substrates very difficult.

Genomic DNA is obtained by standard methods from DNA from cell, tissue or other test samples. This standard methodology can be found in references such as Fritsch and Maniatis eds. , Molecular Cloning: A Laboratory Manual, 1989.

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 are inserted by inserting "wobbles", i.e. H. Positions where there are different bases can still be extended. Furthermore, these binding sites are at certain distances from each other.

The distribution of the DNA in the interphase chroatin, 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 attach regions (MAR) or scaffold attach ent regions (SAR). Tacking has a significant impact on the

Transcription or replication. These MAR fragments have no conservative sequences, but consist of 70% A and T and are close to free-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 e.g. inhibit the effect of the enhancer on the promoter if they are between the enhancer and the promoter or, if they are between heterochromatin and a gene, protect the active gene from the influence of 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 (spacing 100 bp each). The protein BEAF-32 binds to ses'. These insulators can be on either side of the gene.

The object of the present invention is to provide a method which is particularly suitable for the simultaneous detection of cytosine methylations and SNPs in genomic DNA samples. It should preferably be possible to examine a large number of fragments at the same time.

The object is achieved according to the invention by a method for the detection of 5-methylcytosine in genomic DNA samples, the following steps being carried out: (a) a genomic DNA from a DNA sample is reacted chemically with a reagent, wherein 5-methylcytosine and cytosine react differently and thus they have a different base pairing behavior after the reaction show the DNA duplex;

(b) the pretreated DNA is amplified using a polymerase and at least one oligonucleotide (type A) as a primer; (c) a set of oligonucleotides is hybridized to the amplified genomic DNA to form a duplex, this set of oligonucleotides consisting of different species of type B and type C, and said hybridized type B oligonucleotides having their 3 'end directly or adjacent at intervals of up to 10 bases to the positions to be examined for their methylation in the genomic DNA sample and wherein the second oligonucleotide (type C) hybridizes to a second region of the target molecule, so that the 5 'end the second oligonucleotide (type C) is separated by a gap the size of a single nucleotide or up to 10 nucleotides from the 3 'end of the first hybridized oligonucleotide (type B) at the location of said selected position; (d) the oligonucleotide (type B) with a known sequence of n nucleotides is extended by means of a polymerase by a maximum of the number of nucleotides between the 3 'end of the type B oligonucleotide and the 5' end of the type C oligonucleotide lie, the extension being dependent on the methylation status of the respective cytosine in the genomic DNA sample;

(e) the oligonucleotides are incubated in the presence of a ligase, the adjoining first oligonucleotide of type B, which has been lengthened by the polymerase reaction, and the second oligonucleotide of type C being joined, and a ligation product is thereby obtained, provided that in the previous step an extension of the type B oligonucleotide was carried out in such a way that the 3 'end with the 3' hydroxy function of the extended oligonucleotide present is immediately adjacent to the 5 'end of the type C oligonucleotide; (f) it is detected whether a ligation product has formed.

It is preferred according to the invention that the 5 'end of the first oligonucleotide (type B) is immobilized on a solid phase or that the 3' end of the second oligonucleotide (type C) is immobilized on a solid phase.

It is also preferred according to the invention that the amplified products generated in step b are bound to a solid phase at defined points. It is particularly preferred that at least one primer (type A) is bound to a solid phase during the amplification.

It is further preferred that one be different

Arranges amplificates on the solid phase in the form of a rectangular or hexagonal grid.

It is further preferred that different oligonucleotide sequences are arranged on a flat solid phase in the form of a rectangular or hexagonal lattice.

It is also preferred that the markings attached to the extended oligonucleotides can be identified at any position on the solid phase at which an oligonucleotide sequence is located.

It is further preferred according to the invention that the solid phase surface consists of silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver or gold.

It is very particularly preferred that the DNA is treated with a bisulfite solution (= disulfite, bisulfite) before amplification. It is also preferred according to the invention that the amplification is carried out by means of the polymerase chain reaction (PCR).

It is further preferred that the type A oligonucleotides used either contain only the bases T, A and C or else the bases T, A and G and / or that the type B and / or type C oligonucleotides used either contain only the bases T, A and C or the bases T, A and G contain.

It is furthermore particularly preferred that the ligation products and / or the extension products are provided with a detectable label for the detection. It is particularly preferred that the markings are fluorescent markings or that the markings are radionuclides or that the markings are removable mass markings that can be detected in a mass spectrometer.

