EP1534864A2

EP1534864A2 - Method for the detection of nucleic acid sequences by means of crackable probe molecules

Info

Publication number: EP1534864A2
Application number: EP03793600A
Authority: EP
Inventors: Philipp Schatz; Matthias Schuster; Kurt Berlin
Original assignee: Epigenomics AG
Current assignee: Epigenomics AG
Priority date: 2002-09-01
Filing date: 2003-09-01
Publication date: 2005-06-01
Also published as: WO2004022779A3; DE10240746A1; US20060166201A1; WO2004022779A2; AU2003266197A1

Abstract

The invention relates to a method for detecting nucleic acid sequences, which is characterized by the fact that the following steps are carried out: a) at least one nucleic acid is bound onto a solid phase; b) probe molecules which are provided with a crackable bond and a probe molecule-specific mass marker are hybridized in a sequence-specific manner to the nucleic acid; c) the probe molecules that are not hybridized are removed; d) the hybridized probe molecules are brought into contact with a matrix which cracks said crackable bonds and is used as a matrix in a MALDI mass spectrometer; e) the mass markers are detected in the positions in which the nucleic acid is bound.

Description

Method for the detection of nucleic acid sequences using cleavable probe molecules

The present invention relates to a method for the detection of nucleic acid sequences in nucleic acids.

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 can be correlated with the extent and character of the methylation of the genes or the genome.

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, in the case of PCR amplification, the epigenetic information which the 5-methylcytosines carry is completely lost.

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 üracil after subsequent alkaline hydrolysis, which corresponds to the thymidine in its base pairing behavior. 5 In contrast, met ylcytosine 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 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 (bisulfite only reacts on single-stranded DNA) was prevented and all precipitation and purification steps were replaced by rapid dialysis (Olek A, Oswald J, Walter J. A modified and identified method for bisulphite based cytosine methylation analysis. Nucleic Acids Res. 1996 DEC 15; 24 (24): 5064-6) With this method, individual cells can be placed under what the potential of the method illustrates. However, only individual regions up to approximately 3000 base pairs in length have so far been examined; a global examination of cells for thousands of possible methylation analyzes is not possible. However, even this method cannot reliably analyze very small fragments from small sample quantities. These are lost despite the diffusion protection through the matrix.

An overview of the other known options for detecting 5-methylcytosine can be found in the following overview article: Rein T, DePamphilis ML, Zorbas H. Identifying 5-methylcytosine and related modifications in DNA genomes. Nucleic Acids Res. 1998 May 15; 26 (10) .2255-64. The bisulfite technique has so far been used with a few exceptions (e.g. Zeschnigk M, Lieh C, Buiting K, Dörfler W, Horsthemke B. A single-tube PCR test for the diagnosis of Angelman and Prader-Willi syndrome based on allelic methylation differences at the SNRPN locus. Eur J Hu Genet. 1997 Mar-Apr; 5 (2): 94-8) only used in research. However, short, specific pieces of a known gene are always amplified after bisulfite treatment and either completely sequenced (Olek A, Walter J. The pre-i plantation ontogeny of the H19 methylation imprint. Nat Genet. 1997 Nov.; 17 ( 3): 275-6) or individual cytosine positions by a "primer extension reaction" (Gonzalgo ML, Jones PA. Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Res. 1997 Jun. 15; 25 (12): 2529-31, WO-A 95/00669) or an enzyme cut (Xiong Z, Laird PW. COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res. 1997 Jun 15; 25 (12): 2532-4) and detection by hybridization has also been described (Olek et al., WO-A 99/28498).

A newer method is also the detection of cytosine methylation by means of a Taqman PCR, which has become known as MethylLight (WO-A 00/70090). With this

It is possible to detect the methylation status of individual or fewer positions directly in the course of the PCR, so that a subsequent analysis of the products is unnecessary.

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, Molecular Cloning: A Laboratory Manual, 1989. Mass-labeled oligonucleotides (www.quiagengenomics.com), which are easy to prepare and do not fragment, have been used in many cases for labeling amplicons.

As mass markings e.g. Trityl groups with different masses used (Shchepinov, M.S., Southern E.M. Trityl mass-tags for encoding in combinatorial oligonucleotide synthesis (1999), Nucleic Acids Symposium Series 42: 107-108)

Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-TOF) is a very powerful development for the analysis of biomolecules (Karas M, Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons Chem. 1988 Oct. 15; 60 (20): 2299-301). An analyte is embedded in a light-absorbing matrix. The matrix is vaporized by a short laser pulse and the analyte molecule is 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. The flight time is converted into the mass of the ions.

