Methods of Identification Using Methylation of CPG

Abstract

The present invention relates to the materials and methods for the identification of methylated nucleotides in samples of genomic DNA. The present invention also relates to methods of diagnosis of specific conditions by identification of specific methylated nucleotides.

1 . A method of detecting methylated DNA at a specific locus in a sample, comprising:
 treating the sample with a methyl-active cleavage method that cleaves DNA at a consistent site in the presence of methylated DNA at the specific locus in the sample,   adding primers which compliment DNA at or near the specific locus,   subjecting the sample to a polymerase chain reaction and generating an amplification product when there is a successful methyl-active cleavage, but no amplification when there is no successful cleavage, and   detecting the presence of the amplification product, indicating the presence of methylated DNA at the specific locus in the sample.   
   
   
       2 . The method of  claim 1 , wherein the sample comprises genomic DNA. 
   
   
       3 . The method of  claim 1 , wherein the specific locus is a promoter region of a known gene. 
   
   
       4 . The method of  claim 3 , wherein the sample comes from a patient, and wherein the presence of methylated DNA at the promoter region of the known gene indicates the presence cancerous cells in the patient. 
   
   
       5 . The method of  claim 1 , wherein the methyl-active cleavage method that cleaves DNA at a consistent site in the presence of methylated DNA is a methyl-active restriction enzyme. 
   
   
       6 . The method of  claim 1 , wherein the methyl-active cleavage method that cleaves DNA at a consistent site in the presence of methylated DNA is treatment with 5-methyl deoxycytidine glycosylase/lyase. 
   
   
       7 . A method of detecting cancer in a patient by detecting methylated DNA at a specific locus in a sample from the patient, comprising:
 treating the sample with a methyl-active cleavage method that cleaves DNA at a consistent site in the presence of methylated DNA at the specific locus in the sample,   adding primers which compliment DNA at or near the specific locus,   subjecting the sample to a polymerase chain reaction and generating an amplification product when there is methylated DNA at the specific locus in the sample,   detecting the presence of the amplification product, indicating the presence of methylated DNA at the specific locus in the sample, and   detecting cancer in the patient from the presence of the amplification product.   
   
   
       8 . The method of  claim 7 , wherein the sample comprises genomic DNA. 
   
   
       9 . The method of  claim 7 , wherein the specific locus is a promoter region of a known gene. 
   
   
       10 . The method of  claim 7 , wherein the methyl-active cleavage method that cleaves DNA at a consistent site in the presence of methylated DNA is a methyl-active restriction enzyme. 
   
   
       11 . The method of  claim 7 , wherein the methyl-active cleavage method that cleaves DNA at a consistent site in the presence of methylated DNA is treatment with 5-methyl deoxycytidine glycosylase/lyase. 
   
   
       12 . A method of detecting cancer in a patient by detecting methylated DNA at specific locus in genomic DNA in a sample from the patient comprising
 treating the genomic DNA with a methyl-active restriction enzyme to create a cleavage product,   adding primers which compliment DNA at or near the specific locus,   subjecting the sample to a polymerase chain reaction to obtain an amplification product when there is methylated DNA at the specific locus,   detecting the presence of the amplification product which indicates the presence of methylated DNA at the specific locus, and   detecting cancer in a patient by detecting the presence of the amplification product.   
   
   
       13 . The method of  claim 12 , wherein the specific locus is a promoter region of a known gene. 
   
   
       14 . The method of  claim 12 , wherein the methyl-active restriction enzyme is  E. coli  McrBC. 
   
   
       15 . The method of  claim 12 , wherein the method further comprises the steps of
 creating blunt ends on the cleavage product,   ligating the ends of the cleavage product to create a closed circle of the cleavage product, and   wherein the primers are oriented such that the amplification product can only result from a closed ligated circle of the cleavage product.   
   
   
       16 . A method of detecting cancer in a patient by detecting methylated DNA at specific locus in genomic DNA in a sample from the patient comprising
 treating the genomic DNA with a methyl-active restriction enzyme to create a cleavage product,   creating blunt ends on the cleavage product,   ligating the ends of the cleavage product to create a closed circle of the cleavage product,   adding primers which compliment DNA at or near the specific locus, wherein the primers are oriented such that an amplification product can only result from a closed ligated circle of the cleavage product   subjecting the sample to a polymerase chain reaction to obtain an amplification product,   detecting the presence of the amplification product which indicates the presence of methylated DNA at the specific locus, and   detecting cancer in a patient by detecting the presence of the amplification product.