It is also preferred that the elongated oligonucleotides and ligation products can be detected in total in the mass spectrometer and are therefore clearly labeled by their mass. It is further preferred according to the invention that a fragment of the elongated and / or ligated oligonucleotides can be detected in the mass spectrometer.

It is further preferred in the method according to the invention that the fragment is generated by digestion with one or more exo- or endonucleases.

It is particularly preferred here that the fragments generated have a single positive or negative net charge for better detectability in the mass spectrometer. In the method according to the invention it is further preferred according to the invention that the detection of the extended oligonucleotides and / or the ligation products is carried out and visualized by means of matrix assisted laser desorption / ionization mass spectrometry (MALDI) or by means of electrospray mass spectrometry (ESI).

A method is also preferred in which the polymerases are heat-resistant DNA polymerases and / or the ligases are thermostable ligases.

A method is also preferred in which, in addition to DNA methylation, SNPs are also detected and visualized.

A method is also preferred, the nucleotides used being terminating (type D 2) and / or chain-extending nucleotides (type D 1). It is particularly preferred here that the chain-terminating nucleotide (type D 2) is selected from a group which either contains bases T and C or bases G and A and / or the chain-extending nucleotides (type D 1) are selected from a group which contains either the nucleobases A, T and C or the bases G and A and T.

It is also preferred that the fluorescently labeled dCTP derivative is Cy3-dCTP or Cy5-dCTP.

It is also preferred according to the invention that the amplification of several DNA sections is carried out in one reaction vessel.

The method according to the invention is very particularly preferred, the genomic DNA being obtained from a DNA sample in step a), sources of DNA e.g. B. cell lines, blood, sputum, stool, urine, brain Spinal fluid, paraffin-embedded tissue, histological slides, and all possible combinations thereof.

It is also preferred according to the invention that the

Forms the 3 'end of the type B oligonucleotide extended in step d by the nucleotide 2' deoxyadenosine, which enables the ligation in step e by its 3 'hydroxyl function, whereas a 2', 3 'dideoxyguanosine at the 3' end of the oligonucleotide does not lead to a ligation product and / or that the 3 'end of the oligonucleotide of type B extended in step d is formed by the nucleotide 2' deoxyguanosine, which enables the ligation in step e by its 3'-hydroxy function, o - On the other hand, a 2 ', 3' dideoxyadenosine at the 3 'end of the oligonucleotide does not lead to a ligation product and / or that the 3' end of the type B oligonucleotide extended in step d is formed by the nucleotide thymidine, which is formed by its 3 'hydroxyl function enables the ligation in step e, whereas a 2', 3 'dideoxy cytidine at the 3' end of the oligonucleotide does not lead to a ligation product and / or that the 3 'end of the in step d forms an extended oligonucleotide of type B by the nucleotide 2 '-deoxycytidine, which enables the ligation in step e by its 3' -hydroxy function, whereas a 2 ', 3' -dideoxythymidine at the 3 'end of the oligonucleotide does not lead to a ligation product ,

Furthermore, it is preferred according to the invention that methylation analyzes of the upper and lower DNA strand are carried out simultaneously.

Finally, a method according to the invention is preferred, wherein steps ce are carried out in solution and the ligated oligonucleotides are applied to a solid phase for detection. A method for the detection of methylcytosine in genomic DNA samples is thus described:

The method involves the amplification, hybridization and extension reaction of an entire DNA or a fragment thereof. The method can be used for the detection of methylcytosine and also of single nucleotide polymorphisms (SNPs) and mutations.

The genomic DNA to be analyzed is preferably obtained from conventional sources for DNA, such as. B. cell lines, blood, sputum, stool, urine, brain spinal fluid, paraffin-embedded tissue, histological see slides and all possible combinations thereof.

In the first step of the process, the DNA used is preferably treated with bisulfite, (= disulfite, hydrogen sulfite) or another chemical in such a way that none of the methylated cytosine bases at the 5 ~ position of the base are changed in such a way that a different bases are formed with regard to the base pairing behavior, while the cytosines methylated in the 5-position remain unchanged. If bisulfite is used, an addition takes place on the unmethylated cytosine bases. The subsequent alkaline hydrolysis then leads to the conversion of unmethylated cytosine nucleobases into uracil. The genomic DNA used is preferably fragmented before chemical treatment with a restriction endonuclease.

