Method for determining the degree of methylation of defined cytosines in genomic dna in the sequence context 5'-cpg-3'

Abstract

A method is described for the detection of the degree of methylation of a specific cytosine in the sequence context 5′-CpG-3′ of a genomic DNA sample. In the first step, the genomic DNA is chemically treated in such a way that the cytosine bases are converted to uracil, but not the 5-methylcytosine bases. Then segments of the genomic DNA which contain the said specific cytosine are amplified, whereby the amplified products are given a detectable label and in the following steps the extent of hybridization of the amplified products on two classes of oligonucleotides is determined by detection of the label of the amplified products, and a conclusion is made on the extent of methylation of said specific cytosine in the genomic DNA sample from the ratio of the labels detected on the two classes of oligonucleotides as a consequence of the hybridization.

1 . A method for the detection of the degree of methylation of a specific cytosine in the sequence context 5′-CpG-3′ of a genomic DNA sample, is hereby characterized in that 
 a) the genomic DNA is chemically treated, whereby the cytosine bases are converted to uracil, but not the 5-methylcytosine bases;  
 b) segments of the genomic DNA, which contain said specific cytosine, are amplified, whereby the amplified products are given a detectable label;  
 c) the amplified products are hybridized to two classes of oligonucleotides and/or PNA oligomers, each of which class has at least one member;  
 d) the extent of hybridization of the amplified products to the two classes of oligonucleotides and/or PNA oligomers is determined by detection of the label of the amplified products;  
 e) a conclusion is made on the extent of methylation of said specific cytosine in the genomic DNA sample from the ratio of the labels detected for the two classes of oligonucleotides and/or PNA oligomers as a consequence of the hybridization.  
 
     
     
         2 . The method according to  claim 1 , further characterized in that a hybridization of the amplified products is conducted in step c) on two classes of oligomers (oligonucleotides and/or PNA oligomers), each of which class has at least one member, whereby the oligomers of the first class preferably hybridize to the sequence which arises after the chemical treatment of the genomic DNA, if said specific cytosine was present in the methylated state in the genomic DNA and whereby the oligomers of the second class preferably hybridize to the sequence which arises after the chemical treatment of the genomic DNA, if said specific cytosine was present in the unmethylated state in the genomic DNA.  
     
     
         3 . The method according to  claim 1 , further characterized in that a hybridization of the amplified products is conducted in step c) on two classes of oligomers (oligonucleotides and/or PNA oligomers), each of which class has at least one member, whereby the oligomers of the first class preferably hybridize to the sequence which arises after the chemical treatment of the genomic DNA, if said specific cytosine was present in the methylated state in the genomic DNA and less preferably hybridize to the sequence which arises after the chemical treatment of the genomic DNA, if said specific cytosine was present in the unmethylated state in the genomic DNA, and whereby the oligomers of the second class hybridize to the amplified product to be investigated essentially independently of the degree of methylation of said specific cytosine in the genomic DNA.  
     
     
         4 . The method according to  claim 1 , further characterized in that a hybridization of the amplified products is conducted in step c) on two classes of oligomers (oligonucleotides and/or PNA oligomers), each of which class has at least one member, whereby the oligomers of the first class preferably hybridize to the sequence which arises after the chemical treatment of the genomic DNA, if said specific cytosine was present in the unmethylated state in the genomic DNA and less preferably hybridize to the sequence which arises after the chemical treatment of the genomic DNA, if said specific cytosine was present in the methylated state in the genomic DNA, and whereby the oligomers of the second class hybridize to the amplified product to be investigated essentially independently of the degree of methylation of said specific cytosine in the genomic DNA.  
     
     
         5 . The method according to  claim 1 , further characterized in that the method is conducted not only with the genomic DNA sample, but also logically with standard DNA in which it is known whether the cytosine at said specific position is present in methylated or unmethylated state, whereby the ratios of the labels detected on the two classes of oligonucleotides, which are measured each time with the unmethylated standard DNA according to  claim 1 , serve as a calibration value for a degree of methylation of 0, and correspondingly, the ratios of the labels detected on the two classes of oligonucleotides, which are measured each time with the methylated standard DNA according to  claim 1 , serve as a calibration value for a degree of methylation of 1, and these calibration values are used for the determination of the degree of methylation of the genomic DNA sample.  
     
     
         6 . The method according to  claim 5 , further characterized in that additional known standard DNA samples, each of which has any known degree of methylation of said specific cytosine, are used for calibration.  
     
     
         7 . The method according to  claim 5 , further characterized in that the DNAs used as the standard are each labeled differently and the amplified product from the genomic DNA is provided in turn with a label that is different therefrom.  
     