Technical innovations in the hardware have significantly improved the method. It is worth mentioning the “laid extraction” method (DE). For DE, the acceleration voltage is switched on with a delay to the laser pulse and an improved resolution of the signals is achieved because the number of shocks is reduced. MALDI-TOF spectroscopy is excellently suited 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 backbone that is often negatively charged, the ionization process through the matrix is much more inefficient. In MALDI-TOF spectroscopy, the choice of the matrix plays an eminently important role. For the desorption of peptides, some very powerful matrices have been found 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). Coupling a "charge tag" to this modified DNA results in an increase in sensitivity by the same amount as is found for peptides. Another advantage of "charge tagging" is the increased stability of the analysis against impurities, which Completely complicate the detection of unmodified substrates. PNAs and methylphosphonate oligonucleotides have been examined with MALDI and can thus be analyzed. This technology is currently able to distinguish molecules with a mass difference of 1 Da in the mass range from 1,000 to 4,000 Da. Due to the natural distribution of isotopes, most biomolecules are already about 5 Da wide. Technically, this mass spectrometric method is ideally suited for the analysis of biomolecules. Reasonably, the products to be analyzed that are to be differentiated must be at least 5 Da apart. In this mass range, 600 molecules could be distinguished.

In addition to DNA, PNA and LNA have also been described many times as probe molecules in the literature. The PNA is a synthetic nucleic acid analogue, where the sugar-phosphate backbone is replaced by a peptide-like polyamide. Instead of 5 'and 3' ends, PNAs have N and C ends. Like the LNAs (Locked Nucleic Acids) (see www.cureon.com/technology/aboutlna), the PNAs have a high stability towards nucleases and a high binding affinity towards complementary DNA.

Described are photo-cleavable building blocks for MALDI-TOF measurements that allow light-controlled release of the samples (Olejnik et al., 1998, Nucleic Acids Res., 3572-3576). Koster et al. (WO-A 98/20166) suggested the use of cleavable bonds. For this purpose, prime oligonucleotides were immobilized, which were then hybridized with genomic DNA. After the subsequent extension reaction, the resulting products were specifically cleaved from the surface and analyzed by mass spectrometry. Again similarly, the use of photolytically cleavable oligonucleotide probes on an array has been proposed (Jaschke, A., Hausch, F.

EP1138782), a multiplex sequence-dependent modification cation of the oligonucleotide probes is carried out and the masses of the modified probes are measured directly on the array. The mixture of the target sequences is separated by the defined positions of the probes on the array.

Matrix-induced fragmentation of P3 '-N5' phosphoramidate-containing DNA is described in the literature (Shchepinov, M., Denissenko, M., 2001, Nucleic Acids Res., 3864-3872). The P-N bond can be split under acidic conditions.

task

The object of the present invention is to provide a method for the detection of nucleic acid sequences. For this purpose, probe molecules are to be hybridized in a sequence-specific manner to one or more solid phases immobilized nucleic acids. These probe molecules are to be provided with a cleavable bond and a specific mass label. The hybridized probe molecules are then to be contacted with a substance or a mixture of substances which cleaves the cleavable bonds and at the same time serves as a matrix in a MALDI mass spectrometer. The mass markings should finally be detected at the positions on the solid phase at which the nucleic acids were bound.

DESCRIPTION OF THE INVENTION A method for the detection of nucleic acid sequences is described. This procedure is characterized by the following steps:

In the first step of the method, at least one nucleic acid sample is bound to a solid phase. In the next step, the probe molecules become sequence-specific hybridizes to the nucleic acid sample, the probe molecules being provided with a cleavable bond and a mass label which is specific for the probe molecule. The non-hybridized probe molecules are then removed. In another

Step of the method, the hybridized probe molecules are contacted with a matrix which cleaves the cleavable bonds and at the same time serves as a matrix in a MALDI mass spectrometer. The mass label is detected in the last step of the procedure at the positions at which the nucleic acid sample was bound.

This method is described in detail below: A nucleic acid is preferably composed of cell lines, blood, sputum, stool, urine, brain-spinal cord fluid, tissue embedded in paraffin, for example tissue from the eyes, intestine, kidney, brain, heart, prostate, lung , Breast or liver, histological slides or any combination of these.

The nucleic acid is amplified, this preferably being done by means of enzymatic pri extension, PCR, rolling circle amplification, ligase chain reaction or other methods.

In a particularly preferred variant of the method, the amplification of several different fragments is carried out in one reaction vessel.

At least one nucleic acid is bound to a solid phase, which can preferably also serve as a sample carrier for a mass spectrometer. The solid phase or solid phase surface particularly preferably consists of non-conductive materials such as glass. Also particularly preferred are conductive materials such as such as Teflon, Silicon or Black Conductive Polypropylene. Other materials such as polystyrene, aluminum, steel, iron, copper, nickel, silver or gold are also preferred.

According to the invention, the binding of the nucleic acid to the surface can be both covalent (for example by a primer which carries a thiol at the 5 'end and is bound to a surface activated with bromoacetic acid) and non-covalently by van der Waals forces or hydrogen bonds (for example by heat mobilization or incubation).