In the second step of the method, the pretreated DNA is preferably amplified using a heat-resistant polymerase and at least one primer (type A). This primer can preferably contain 10-40 base pairs. In a particularly preferred variant of the method, the amplification is carried out using type A primers by means of the polymerase chain reaction (PCR).

In a preferred variant of the method, the amplification of several DNA fragments is carried out in one reaction vessel. This can either be a so-called multiplex PCR, in which different primers each generate defined fragments. Various defined amplifications are carried out in one reaction vessel. In a further, particularly preferred variant of the method, primers amplify several fragments in a targeted and reproducible manner. This can be achieved, for example, by binding the fragments to repetitive elements in the genome, for example. In a particularly preferred variant of the method, the primers bind to transcription factor binding sites, to promoters or other regulatory elements in genes. In a particularly preferred variant of the method, the

Amplification takes place by extending primers that are bound to a solid phase. A multiplex PCR in the broader sense can be carried out by binding different primers to different, defined locations of a solid phase. In these versions of the amplification, the primer complementary to one strand (e.g. forward primer) is always immobilized and the primer complementary to the opposite strand (e.g. reverse primer) is always in solution.

In a further preferred variant of the second method step, the solid phase is flat, the different oligonucleotide sequences being arranged in the form of a rectangular or hexagonal lattice. As a result, the different amplificates on the solid phase in the form of a right-angled or hexagonal grid are arranged. As already described above, in this case several amplificates are generated directly on the solid phase.

The solid phase surface preferably consists of silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver or gold.

In a particularly preferred variant of the method, the oligonucleotides of type A either contain only the bases T, A and C or only the bases T, A and G.

In the third step of the method, a set of oligonucleotides consisting of two types of oligonucleotides, a first (type B) and a second (type C) oligonucleotide is hybridized to a selected position of the amplified genomic DNA. The first oligonucleotide (type B) is a primer which hybridizes to a first region of the target sequence to be investigated in such a way that its 3 'end immediately or at intervals of up to 10 bases adjoins the positions which are relevant for their methylation in of the genomic DNA sample are to be examined. The second oligonucleotide (type C) hybridizes to a second region of the target molecule so that the 5 'end of the second oligonucleotide (type C) is separated by a gap the size of a single nucleotide or up to 10 nucleotides from the 3' end of the first hybridized oligonucleotide (type B) is separated at the location of said selected position.

The type B oligonucleotides hybridized to the amplificates can be connected to a solid phase at their 5 'end or at another base or via their backbone, but not via their 3' end. Binding preferably takes place via the 5 'end. In a preferred variant, the solid phase is flat, the different olives gonucleotide sequences (type B) are arranged in the form of a rectangular or hexagonal grid.

The type C oligonucleotides hybridized to the amplificates can be connected to a solid phase at their 3 'end or at another base or via their backbone, but not via their 5' end. Binding preferably takes place via the 3 'end. In a preferred variant, the solid phase is flat, the different OHgonucleotide sequences (type C) being arranged in the form of a rectangular or hexagonal lattice. The 5 'end of the type C oligonucleotide must be phosphorylated.

In summary, there are preferably the following different options for immobilization on a solid phase:

1. The 5 'end of the first oligonucleotide (type B) is immobilized on a solid phase.

2. The 3 'end of the second oligonucleotide (type C) is immobilized on a solid phase.

3. The amplification is already carried out on a solid phase, the 5 'end of a primer preferably being connected to the solid phase.

4. The amplification, hybridization, extension reaction and ligation are preferably carried out in solution and the ligation product is first applied to the solid phase for detection.

The solid phase surface preferably consists of silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver or gold. In a particularly preferred variant of the method, the oligonucleotides of type B and / or type C contain either only the bases T, A and C or only the bases T, A and G.

In the fourth process step, the oligonucleotide (type B) with a known sequence of n nucleotides is extended with a heat-resistant polymerase by at most the number of nucleotides between the 3 'end of the type B OHgonucleotide and the 5' end of the type oligonucleotide C. At least one nucleotide preferably carries a detectable label. In a particularly preferred variant of the method, either the type B oligonucleotide or the type C oligonucleotide bears a detectable label. The type of extension depends on the methylation status of the respective cytosine in the genomic DNA sample, or on any SNPs, point mutations or deletions, insertions and inversions that are present.

