Method for distinguishing 5-position methylation changes of cytosine bases and cytosine-to-thymine mutations and for detecting single nucleotide polymorphisms (SNPs) or point mutations in genomic DNA

Abstract

A method is described for distinguishing 5-position methylation changes of cytosine bases and cytosine-to-thymine mutations and for the detection of single nucleotide polymorphisms (SNPs) or point mutations in genomic DNA, in which: a) a genomic DNA sample is treated with sulfite or disulfite in such a way that all of the cytosine bases that are not methylated in the 5-position of the base are changed in such a way that a base is formed that is different in its base-pairing behavior, whereas the cytosines methylated at the 5-position remain unchanged, and b) an aliquot of the same genomic DNA sample is quantitatively methylated with Sss1 or another methyltransferase prior to the chemical treatment according to a) and c) both of the DNA samples treated in this way are investigated for the presence of cytosine by means of the same analytical method, and d) the cytosine positions that are determined are matched with a reference DNA sequence.

1 . A method for distinguishing 5-position methylation changes of cytosine bases and cytosine-to-thymine mutations and for the detection of single nucleotide polymorphisms (SNPs) or point mutations in genomic DNA, characterized in that: 
 a) a genomic DNA sample is treated with sulfite or disulfite in such a way that all of the cytosine bases that are unmethylated at the 5-position of the base are changed, so that a base different in its base-pairing behavior arises, whereas the cytosines methylated at the 5-position remain unchanged and    b) an aliquot of the same genomic DNA sample prior to the chemical treatement according to a) is methylated quantitatively with Sss1 or another methyltransferase and    c) both of the DNA samples treated in this way are investigated for the presence of cytosine by means of the same analytical method and    d) the determined cytosine positions are matched to a reference DNA sequence.    
     
     
         2 . The method according to  claim 1 , further characterized in that it is determined by means of comparing the individual cytosine positions of both samples treated in different ways with the reference sequence whether cytosine is not detectable at a specific position and whether this is due to the fact that cytosine is present in the unmethylated state in the genomic DNA or whether it is present but changed due to a mutation or a polymorphism and thus is not present in the genomic DNA.  
     
     
         3 . The method according to  claim 1 , further characterized in that the two DNA samples or parts of these DNA samples are amplified by a cyclical process, namely polymerase chain reaction or a comparable process prior to the detection of the cytosine base.  
     
     
         4 . The method according to  claim 3 , further characterized in that more than 10 different fragments of the treated genomic DNA are produced in one amplification batch.  
     
     
         5 . The method according to  claim 1 , further characterized in that those primers are used for the amplification of the genomic DNA samples, which contain so-called consensus sequences or such sequences important for gene regulation and which thus predominantly bind to regulating or coding sequences.  
     
     
         6 . The method according to  claim 1 , further characterized in that cytosine is detected in the specific context 5′-CpG-3′.  
     
     
         7 . The method according to  claim 1 , further characterized in that the DNA is provided with one or more detectable label(s) in the amplification by incorporation of nucleotide building blocks or oligonucleotides provided with a detectable label.  
     
     
         8 . The method according to  claim 7 , further characterized in that the label is detected by fluorescence or chemiluminescence.  
     
     
         9 . The method according to  claim 1 , further characterized in that cytosine is detected by means of hybridizing with oligomers specific for “sequence context-cytosine-sequence context”, which [oligomers] are attached in a defined arrangement onto one or more surfaces.  
     
     
         10 . The method according to  claim 9 , further characterized in that for each cytosine to be detected in its sequence-specific context, at least one oligomer complementary to the sequence context is attached onto the surface, which contains guanine complementary to the cytosine to be detected, and another oligomer, which contains the base that is complementary to the base to which unmethylated cytosines are converted by the chemical reaction at the site of the cytosine to be detected.  
     
     
         11 . The method according to  claim 9 , further characterized in that for cytosine positions to be detected, those oligomers are attached, which bind specifically each time to the methylated and unmethylated positions both on the plus strand as well as on the minus strand or/and hybridize specifically to the complementary strands that are formed by amplification.  
     
     
         12 . The method according to  claim 9 , further characterized in that additional oligomers are attached onto the surface, which [oligomers] bind specifically each time to the sequence “sequence context-thymine-sequence context” and/or which detect cytosine and the base that is formed by chemical treatment in the plus strand, the minus strand and the strands forming by amplification of the compelementary strands that arise.  
     
     
         13 . The method according to  claim 9 , further characterized in that signals that are specific for the methylated or unmethylated or mutated [positions] in the original genomic sample are detected by the oligomers at points of the surface(s).  
     
     
         14 . The method according to  claim 13 , further characterized in that the absolute degree of methylation and/or the homozygotic or heterozygotic status is determined by a comparison of the detected signals.  
     
     
         15 . The method according to  claim 1 , further characterized in that the amplified fragments of both samples are attached onto a surface and hybridized with sequence-specific oligomers provided with a detectable label on these surfaces.  
     
     
         16 . The method according to  claim 15 , further characterized in that the analysis of the hybridized sequence-specific oligomers is conducted by means of mass spectrometry and preferably with a MALDI mass spectrometer.  
     
     
         17 . The method according to  claim 15 , further characterized in that the analysis of the hybridized sequence-specific oligomers is conducted by means of fluorescence or chemiluminescence.  
     
     
         18 . The method according to  claim 1 , further characterized in that the detection of cytosine is made in the sequence context by a polymerase reaction, which is stopped specifically upon reaching a cytosine base in the template, and the lengths of the fragments that are formed are measured.  
     
     
         19 . The method according to  claim 18 , further characterized in that [for] the oligomers that are utilized for initiating the primer-dependant polymerase reaction, each different sequence is fixed at a different site onto a surface and the polymerase reaction is conducted on this surface.  
     
     
         20 . The method according to  claim 18 , further characterized in that the oligomers that are utilized for initiating the primer-dependent polymerase reaction, are stripped from the surface by a chemical reaction or by light.  
     
     
         21 . The method according to  claim 18 , further characterized in that for the termination of the polymerase reaction at the position of a cytosine or—in the counterstrand—of a guanine, a nucleotide building block is used, which permits a detection via a chemical modification, for example, by fluorescence, chemiluminescence or the binding of an antibody.  
     
     
         22 . The method according to  claim 18 , further characterized in that the termination at the position of a cytosine or—in the counterstrand—of a guanine is detected by means of a length measurement of the fragments that arise by gel electrophoresis, particularly capillary electrophoresis.  
     
     
         23 . The method according to  claim 18 , further characterized in that the length measurement of the fragments that form is conducted by mass-spectrometric analysis and preferably in a MALDI mass spectrometer.  
     
     
         24 . The method according to  claim 1 , further characterized in that the reference DNA sequence originates from a database, namely from the human genome project.  
     
     
         25 . A kit containing reference DNA and/or chemicals and other materials for conducting the bisulfite reaction and/or the amplification and/or a methyltransferase and/or documentation for conducting the method.