A plurality of nucleic acids are preferably arranged on a solid phase surface in the form of a rectangular or hexagonal grid. Alternatively, it is preferred that at least one nucleic acid is arranged on a plurality of surfaces. These solid phases can particularly preferably be used in the sample holder of a MALDI mass spectrometer.

The nucleic acids to be detected are preferably DNA sequences and, in particular, sequences which can be changed between different samples and which contain SNPs, point mutations, deletions, inversions or insertions. The nucleic acid sequences to be detected are particularly preferably chemically pretreated DNA sequences which serve for the detection of DNA methylation at certain CpG positions.

The chemical treatment is particularly preferably carried out with a bisulfite (= disulfite, hydrogen sulfite). All cytosines that are not in the context of CpG are converted into thymidines, which enables the investigation of individual CpG positions. 1 U

The chemical treatment is preferably carried out after embedding the DNA in agarose. It is likewise preferred that a reagent denaturing the DNA duplex and / or a radical scavenger is present during the chemical treatment.

The probe molecules required for binding to nucleic acids to be detected are often produced combinatorially in the prior art in the form of libraries (EP1036202), which are also preferably used in the method according to the invention.

The probe molecules are particularly preferably provided with a cleavable bond and a mass marking which is specific for the respective probe molecule. Such markings can be, for example, 6-

Triethylammoniumhexyryl-, 6-Trimethylammoniumhexyryl- or the acid labile monomethoxytrityl or 4-methyltrityl protective groups.

The mass of a marking preferably differs from the mass of all other markings used in an experiment by at least 1 Da. The probe molecules preferably comprise at least one CG, TG or CA dinucleotide.

The different mass can particularly preferably also be the result of an enzymatic reaction. Here, the probe is synthesized with a mass label and then modified enzymatically, whereby the mass changes.

The mass marking is particularly preferably only connected to the probe molecule as a result of an enzymatic modification. In this case, the probe is manufactured chemically without mass marking and then enzymatically provided with a mass marking. The probe is preferably chemically provided with a mass label and not changed enzymatically. In addition, it is preferred according to the invention that the probe provided with a mass marking is changed chemically. This can be done for example with acid or with a treatment according to Maxam and Gilbert.

These probe molecules, which particularly preferably consist of DNA or modified DNA, are hybridized to the nucleic acids in a sequence-specific manner. The probe molecules RNA, LNA, PNA or corresponding hybrids thereof are preferably also combined with DNA or modified DNA. The non-hybridized probe molecules are removed. The remaining hybridized probe molecules are preferably modified enzymatically after the hybridization by primer extension or ligation. For example, thermosquenase or Taq poly erase are suitable as enzymes for the primer extension, whereas ampligase DNA ligase, Pfu or Taq DNA ligase are used for the ligation.

The amplification products are preferably hybridized to two classes of probe molecules, each with at least one member, the probe molecules of the first class preferably hybridizing to the sequence which results from the chemical treatment of the genomic DNA when a cytosine to be examined is methylated in the genomic DNA and wherein the probe molecules of the second class preferentially hybridize to the sequence which results from the chemical treatment of the genomic DNA if a cytosine to be examined were present unmethylated in the genomic DNA.

Hybridization to two classes of probe molecules, each with at least one member, is preferred, the probe molecules of the first class preferably hybridizing to the sequence which results from the chemical treatment of the genomic DNA if a cytosine to be examined is methylated in the genomic DNA and less preferably to the sequence which results from the chemical treatment of the genomic DNA, if a cytosine to be examined is unmethylated in the genomic DNA and the oligomers of the second class hybridize to the amplificate to be examined essentially independently of the methylation of said particular cytosine in the genomic DNA.

Hybridization is preferably carried out on two classes of probe molecules, each with at least one member, the probe molecules of the first class preferably hybridizing to the sequence that results from the chemical treatment of the genomic DNA if a cytosine to be examined was present in the genomic DNA unmethylated and less preferably to the sequence which results from the chemical treatment of the genomic DNA, if a cytosine to be investigated were methylated in the genomic DNA and the oligomers of the second class of the amplificate to be examined are essentially independent of the methylation of the particular cytosine in question hybridize the genomic DNA.

The non-hybridized probe molecules are then removed.

The hybridized probe molecules are contacted with a matrix (eg by spraying, pipetting, spotting), which cleaves the cleavable bonds and at the same time serves as a matrix in a MALDI mass spectrometer. For example, a 2 ', 4', 6 'trihydroxyacetophenone (THA) matrix or 3-HPA (3-hydroxypicolinic acid) matrix can be used, the THA matrix according to the invention being diluted with ter acid such as TFA (trifluoroacetic acid) is added. The detection limit can be significantly reduced by the size of the oligonucleotide gap product. Examples of protective groups which can be removed from acid are monomethoxytrityl or 4-methyltrityl.