In a preferred variant of the method, the nucleotides used are terminating (type D2) and / or chain-extending nucleotides (type D1). The terminating nucleotide (type D2) is a 2 ', 3' dideoxynucleotide and the chain-extending nucleotide is a 2'-

Deoxynucleotide. In a particularly preferred variant of the method, the nucleobases of type Dl are selected from a group which contain bases T, A and C or bases T, A and G. In a further, particularly preferred variant of the method, the nucleobases of type D2 are selected from a group which contains either bases T and C or bases G and A.

The extended type B oligonucleotides are preferably labeled using absorbing dyes and / or chemiluminescence and / or radioactive Isotopes and / or via fluorescent labels which are introduced via the nucleotides added in the fourth method step or else via the oligonucleotides of type B or C. In principle, in the case of labeling the oligonucleotides, the immobilized oligonucleotide has no label. Labeling via the molecular mass of the elongated and ligated oligonucleotide is also preferred.

These hybridized oligonucleotides are in the fifth

Process step incubated in the presence of a preferably thermostable ligase in order to connect the adjacent hybridized first (type B) and second (type C) oligonucleotide and thereby to obtain a ligation product, provided that in the previous step the oligonucleotide was extended such that a Deoxynucleoside directly adjacent to the 5 'end of the type C oligonucleotide.

In the sixth method step it is detected whether a ligation product has formed. For this purpose, the extended oligonucleotides at each position of the solid phase, at which an oligonucleotide sequence is located, are examined for the presence of a label.

In a particularly preferred variant of the method, the detection of the extended oligonucleotides takes place via their fluorescence. Different ligation products preferably have different fluorescence properties, which can be achieved, for example, by built-in nucleotides labeled with different dyes.

In a preferred variant of the method, fragments of the extended oligonucleotide are generated by digestion with one or more exo- or endonucleases. In a particularly preferred variant of the method, the markings of the nucleotides are detachable mass markings which can be detected in a mass spectrometer.

Detachable mass markings, the extended oligonucleotides as a whole or fragments thereof are, in a particularly preferred variant of the method, using matrix-assisted laser desorption / ionization

Mass spectrometry (MALDI-MS) or by means of electron spray mass spectrometry (ESI) detected and visualized based on their unique mass.

The fragments detected in the mass spectrometer preferably have a single positive or negative net charge.

In a particularly preferred variant of the method, SNPs (single nucleotide polymorphisms) and cytosine methylations are analyzed in one experiment.

In a particularly preferred variant of the method, the lower and the upper strand of the DNA sample after the chemical pretreatment are analyzed in an experiment in order to ensure internal experimental control.

The invention further relates to a kit which contains chemicals and aids for carrying out the bisulfite

Contains reaction and / or the amplification, the hybridization, the extension reaction and the ligase reaction and / or polymerases and / or the documentation for performing the method.

The following examples illustrate the invention. Example 1 :

The following example relates to a fragment of exon 23 of the factor VIII gene in which a specific CG position is to be examined for methylation.

In the first step, the fragment is amplified by type A primers, namely by ATTATGTTGGAGTAGTAGAGTTTAAATGGTT (SEQ-ID No .: 1) and ACTTAACACTTACTATTTAAATCACAACCCAT (SEQ-ID No .: 2). The amplified DNA is hybridized to an oligonucleotide of type B (for example ATGTTGGATGTTGTTGAG (SEQ-ID or 3)) and a 5 '-phosphorylated oligonucleotide of type C (for example GTATAAAGTAAATTAGAAGGAAGAT (SEQ-ID No .: 4)). The extension reaction is then carried out with 2 ', 3' -dideoxycytidine triphosphate (ddCTP, as type D2), thymidine triphosphate (dTTP, as type D1) and 2'-deoxyadenosine triphosphate (dATP, as type D1). It is formed when a methylated cytosine was present, the extension product ATGTTGGATGTTGTTGAGAAAC (SEQ ID No .: 5), which carries no hydroxyl at the 3 'end, while in the presence of a non-methylated cytosine in the to be tested sequence, the extension product ATGTTGGATGTTGTTGAGAAA2 ⁷ (SEQ -ID NO .: 6) with a 3'-0H function. In the following ligation reaction, therefore, only the ligation product can

