Highly sensitive method for the detection of cytosine methylation patterns

Abstract

The present invention concerns a method for the detection of cytosine methylation in DNA samples, wherein the following steps are conducted: (a) a genomic DNA sample, which comprises the DNA to be investigated and background DNA, is chemically treated in such a way that all of the unmethylated cytosine bases are converted to uracil, whereas the 5-methylcytosine bases remain unchanged; (b) the chemically treated DNA sample is amplified with the use of at least 1 primer oligonucleotide as well as a polymerase, whereby the DNA to be investigated is preferred as the template over the background DNA, and (c) the amplified products are analyzed and the methylation status in the DNA to be investigated is concluded from the presence of an amplified product and/or from the analysis of additional positions.

1 . A method for the detection of cytosine methylation in DNA samples, characterized in that the following steps are conducted:
 treating a genomic DNA sample, which comprises the DNA to be investigated as well as background DNA chemically in such a way that all of the unmethylated cytosine bases are converted to uracil, while the 5-methylcytosine bases remain unchanged;   amplifying the chemically treated DNA sample with the use of a polymerase and at least 2 primer oligonucleotides binding to positions wherein degree of methylation of said positions in the DNA to be investigated is different from that in the background DNA; and   analyzing the amplified products and the methylation status in the DNA to be investigated is concluded.   
     
     
         2 . The method according to  claim 1 , further characterized in that the sample DNA is obtained from serum or other body fluids of an individual. 
     
     
         3 . The method according to  claim 1 , further characterized in that the DNA samples are obtained from cell lines, blood, sputum, stool, urine, serum, cerebro-spinal fluid, tissue embedded in paraffin said tissue being selected from the group consisting of eyes, intestine, kidneys, brain, heart, prostate, lungs, breast or liver, histological slides and any combination thereof. 
     
     
         4 . The method according to  claim 1 , further characterized in that the chemical treatment is conducted with a bisulfite. 
     
     
         5 . (canceled) 
     
     
         6 . The method according to  claim 4 , further characterized in that a reagent denaturing the DNA duplex and/or a radical trap is present in the chemical treatment. 
     
     
         7 - 23 . (canceled) 
     
     
         24 . The method according to  claim 1 , further characterized in that the background DNA is present in 100 times the concentration of the DNA to be investigated. 
     
     
         25 . The method according to  claim 1 , further characterized in that the background DNA is present in 1000 times the concentration of the DNA to be investigated. 
     
     
         26 . The method according to  claim 1 , further characterized in that the analysis is conducted by means of hybridization to oligomer arrays. 
     
     
         27 . The method according to  claim 26 , further characterized in that the oligomers hybridize to the DNA to be analyzed over a 12-22 base long segment and they contain a CG, TG or CA dinucleotide. 
     
     
         28 . The method according to  claim 26 , further characterized in that the methylation status of more than 20 methylation positions of the DNA to be analyzed is detected in one experiment. 
     
     
         29 . The method according to  claim 26 , further characterized in that the methylation status of more than 60 methylation positions of the DNA to be analyzed is detected in one experiment. 
     
     
         30 . The method according to  claim 1 , further characterized in that the analysis is conducted by length measurement of the amplified DNA to be investigated, whereby methods for length measurement include gel electrophoresis, capillary gel electrophoresis, chromatography and mass spectrometry. 
     
     
         31 . The method according to  claim 1 , further characterized in that the analysis is conducted by sequencing. 
     
     
         32 . (canceled) 
     
     
         33 . The method according to  claim 1 , further characterized in that the presence of a disease or medical condition of the patient is concluded from the degree of methylation at the positions investigated. 
     
     
         34 . The method according to  claim 1 , further characterized in that the amplified products themselves are provided with a detectable label for detection. 
     
     
         35 . The method according to  claim 34 , further characterized in that the labels are fluorescent labels. 
     
     
         36 - 37 . (canceled) 
     
     
         38 . The method according to  claim 1 , further characterized in that one of the primers is bound to a solid phase in the amplification. 
     
     
         39 . (canceled) 
     
     
         40 . (canceled) 
     
     
         41 . (canceled) 
     
     
         42 . (canceled)