Method for Enriching Methylated CpG Sequences

Abstract

Compositions and methods are provided for facilitating the enrichment of single-stranded DNA containing methylated CpG in a mixture containing methylated and unmethylated DNA. The compositions relate to methylation-binding protein domains that selectively bind to methylated single strand DNA. In embodiments of the invention, the methylated DNA is eluted in 0.4M-0.6M NaCl while the unmethylated single strand DNA is eluted in less than 0.4M salt. The ability to readily enrich for methylated DNA permits high throughput sequencing of the methylated DNA and identification of abnormal methylation patterns associated with disease.

1 .- 16 . (canceled) 
     
     
         17 . A composition comprising:
 a first polypeptide comprising a sequence having at least 90% amino acid sequence homology with SEQ ID NO:3; and   a mixture containing methylated and unmethylated polynucleotides, wherein the polynucleotides are single-stranded.   
     
     
         18 . The composition of  claim 17 , further comprising a second polypeptide fused to the first polypeptide. 
     
     
         19 . The composition of  claim 17 , wherein the first polypeptide is immobilized on a solid substrate. 
     
     
         20 . The composition of  claim 18 , wherein the second polypeptide is a substrate-binding domain. 
     
     
         21 . The composition of  claim 20 , wherein the second polypeptide is maltose-binding protein. 
     
     
         22 . The composition of  claim 17 , wherein the first polypeptide is selected from the group consisting of: human UHRF1 and mouse NP95 SRA. 
     
     
         23 . The composition of  claim 17 , further comprising a low concentration of salt. 
     
     
         24 . The composition of  claim 23 , wherein a low concentration of the salt is less than 0.4 M salt. 
     
     
         25 . The composition of  claim 17 , further comprising salt at a concentration of 0.4 M-0.6 M salt. 
     
     
         26 . The composition of  claim 25 , wherein the salt is NaCl. 
     
     
         27 . The composition of  claim 17 , wherein the methylated polynucleotides contains hemi-methylated CpG. 
     
     
         28 . A method, comprising:
 (a) comparing the methylation pattern for selected polynucleotide sequences in both pre-identified immortalized eukaryotic cells and non-immortalized eukaryotic cells by differential binding of methylated polynucleotides to the first polypeptide of  claim 17 ;   (b) determining the presence of abnormal methylation patterns associated with alteration of tumor suppressor function; and   (c) utilizing the abnormal methylation patterns as a diagnostic tool for determining whether any eukaryotic cells in a sample are immortalized.   
     
     
         29 . The method according to  28 , wherein the methylated polynucleotide contains hemi-methylated CpG. 
     
     
         30 . The method according to  claim 28 , wherein step (a) further comprises forming single-stranded DNA for differential binding of the hemi-methylated CpG-containing polynucleotide.