ATGTTGGATGTTGTTGAGAAAΪ 'GTATAAAGTAAATTAGAAGGAAGAT (SEQ ID No.: 7). If the oligonucleotide of type C GTATAAAGTAAATTAGAAGGAAGAT (SEQ-ID No .: 4) is now fluorescence-labeled, a fluorescent label is only inserted if there is an unmethylated cytosine in the DNA sample to be examined. Conversely, a control can be performed using the same sequences, but with a different set of triphosphates. Analogously to the example above, in the extension reaction 2 ', 3' -dideoxothymidine triphosphate (ddTTP, as type D2), 2'-deoxycytidine triphosphate (dCTP, as type Dl) and 2'-deoxy-adenosine triphosphate (dATP, as type Dl) carried out, after the ligase reaction, the reverse only results in the incorporation of a label if a methylated cytosine has been present in the DNA sample to be examined.

Example 2:

In the first step, the fragment is amplified by type A primers, namely by ATTATGTTGGAGTAGTAGAGTTTAAATGGTT (SEQ-ID No .: 1) and

ACTTAACACTTACTATTTAAATCACAACCCAT (SEQ ID No .: 2). The 5 'end of the amplified DNA is attached to an oligonucleotide of type B immobilized on a solid phase surface (for example ATGTTGGATGTTGTTGAG (SEQ-ID No.: 3)) and a 5' -phosphorylated oligonucleotide of type C (for example GTATAAAGTAAATTAGAAGGAAGAT (SEQ-ID No .: 4)) hybridized. The extension reaction is then carried out with 2 ', 3' -dideoxycytidine triphosphate (ddCTP, as type D2), thymidine triphosphate (dTTP, as type D1) and 2'-deoxyadenosine triphosphate (dATP, as type D1). When a methylated cytosine was present, the solid phase-bound extension product ATGTTGGATGTTGTTGAGAAAC (SEQ-ID No .: 5), which has no hydroxyl function at the 3 'end, is formed, while when there is an unmethylated cytosine in the the sequence the extension product

ATGTTGGATGTTGTTGAGAAAT (SEQ-ID No.: 6) with a 3 'OH function is created. In the now following ligation reaction, therefore, only the solid phase-bound ligation product ATGTTGGATGTTGTTGAGAAAT GTATAAAGTAAATTAGAAGGAAGAT (SEQ ID No .: 7) can be formed. If the oligonucleotide of type C GTATAAAGTAAATTAGAAGGAAGAT (SEQ-ID No.: 4) is now fluorescence-labeled, a fluorescent label is only inserted if there is an unmethylated cytosine in the DNA sample to be examined.

Conversely, a control can be performed using the same sequences, but with a different set of triphosphates. Analogously to the example above, in the extension reaction 2 ', 3' -dideoxy-thy idin triphosphate (ddTTP, as type D2), 2'-deoxy-cytidine triphosphate (dCTP, as type Dl) and 2'-deoxy-adenosine triphosphate (dATP, as Type Dl) carried out, after the ligase reaction, the reverse only results in the incorporation of a label if a methylated cytosine has been present in the DNA sample to be examined.

Example 3: The following example relates to a fragment of exon 23 of the factor VIII gene in which a specific CG position is to be examined for methylation.

In the first step, the fragment is amplified by type A primers, namely by

ATTATGTTGGAGTAGTAGAGTTTAAATGGTT (SEQ-ID No .: 1) and ACTTAACACTTACTATTTAAATCACAACCCAT (SEQ-ID o .: 2). The amplified DNA is attached to an oligonucleotide of type B (for example ATGTTGGATGTTGTTGAG (SEQ-ID No .: 3)) and a 5 '-phosphorylated oligonucleotide of type C (both for example, GTATAAAGTAAATTAGAAGGAAGAT (SEQ-ID No .: 4)) hybridizes, the latter having its 3 'end bound to a solid phase surface. The extension reaction is then carried out using 2 ', 3' - dideoxycytidine triphosphate (ddCTP, as type D2), thymidine triphosphate (dTTP, as type D1) and 2'-deoxyadenosine triphosphate (dATP, as type D1). If a methylated cytosine was present, the solid phase-bound extension product ATGTTGGATGTTGTTGAGAAAC (SEQ-ID No.: 5), which has no hydroxyl function at the 3 'end, while the presence of an unmethylated cytosine in the sequence to be investigated results in the extension product ATGTTGGATGAAT -ID No .: 6) with a 3 'OH function. In the now following ligation reaction, therefore, only the solid phase-bound ligation product ATGTTGGATGTTGTTGAGAAAT GTATAAAGTAAATTAGAAGGAAGAT (SEQ ID No .: 7) can be formed. If the oligonucleotide of type B ATGTTGGATGTTGTTGAG (SEQ-ID No .: 3) is now fluorescence-marked, a fluorescent label is only inserted if there is a non-methylated cytosine in the DNA sample to be examined.