The oligonucleotides can also be cleaved in a structure-specific manner by adding a flap endonuclease such as Cleavage VIII. Endonucleases can also be used for cleavage, which cleave the probe from the center, for example mung bean nuclease or T7 endonuclease I. Furthermore, sequence-specific endonucleases can be used for cleavage, for example the enzymes Tsp 5091 or Msel can be used for this , In addition, the digestion with a 3'-

Endonuclease possible, preferably after adding acidic matrix. Exonucleases are also used for cleavage. A 3 '-5' exonuclease such as exonuclease I cleaves the single-stranded probe from the 3 'end to a modification (e.g. phosphothioate). A 5 '-3' exonuclease such as T7 exonuclease accordingly cleaves the single-stranded probe from the 5 'end until a modification.

The mass labels are detected at the positions at which the nucleic acid was bound. This detection is particularly preferably carried out using MALDI-TOF mass spectrometry. The detection limit is decisively reduced by the preferred loading of the mass marking of simply positive or simply negative.

The method described above is preferably used for the diagnosis and / or prognosis of adverse events for patients or individuals, these adverse events being at least one of the following Include categories: adverse drug effects; Cancers; CNS malfunction, damage or illness; Symptoms of aggression or behavioral disorders; clinical, psychological and social consequences of brain damage; psychotic disorders and personality disorders; Dementia and / or associated syndromes; cardiovascular disease, malfunction and damage; Malfunction, damage or disease of the gastrointestinal tract; Malfunction, damage or disease of the respiratory system; Injury, inflammation, infection, immunity and / or convalescence; Malfunction, damage or illness of the body as a deviation in the development process; Malfunction, damage or disease of the skin, muscles, connective tissue or bones; endocrine and etabolic malfunction, damage or disease; Headache or sexual malfunction.

The method described above is preferably used to distinguish cell types or tissues or to study cell differentiation.

Also preferred is a kit comprising a solid phase for immobilizing the nucleic acid, probe molecules, components for carrying out the mass spectrometric

Measurement and instructions for performing the procedure.

The following examples illustrate the process according to the invention:

Performing the methylation analysis in the MDRI gene using a cleavable probe

Example: Linking the target sequence to the solid phase In the first step, a genomic sequence is treated using bisulfite (hydrogen sulfite, disulfite) in such a way that all cytosines not methylated at the 5-position of the base are changed in such a way that a base which is different with regard to the base pairing behavior is formed, while the in the 5-position methylated cytosines remain unchanged. If bisulfite is used for the reaction, an addition takes place at the unmethylated cytosine bases. In addition, a denaturing reagent or solvent and a radical scavenger must be present. Subsequent alkaline hydrolysis then leads to the conversion of unmethylated cytosine nucleobases into uracil. This converted DNA is used to detect methylated cytosines. In the second process step, the treated nucleic acid is diluted with water or an aqueous solution. Desulfonation of the DNA is then preferably carried out. In the third step of the method, the nucleic acid is amplified in a polymerase chain reaction, preferably with a heat-resistant DNA polymerase. The PCR reactions were carried out in a thermal cycler (Eppendorf GmbH). 40 ng DNA, 0.07 μmol / l of each primer oligonucleotide 1 mmol / 1 dNTPs and four units of Hotstar Taq were used for a 100 μl mixture. The other conditions were chosen according to the manufacturer's instructions. For the PCR, denaturation was first carried out for 15 minutes at 96 ° C., then 40 cycles (60 seconds at 96 ° C., 75 seconds at 56 ° C. and 75 seconds at 65 ° C.) and a final EL water or an aqueous solution , Desulfonation of the DNA is then preferably carried out. In the third step of the method, the nucleic acids are amplified in a polymerase chain reaction, preferably with a heat-resistant DNA polymerase. The PCR reactions were carried out in a Thermocyc-1er (Eppendorf GmbH). It was for one

100 μl batch 40 ng DNA, 0.07 μmol / l from each primeroli gonucleotide 1 mM dNTPs and four units of HotstarTaq were used. The other conditions were chosen according to the manufacturer's instructions. For the PCR, denaturation was first carried out for 15 minutes at 96 ° C, then 40 cycles (60 seconds at 96 ° C, 75 seconds at 56 ° C and

75 seconds at 65 ° C) and a final elongation of 10 minutes at 72 ° C. The presence of the PCR products was checked on agarose gels. One of the two primer oligonucleotides was modified at its 5 'end thiol (in the following example 8 instead 5' end phosphate).

