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.

What is claimed is: 
     
         1 . An isolated first polypeptide comprising a sequence having at least 90% amino acid sequence homology with SEQ ID NO:3 and capable of binding single-stranded methylated polynucleotides. 
     
     
         2 . The isolated first polypeptide according to  claim 1 , further comprising a second polypeptide fused to the first polypeptide. 
     
     
         3 . The isolated first polypeptide according to  claim 1 , wherein the first polypeptide is immobilized on a solid substrate. 
     
     
         4 . The isolated first polypeptide according to  claim 2 , wherein the second polypeptide is a substrate-binding domain. 
     
     
         5 . The isolated first polypeptide according to  claim 4 , wherein the second polypeptide is maltose-binding protein. 
     
     
         6 . The isolated first polypeptide according to  claim 1 , capable of binding to a methylated CpG in a single-stranded polynucleotide. 
     
     
         7 . The isolated first polypeptide according to  claim 1 , selected from the group consisting of: human UHRF1 and mouse NP95 SRA. 
     
     
         8 . The use of the polypeptide comprising a sequence having at least 90% amino acid sequence homology with SEQ ID NO:3 for differentially binding a single-stranded methylated polynucleotide in a mixture of polynucleotides. 
     
     
         9 . The use of a polypeptide according to  claim 8 , wherein differential binding occurs in a low salt solution. 
     
     
         10 . A method for enriching for CpG methylated polynucleotides from a mixture containing methylated and unmethylated polynucleotides, comprising:
 allowing the polynucleotides in the mixture to bind to the first polypeptide described in  claim 1 ;   eluting the unmethylated polynucleotide from the isolated polypeptide in a solution containing a low concentration of a salt; and   eluting the methylated polynucleotide from the isolated polypeptide in a solution containing a high concentration of a salt.   
     
     
         11 . A method according to  claim 10 , wherein a low concentration of the salt is less than 0.4 M salt. 
     
     
         12 . A method according to  claim 10 , wherein a high concentration of the salt is 0.4 M-0.6 M salt. 
     
     
         13 . A method according to  claim 11  or  claim 12 , wherein the salt is NaCl. 
     
     
         14 . 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 1 ;   (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.   
     
     
         15 . The method according to  14 , wherein the methylated polynucleotide contains hemi-methylated CpG. 
     
     
         16 . The method according to  claim 15 , wherein step (a) further comprises forming single-stranded DNA for differential binding of the hemi-methylated CpG-containing polynucleotide. 
     
     
         17 . The use of the polypeptide according to  claim 8 , wherein the methylated polynucleotide contains methylated CpG for binding to the polypeptide, and the polypeptide is immobilized on a solid substrate. 
     
     
         28 . The use of the polypeptide according to  claim 9 , wherein the polypeptide is a first polypeptide, the first polypeptide being immobilized on a solid substrate through a second polypeptide fused to the first-polypeptide.