Conversely, a control can be made using the same sequences, but with a different set of

Triphosphates are carried out. Analogously to the example above, in the extension reaction 2 ', 3' -dideoxythymidine triphosphate (ddTTP, as type D2), 2'-deoxycytidine triphosphate (dCTP, as type D1) and 2'-deoxy-adenosine triphosphate (dATP, as type Dl) carried out, after the ligase reaction the reverse only results in the incorporation of a label if a methylated cytosine has been present in the DNA sample to be examined.

Claims

claims

1. A method for the detection of 5-methylcytosine in genomic DNA samples, characterized in that one carries out the following steps:

(a) a genomic DNA from a DNA sample is reacted chemically with a reagent, 5-methylcytosine and cytosine reacting differently and thus showing a different base pairing behavior in the DNA duplex after the reaction;

(b) the pretreated DNA is amplified using a polymerase and at least one oligonucleotide (type A) as a primer;

(c) a set of oligonucleotides is hybridized to the amplified genomic DNA to form a duplex, this set of oligonucleotides consisting of different species of type B and type C, and said hybridized type B oligonucleotides having their 3 'end directly or adjacent at intervals of up to 10 bases to the positions to be investigated for their methylation in the genomic DNA sample and wherein the second oligonucleotide (type C) hybridizes to a second region of the target molecule so that the 5 ' -End of the second oligonucleotide (type C) is separated by a gap the size of a single nucleotide or up to 10 nucleotides from the 3 'end of the first hybridized 0-ligonucleotide (type B) at the location of said selected position;

(d) the oligonucleotide (type B) with a known sequence of n nucleotides is extended by means of a poly erase by at most the number of nucleotides between the 3 'end of the type B oligonucleotide and the 5' end of the type C oligonucleotide, the extension depending on the methylation status of the respective cytosine in the genomic DNA sample;

(e) the oligonucleotides are incubated in the presence of a ligase, the adjoining first oligonucleotide of type B, which has been lengthened by the polymerase reaction, and the second oligonucleotide of type C being joined, and a ligation product is thereby obtained if an extension of the Type B oligonucleotide was carried out in such a way that the 3 'end with the 3'

Hydroxy function of the extended oligonucleotide immediately adjacent to the 5 'end of the type C oligonucleotide;

(f) it is detected whether a ligation product has formed.

2. The method according to claim 1, characterized in that the 5 'end of the first oligonucleotide (type B) is immobilized on a solid phase.

3. The method according to claim 1, characterized in that the 3 'end of the second oligonucleotide (type C) is immobilized on a solid phase.

A method according to claim 1, characterized in that the amplificates generated in step b are bound to a solid phase at defined locations.

5. The method according to claim 4, characterized in that at least one primer (type A) is bound to a solid phase in the amplification.

6. The method according to claim 1, 4 or 5, characterized in that different amplificates are arranged on the solid phase in the form of a rectangular or hexagonal grid.

7. The method according to any one of claims 2 to 6, characterized in that different oligonucleotide sequences are arranged on a flat solid phase in the form of a rectangular or hexagonal grid,

8. The method according to any one of claims 2 to 7, characterized in that the markings attached to the extended oligonucleotides can be identified at any position of the solid phase at which an oligonucleotide sequence is located.

9. The method according to any one of claims 2 to 8, characterized in that the solid phase surface consists of silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver or gold.

10. The method according to any one of the preceding claims, characterized in that the treatment of the DNA before amplification with a bisulfite solution (= disulfite, hydrogen sulfite) is carried out.

11. The method according to any one of the preceding claims, characterized in that the amplification is carried out by means of the polymerase chain reaction (PCR).