In the present case, cytosines from the potential promoter region of the MDRI gene are examined. With sequences of this gene, a patient's response to a

Chemotherapy can be followed. For this purpose, a defined fragment with a length of 242 bp is amplified with the specific primer oligonucleotides SH-TAA GTA TGT TGA AGA AAG ATT ATT GTA G (Seq. ID 1.) and CGC ATC AAC TAA ATC ATT AAA A (Seq. ID 2.). With its thiol modification, this amplificate is bound to a polylysine solid phase treated with bromoacetic acid. Polylysine-coated glass slides are cleaned beforehand in ultrasound and activated for 1 h in 20 mmol / 1 bromoacetic acid, 20 mmol / 1 dicylohexylcarbodiimide, 2 mmol / 1 4- (dimethylamino) pyridine solution. Binding with the PCR product takes place in a 0.18 mol / 1 triscarboxyethylphosphine, 200 mmol / 1 NaH2P04 buffer solution for 2 hours in a 25 ° C humid chamber. The PCR products are denatured with 0.05 mol / 1 NaOH and then analyzed.

Example 1:

The single-stranded PCR product was hybridized with an oligonucleotide to form a duplex structure. Acid-labile modified oligonucleotides were used for this: 5 '-TAT AAA CAC GTC TTT CApnA-Amino-3 ^λ (Seq. ID 3.) or 5 '-TAT AAA CAC ATC TTT CapnA-Amino-3 ^Λ (Seq. ID 4.) with the cytosine to be detected at position 198 of the amplificate. The adenosine at the penultimate position of both oligonucleotides is a 5 'amino adenosine which is easily hydrolyzed by acid. A 6-triethylammonium hexyryl-N-hydroxysuccinimidyl ester (199 Da) (CT1) or a 6-trimethylammonium hexyryl-N-hydroxysuccinimidyl ester (129 Da) (CT2) is coupled to the amino function at the 3 'end beforehand. This means that the masses of the two smaller fission products differ by 70 Da. The methylated cytosine is detected with the oligonucleotide (Seq. ID 3.), whereas the unmethylated state, which is represented by a thymine, is detected with the oligonucleotide (Seq. ID 4.). Both oligonucleotides hybridize on the complementary strand. The acid-labile cleavage site is hydrolyzed by applying 350 mmol / l of 3-HPA in acetonitrile with 1.5% trifluoroacetic acid. The detection of the hybridization product is based on the detection of the mass of the cleavage products using MALDI-TOF mass spectrometry. The detection limit is decisively reduced by the size of the oligonucleotide cleavage product and the defined charge of simply positive. A hybridization reaction of the amplified DNA with the probe (Seq. ID3., Seq. ID 4.) occurs only if a methylated cytosine is present at this point in the bisulfit-treated DNA. The methylation status of the respective cytosine to be examined thus decides on the hybridization product and thus on the mass detected.

By installing the PN binding directly at the 3 ^Λ end, you also have the advantage of being able to use an unloaded mass marker. For example, a peptide can then be coupled.

Example 2 The single-stranded PCR product was hybridized with an oligonucleotide to form a duplex structure. Modified oligonucleotides were used for this: 5'Amino-TAT AAA CAC GTC TTT CAA (Seq. ID 5.) or

5'Amino-TAT AAA CAC ATC TTT CAA (Seq. ID 6.) A 6-triethylammonium hexyryl-N-hydroxysuccinimidyl ester (199 Da) (CTI) or a 6- is added to the amino function at the 5 'end. Trimethylammoniumhexyryl-N-hydroxysuccinimidyl ester (129 Da) (CT2) coupled. This means that the masses of the two smaller fission products differ by 70 Da. The methylated cytosine is detected with the oligonucleotide (Seq. ID 5.), whereas the unmethylated state, which is represented by a Thy in, is detected with the oligonucleotide (Seq. ID 6.). Both oligonucleotides hybridize on the complementary strand. The oligonucleotides are then subjected to Maxam and Gilbert treatment. By applying dimethyl sulfate and heating in an alkaline pH, all adenosines and guanosines are split. The detection of the hybridization product is based on the detection of the mass of the cleavage products using MALDI-TOF mass spectrometry. In this case, a mass of (498 Da + 199 Da (CTl + dT), or + 129 Da (CT2 + dT)) 697 or 627 Da is observed. The detection limit is significantly reduced by the size of the oligonucleotide cleavage product. A hybridization reaction of the amplified DNA with the probe (Seq. ID 5., Seq. ID 6.) occurs only if a methylated cytosine has been present at this point in the bisulfite-treated DNA. The methylation status of the respective cytosine to be examined thus decides on the hybridization product and thus on the mass detected. Splits can be ridin the cytosine and thymidine with hydrazine and pipe- then a 3 ^Λ -modified Oligonukleo- be examined tidpärchen. Would result as fission products in this case dAdA-CTl and dAdA-CT2. However, all possible fission products are loaded several times.