     
         8 . The method according to  claim 1 , further characterized in that amplified products that are derived from different genomic DNA samples are provided with different labels.  
     
     
         9 . The method according to  claim 1 , further characterized in that amplified products originating from the same genomic DNA samples are provided with different labels in order to achieve an increase of measurement accuracy by an averaging of the values obtained from different detection methods.  
     
     
         10 . The method according to  claim 1 , further characterized in that said labels are fluorescent labels.  
     
     
         11 . The method according to  claim 1 , further characterized in that the chemical treatment is conducted with a solution of a bisulfite (=hydrogen sulfite, disulfite).  
     
     
         12 . The method according to  claim 1 , further characterized in that oligonucleotides are used for the amplification, which comprise a sequence segment of a chemically pretreated DNA which is at least 18 bases long of one of the sequences Seq. ID 1 to Seq. ID 40712.  
     
     
         13 . The method according to  claim 1 , further characterized in that in a hybridization step, oligonucleotides and/or peptide nucleic acid (PNA) oligomers are used, which hybridize to a sequence segment that is at least 9 bases long of a chemically pretreated DNA according to one of the sequences Seq. ID 1 to Seq. ID 40712 or correspond to this sequence, whereby the base sequence contains at least one CpG dinucleotide and the CpG dinucleotide is found in approximately the middle third of the oligomer.  
     
     
         14 . The method according to  claim 1 , further characterized in that said label is detected by its chemiluminescence, its UV absorption or fluorescence polarization.  
     
     
         15 . The method according to  claim 1 , further characterized in that the labels are radionuclides.  
     
     
         16 . The method according to  claim 1 , further characterized in that the labels are removable mass labels, which are detected in a mass spectrometer.  
     
     
         17 . The method according to  claim 1 , further characterized in that the PCR products as a whole or their characteristic fragments are detected in the mass spectrometer and thus are clearly characterized by their mass.  
     
     
         18 . The method according to  claim 1 , further characterized in that the oligomers (oligonucleotides and/or PNA oligomers) of one class contain the sequence 5′-CG-3′.  
     
     
         19 . The method according to  claim 1 , further characterized in that the oligomers (oligonucleotides and/or PNA oligomers) of one class contain the sequence 5′-TG-3′ and/or the sequence 5′-CA-3′.  
     
     
         20 . The method according to claims  18  or  19 , further characterized in that the oligomers (oligonucleotides and/or PNA oligomers) of the first class contain the sequence 5′-CG-3′ and the oligomers of the second class contain the sequence 5′-TG-3′ and/or the sequence 5′-CA-3′.  
     
     
         21 . The method according to  claim 1 , further characterized in that the oligomers of the first and the second classes are immobilized on a common solid phase.  
     
     
         22 . The method according to  claim 11 , further characterized in that the oligonucleotides are arranged on a planar solid phase in a rectangular or hexagonal grid and the site of specific oligonucleotides on the solid phase is correlated with their respective sequence.  
     
     
         23 . The method according to  claim 1 , further characterized in that the oligomers of the first and second classes are immobilized on beads, which are coded with a set of separately detectable labels.  
     
     
         24 . The method according to  claim 1 , further characterized in that step b) is conducted in two sub-steps as follows: 
 a) a PCR pre-amplification with at least one pair of primers of different sequence which hybridize nonspecifically to a DNA sample pretreated according to  claim 1  and thus produce more than one amplified product in the PCR step;    b) a PCR amplification of the product formed in the pre-amplification, with primers of different sequence, which are each identical or inversely complementary to a segment of the DNA sample ((+) strand or (−) strand) that has been pretreated according to  claim 1 , and hybridize specifically to the DNA to be amplified.    
     
     
         25 . The method according to  claim 1 , further characterized in that the amplification of several DNA segments is conducted in one reaction vessel.  
     
     
         26 . The method according to  claim 1 , further characterized in that a heat-stable DNA polymerase is used for the amplification.  
     
     
         27 . The method according to  claim 1 , further characterized in that the primer oligonucleotides used for the amplification contain either only the bases T, A and C or the bases T, A and G.  
     
     
         28 . The method according to  claim 1 , further characterized in that at least 10 CpG positions in different sequence context are analyzed simultaneously.  
     
     
         29 . The method according to  claim 1 , further characterized in that at least 50 CpG positions in different sequence context are analyzed simultaneously.  
     
     
         30 . The method according to  claim 1 , further characterized in that at least 100 CpG positions in different sequence context are analyzed simultaneously.  
     