12. The method according to any one of the preceding claims, characterized in that the oligo- used Type A nucleotides contain only bases T, A and C or bases T, A and G.

13. The method according to any one of the preceding claims, characterized in that the oligonucleotides of type B and / or type C used either contain only bases T, A and C or bases T, A and G.

14. The method according to any one of the preceding claims, characterized in that the ligation products and / or the extension products for the detection are provided with a detectable marking.

15. The method according to any one of the preceding claims, characterized in that the markings are fluorescent markings.

16. The method according to any one of claims 1 to 14, characterized in that the markings are radionuclides.

17. The method according to any one of claims 1 to 14, characterized in that the markings are detachable mass markings which can be detected in a mass spectrometer.

18. The method according to any one of claims 1 to 14, characterized in that the extended oligonucleotides and ligation products are detectable in total in the mass spectrometer and are thus clearly marked by their mass.

19. The method according to any one of claims 1 to 14, characterized in that in each case a fragment of the extended can be detected and / or ligated oligonucleotides in the mass spectrometer.

20. The method according to claim 19, characterized in that the fragment is generated by digestion with one or more exo- or endonucleases.

21. The method according to claim 19 or 20, characterized in that for better detectability in the mass spectrometer, the fragments generated have a single positive or negative net charge.

22. The method according to any one of the preceding claims, characterized in that the detection of the extended oligonucleotides and / or the ligation products is carried out and visualized by means of matrix assisted laser desorption / ionization mass spectrometry (MALDI) or by means of electrospray mass spectrometry (ESI).

23. The method according to any one of the preceding claims, wherein the polymerases are heat-resistant DNA polymerases and / or the ligases are thermostable ligases.

24. The method according to any one of the preceding claims, wherein in addition to DNA methylation, SNPs are also detected and visualized.

25. The method according to any one of the preceding claims, wherein the nucleotides used terminating (type

D 2) and / or chain-extending nucleotides (type D 1).

26. The method according to claim 25, wherein the chain-terminating nucleotide (type D 2) is selected from a group which contains either bases T and C or bases G and A.

27. The method according to claim 25 or 26, wherein the chain-extending nucleotides (type D 1) is selected from a group which either contains the nucleobases A, T and C or the bases G and A and T.

28. The method according to claim 27, characterized in that the fluorescently labeled dCTP derivative is Cy3-dCTP or Cy5-dCTP.

29. The method according to any one of the preceding claims, characterized in that one carries out the amplification of several DNA sections in a reaction vessel.

30. The method according to claim 1, characterized in that in step a) the genomic DNA was obtained from a DNA sample, sources of DNA z. B. cell lines, blood, sputum, stool, urine, cerebrospinal fluid, paraffin-embedded tissue, histological slides and all possible combinations thereof.

31. The method according to any one of the preceding claims, characterized in that the 3 'end of the oligonucleotide of type B extended in step d is formed by the nucleotide 2' deoxyadenosine which through its 3 'hydroxy function forms the ligation in

Step e enables, whereas a 2 ', 3' dideoxy guanosine at the 3 'end of the oligonucleotide does not lead to a ligation product.

32. The method according to any one of the preceding claims, characterized in that the 3 "end of the in Step d forms an elongated oligonucleotide of type B by the nucleotide 2 '-deoxyguanosine, which enables the ligation in step e by its 3' -hydroxy function, whereas a 2 ', 3' -dideoxy-adenosine at the 3 'end of the oligonucleotide does not leads a ligation product.

33. The method according to any one of the preceding claims, characterized in that the 3 'end of the type B oligonucleotide extended in step d is formed by the nucleotide thymidine, which enables the ligation in step e by its 3' hydroxy function, whereas a 2 ', 3' -dideoxycytidine at the 3 '- end of the oligonucleotide does not lead to a ligation product.

34. The method according to any one of the preceding claims, characterized in that the 3 'end of the oligonucleotide of type B extended in step d is formed by the nucleotide 2' deoxycytidine, which enables the ligation in step e by its 3 'hydroxy function , whereas a 2 ', 3' -dideoxothymidine at the 3 'end of the oligonucleotide does not lead to a ligation product.

35. The method according to any one of the preceding claims, that one carries out methylation analyzes of the upper and lower DNA strand simultaneously.

36. The method according to claim 1, characterized in that steps c-e are carried out in solution and the ligated oligonucleotides are applied to a solid phase for the detection.