Example 3: The single-stranded PCR product was hybridized with an oligonucleotide to form a duplex structure. Modified oligonucleotides were used for this: 5-Amino-TAT AAA CAC GTC TTT CAA (Seq. ID 7th) or 5 ^λ - Amino-5 ^Λ -TAT AAA CAC ATC TTT CAA (Seq. ID 8th) To the amino function At the 5 'end, an acid-releasable protective group such as 4-methyltrityl (258 Da) or monomethoxytrityl (289 Da) is coupled beforehand. As a result, the masses of the two smaller fission products differ by 14 Da. The methylated cytosine is detected with the oligonucleotide (Seq. ID 7.), whereas the unmethylated state, which is represented by a thymine, is detected with the oligonucleotide (Seq. ID 8.). Both oligonucleotides hybridize on the complementary strand. The oligonucleotides are covered with an acidic 2 \ 4 \ 6 trihydroxyacetophenone matrix and measured in the MALDI-TOF. The oligonucleotides are separated from their mass labels. The detection of the hybridization product is based on the detection of the mass of the protective group using MALDI-TOF mass spectrometry. The detection limit is decisively reduced by the size of the protective group and the defined charge of +1. A hybridization reaction of the amplified DNA with the probe only occurs if there is a methylated cytosine in the bisulfite-treated DNA at this point (Seq. ID 7th, Seq. ID 8th). The methylation status of the respective cytosine to be examined thus decides on the hybridization product and thus on the mass detected.

Example 4 The single-stranded PCR product was hybridized with an oligonucleotide to form a duplex structure. Modified oligonucleotides were used for this: 5 5 -AminoTmptAT AAA CAC GTC TTT CAA-3 (Seq. ID 9.) or 5 ^Λ -AminoTmptAT AAA CAC ATC TTT CAA-3 (Seq. ID 10.). The mpt is a methyl phosphonate. A 6-triethylammonium hexyryl-N-hydroxysuccinimidyl ester (199 Da) (CT1), or a 6-

10 Trimethylammoniumhexyryl-N-hydroxysuccinimidyl ester (129 Da) (CT2) coupled. This means that the masses of the two smaller fission products differ by 70 Da. The methylated cytosine is detected with the oligonucleotide (Seq. ID 9.), whereas the unmethylated form,

15. which is represented by a thymine with which the oligonucleotide (Seq. ID 10.) is detected. Both oligonucleotides hybridize on the complementary strand. The oligonucleotides are digested with a 3 - endoglycosidase. Evidence of hybridization

The product is based on the detection of the mass of the remaining dT and the charge tag using MALDI-TOF mass spectrometry. The detection limit is decisively reduced by the size of the mass marking and the defined charge of -1. Only if in the bisulfite

25 treated DNA has a methylated cytosine at this point, there is a hybridization reaction of the amplified DNA with the probe (Seq. ID 9th, Seq. ID 10th). The methylation status of the respective cytosine to be examined thus decides on the hybridization

30. product and thus the detected mass. You can also put the methyl phosphonate all the way to the 3 ^Λ end of the oligonucleotide and then you get a single negatively charged molecule even without a charge tag.

35 Example 5: The single-stranded PCR product was hybridized with two consecutive oligonucleotides to form a duplex structure. Modified oligonucleotides were used for this: δ'-TTC AAC TTA TAT AAA CAmtpC-3 * (Seq. ID 11.) and 5 ^Λ TmtpTC TTT CAA AAT TCA CAT-3 (Seq. ID 12.) or 5 GmtpTC TTT CAA AAT TCA CAT-3 (Seq. ID 13.) The abbreviation mtp stands for methylphosphonate. The methylated cytosine is by the ligation of Seq. ID 11th and Seq. ID 12. detected, whereas the unmethylated state, which is represented by a T ymin, by the ligation of Seq. ID 11 and Seq. ID 13 is detected. Both oligonucleotides hybridize on the complementary strand. The oligonucleotides are digested by 3 - endoglycosidase and 5 endoglycosidase. The detection of the ligation product is based on the detection of the mass of the remaining nucleotides (AmtpCp-T tpC or AmtpCp-TmtpC) using MALDI-TOF mass spectrometry. The detection limit is significantly reduced by the size of the products and the defined charge of simply negative. The mass can be shifted further by additional methylphosphonates.