     
         31 . The method according to  claim 1 , further characterized in that at least 500 CpG positions in different sequence context are analyzed simultaneously.  
     
     
         32 . The method according to  claim 1 , further characterized in that at least 1000 CpG positions in different sequence context are analyzed simultaneously.  
     
     
         33 . The method according to  claim 1 , whereby the genomic DNA sample has been obtained from cell lines, blood, sputum, stool, urine, cerebrospinal fluid, tissue embedded in paraffin, for example, tissue from eyes, intestine, kidney, brain, heart, prostate, lung, breast or liver, histological slides or all other possible combinations thereof.  
     
     
         34 . Use of a method according to  claim 1  for the diagnosis and/or prognosis of adverse events for patients or individuals, whereby these adverse events belong to at least one of the following categories: undesired drug interactions; cancer diseases; CNS malfunctions, damage or disease; symptoms of aggression or behavioral disturbances; clinical, psychological and social consequences of brain damage; psychotic disturbances and personality disorders; dementia and/or associated syndromes; cardiovascular disease, malfunction and damage; malfunction, damage or disease of the gastrointestinal tract; malfunction, damage or disease of the respiratory system; lesion, inflammation, infection, immunity and/or convalescence; malfunction, damage or disease of the body as an abnormality in the development process; malfunction, damage or disease of the skin, the muscles, the connective tissue or the bones; endocrine and metabolic malfunction, damage or disease; headaches or sexual malfunctions.  
     
     
         35 . Use of a method according to  claim 1  for the differentiation of cell types or tissues of for investigation of cell differentiation.  
     
     
         36 . A kit, comprising a reagent containing bisulfite, primer oligonucleotides for preparing the amplified products and/or preferably oligonucleotides wherein the labels are fluorescent labels, immobilized on a solid phase, as well as instructions for conducting the method of  claim 1 .  
     
     
         37 . Nucleic acids comprising a sequence segment at least 18 bases long of a chemically pretreated DNA according to one of the sequences Seq. ID 1 to Seq. ID 40712.  
     
     
         38 . An oligomer (oligonucleotide or peptide nucleic acid (PNA) oligomer) for the detection of the cytosine methylation state in chemically pretreated DNA, each containing at least one base sequence with a length of at least 9 nucleotides, which hybridizes to a chemically pretreated DNA (Seq. ID 1 to Seq. ID 40712).  
     
     
         39 . The oligomer according to  claim 38 , wherein the base sequence contains at least one CpG dinucleotide.  
     
     
         40 . The oligomer according to  claim 39 , further characterized in that the cytosine of the CpG dinucleotide is found in approximately the middle third of the oligomer.  
     
     
         41 . A set of oligomers according to  claim 39 , comprising at least one oligomer for at least one of the CpG dinucleotides of one of the sequences of Seq. ID 1 to Seq. ID 40712.  
     
     
         42 . The set of oligomers according to  claim 41  containing at least one oligomer for each of the CpG dinucleotides of one of the sequences of Seq. ID 1 to Seq. ID 40712.  
     
     
         43 . A set of at least two nucleic acids, which are utilized as primer oligonucleotides for the amplification according to  claim 1  of at least one of the sequences Seq. ID 1 to Seq. ID 40712 or segments thereof.  
     
     
         44 . The set of oligonucleotides according to  claim 43 , further characterized in that at least one oligonucleotide is bound to a solid phase.  
     
     
         45 . A set of oligomer probes for the detection of the cytosine methylation state and/or of single nucleotide polymorphisms (SNPs) in chemically pretreated genomic DNA according to one of the sequences Seq. ID 1 to Seq. ID 40712, containing at least ten of the oligomers according to one of  claims 38  to  40 .  
     
     
         46 . A method for the production of an arrangement of different oligomers (an array) fixed on a support material for the analysis of disorders related to the methylation state of the CpG dinucleotides of one of the sequences Seq. ID 1 to Seq. ID 40712, in which at least one oligomer according to one of  claims 2  to  4  is coupled to a solid phase.  
     
     
         47 . An arrangement of different oligomers (an array) according to one of  claims 38  to  40 , which is bound to a solid phase.  
     
     
         48 . The array of different oligonucleotide and/or PNA oligomer sequences according to  claim 47 , further characterized in that these are arranged on a planar solid phase in the form of a rectangular or hexagonal grid.  
     
     
         49 . The array according to  claim 47 , further characterized in that the solid phase surface is comprised of silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver, or gold.  
     
     
         50 . A DNA and/or PNA array for the analysis of disorders related to the methylation state of genes, which contains at least one nucleic acid according to one of  claims 38  to  40 .