Example 6: The single-stranded PCR product was hybridized with two consecutive oligonucleotides to form a duplex structure. Modified oligonucleotides were used for this: 5 -TTC AAC TTA TAT AAA CApnC-3 (Seq. ID 14.) and 5 ^Λ ATmtpC TTT CAA AAT TCA CAT-3 ^Λ (Seq. ID 15.) or

5 ^λ GmtpTC TTT CAA AAT TCA CAT -3 ^Λ (Seq. ID 16.) The abbreviation mtp stands for methylphosphonate. The methylated cytosine is by the ligation of Seq. ID 14. and Seq. ID 15. detected, whereas the unmethylated state, which is represented by a thymine, was identified by the ligation of Seq. ID 14 and Seq. ID 15 is proven. Both oligonucleotides hybridize on the complementary strand. The oligonucleotides are digested by adding acidic 3-HPA (3-hydroxypicolinic acid) matrix with 0.3% TFA (trifluoroacetic acid) and digestion with a 3 ^Λ - endoglycosidase. The detection of the ligation product is based on the detection of the mass of the remaining nucleotides (NH3 + -Cp-Ap-TmtpC or NH3 + -Cp-Gp-TmtpC) using MALDI-TOF mass spectrometry. The detection limit is significantly reduced by the size of the products and the defined charge of simply negative. Instead of the methyl phosphonate, an NP (nitrogen-phosphorus) bond, ie a 3 'amino guaonside or 3' amino thymidine, can also be used in the second oligonucleotide at the second position at the 5 'end. An NH3 + -Cp- ANH3 + is formed.

Example 7:

The single-stranded PCR product was hybridized with two consecutive oligonucleotides to form a duplex structure. The two oligonucleotides overlap. Modified oligonucleotides were used for this:

5 ^Λ -TTC AAC TTA TAT AAA CAC-3 ^λ (Seq. ID 17.) and 5 ^Λ Amino- CATC TTT CAA AAT TCA CAT-3 ^Λ (Seq. ID 18.) or 5 ^Λ Amino- CGTC TTT CAA AAT TCA CAT-3 (Seq. ID 19.). Before the amino function at the 5 'end, a 6-

Triethylammoniumhexyryl-N-hydroxysuccinimidyl ester (199 Da) (CT1), or a 6-trimethylammonium hexanoic acid-N-hydroxysuccinimidyl ester (129 Da) (CT2). The methylated cytosine is by the ligation of Seq. ID 17th and Seq. ID 18. detected, whereas the unmethylated form, which is represented by a thymine, by the ligation of Seq. ID 18 and Seq. ID 19 is proven. Both oligonucleotides hybridize on the complementary strand. The oligonucleotides are added by adding a flap endonuclease (Clevase VIII Endonuclease) cleaved. The detection of the ligation product is based on the detection of the mass of the remaining nucleotides (CT1 + dC or CT2 + dC) using MALDI-TOF mass spectrometry. The detection limit is significantly reduced by the size of the products and the defined charge of simply negative.

Example 8:

Here a defined fragment with a length of 242 bp was amplified with the specific primer oligonucleotides TAA GTA TGT TGA AGA AAG ATT ATT GTA G and phosphate-CGC ATC AAC TAA ATC ATT AAA A. This amplificate was digested with lambda exonuclease, so that the 5 "phosphate-modified strand was digested and only ssDNA (ss = single stranded) was left. This was now attached to an oligonucleotide with the sequence 5-phosphate-TC TTT CAA AAT TCA CAT-Amino-3 ^Λ (Seq. ID 20) hybridized to form a duplex structure, this oligonucleotide is bound to an activated surface via its 3 'amino modification, then a ligation mix was added, the ligase, ligase buffer and one of the following two 0-ligonucleotides contained: 5 '-DMT-ACT-3' (Seq. ID 21) or 5 -MMT-ACG-3 "(Seq. ID 22) (DMT = Dimethyltrityl, MMT = Monomethyltrityl)" The methylated cytosine was by the Seq. ID 20 and Seq. ID 21 verified. The trityl group was split off by adding acidic matrix and its mass was analyzed in the mass spectrometer. In the case of an unmethylated sample, a T was inserted instead of a C during the bisulfite reaction, sequence ID 22 was ligated and detected by its MMT group. By using additional trityl groups, all possible CpG positions in the described ligase reaction can be examined.

Claims

claims

Method for the detection of nucleic acid sequences, characterized in that the following steps are carried out:

a) at least one nucleic acid is bound on a solid phase;

b) probe molecules are hybridized to the nucleic acid in a sequence-specific manner, the probe molecules being provided with a cleavable bond and a mass label which is specific for the probe molecule;

c) removal of the non-hybridized probe molecules;

d) contacting the hybridized probe molecules with a matrix which cleaves said cleavable bonds and at the same time serves as a matrix in a MALDI mass spectrometer;

e) detection of the mass marking at the positions at which the nucleic acid was bound.

2. The method according to claim 1, characterized in that the nucleic acid sequences to be detected are DNA sequences and in particular sequences which can be changed between different nucleic acids and which contain SNPs, point mutations, deletions, inversions or insertions.

3. The method according to claim 1, characterized in that the nucleic acid sequences to be detected are are DNA sequences which have been pretreated in a mixed manner, and in particular sequences which have been treated with bisulfite and which are used to detect DNA methylation at specific CpG positions.

4. The method according to any one of claims 1 to 3, characterized in that the nucleic acid is amplified before binding to the solid phase, which can preferably be done by means of enzymatic primer extension, PCR, rolling circle amplification, ligase chain reaction or other methods.

5. The method according to any one of the preceding claims, characterized in that the solid phase is the sample carrier of a mass spectrometer.

6. The method according to claim 5, characterized in that the solid phase surface consists of silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver or gold.

7. The method according to any one of claims 1 to 4, characterized in that the solid phase can be used in the sample holder of a MALDI mass spectrometer.

8. The method according to any one of the preceding claims, characterized in that several nucleic acids are arranged on the solid phase surface in the form of a rectangular or hexagonal grid.

9. The method according to any one of the preceding claims, characterized in that the probe molecules are DNA or modified DNA.

10. The method according to any one of the preceding claims, characterized in that the probe molecules are LNA, PNA or corresponding hybrids thereof, also with DNA or modified DNA.

11. The method according to claim 9, characterized in that the probe molecules are modified enzymatically after the hybridization by primer extension.

12. The method according to claim 9, characterized in that the probe molecules are modified enzymatically by ligation after the hybridization.

13. The method according to any one of claims 11 or 12, characterized in that the mass marking is only connected to the probe molecule as a result of the enzymatic modification.

14. The method according to any one of the preceding claims, characterized in that the mass marking carries a single positive or a single negative charge.

15. The method according to one of the preceding claims, characterized in that the masses of a marking differ from the masses of all other markings used in an experiment by at least 1 Da.

16. The method according to claim 3, characterized in that the probe molecules comprise at least one CG, TG or CA dinucleotide.

17. The method according to any one of claims 3 or 16, characterized in that in step b) a hybridization of the amplificates on two classes of probe mole- rinsing with at least one member in each case, the probe molecules of the first class preferably hybridizing to the sequence which results from the chemical treatment of the genomic DNA if a cytosine to be investigated was methylated in the genomic DNA and the probe molecules of the second class are preferably present hybridize the sequence which results from the chemical treatment of the genomic DNA if a cytosine to be examined were present unmethylated in the genomic DNA.

18. The method according to any one of claims 3 or 16, characterized in that in step b) there is hybridization to two classes of probe molecules, each with at least one member, the probe molecules of the first class preferably hybridizing to the sequence which results from the chemical treatment of the genomic DNA arises when a cytosine to be examined is methylated in the genomic DNA and less preferably to the sequence resulting from the chemical treatment of the genomic DNA when a cytosine to be examined is unmethylated in the genomic DNA and the oligomers of the second Hybridize class to the amplificate to be examined essentially independently of the methylation of said particular cytosine in the genomic DNA.

19. The method according to any one of claims 3 or 16, characterized in that in step b) there is hybridization to two classes of probe molecules with at least one member each, the probe molecules of the first class preferably hybridizing to the sequence which results from the chemical treatment of the genomic DNA emerges when a cytosine to be examined unmethylates in the genomic DNA and less preferred to the sequence which arises from the chemical treatment of the genomic DNA if a cytosine to be examined is methylated in the genomic DNA and the oligomers of the second class are linked to the one to be examined

The amplificate hybridizes essentially independently of the methylation of said particular cytosine in the genomic DNA.

20. The method according to any one of the preceding claims, wherein the nucleic acid from cell lines, blood, sputum, stool, urine, brain spinal cord fluid, tissue embedded in paraffin, for example tissue from the eyes, intestine, kidney, brain, heart, prostate, lung , Breast or liver, histological slides, or all possible combinations thereof.

21. Use of a method according to one of the preceding claims for the diagnosis and / or prognosis of adverse events for patients or individuals, wherein these adverse events belong to at least one of the following categories: adverse drug effects; Cancers; CNS malfunction, damage or illness; Symptoms of aggression or behavioral disorders; clinical, psychological and social consequences of brain damage; psychotic disorders and personality disorders; Dementia and / or associated syndromes; cardiovascular disease, malfunction and damage; Malfunction, damage or disease of the gastrointestinal tract; Malfunction, damage or disease of the respiratory system; Injury, inflammation, infection, immunity and / or convalescence; Malfunction, damage or illness of the body as a deviation in the development process; Malfunction

Damage or disease to the skin, muscles, y

Connective tissue or the bone; endocrine and metabolic malfunction, damage or illness; Headache or sexual malfunction.

22. Use of a method according to one of the preceding claims for differentiating cell types or tissues or for investigating cell differentiation.

23. Kit comprising a solid phase for immobilizing the nucleic acid, probe molecules and components for carrying out the mass spectrometric measurement and instructions for carrying out a method according to one of the preceding claims.