SELECTIVE ENRICHMENT OF CpG ISLANDS

Abstract

The present invention provides compositions and methods for selectively enriching genomic CpG island (CGI)- and other epigenetically informative CG-rich polynucleotide targets. The method involves co-incubation of denatured or partially denatured polynucleotide fragments containing the CGI- or CG-targeted region(s) of interest with an oligonucleotide capture pool collectively designed to selectively target CGIs. The oligonucleotide capture pool includes a plurality of different oligonucleotides, each oligonucleotide coupled to a capture tag, whereby the oligonucleotide includes a CpG target sequence restricted to 4 to 10 bases. Upon binding, capture oligonucleotides bound to the target fragments are enriched by separating the bound fragments from the unbound fragments. The enriched fragments may be subjected to further analyses, including bisulfite sequencing to generate a methylation profile at the single nucleotide level.

1 - 26 . (canceled) 
     
     
         27 . An oligonucleotide capture pool configured to collectively and selectively enrich for CpG island-containing DNA fragments present in a fragmented pool of genomic DNA fragments, the oligonucleotide capture pool comprising a plurality of different capture oligonucleotides, each capture oligonucleotide in said plurality coupled to a capture tag and comprising a CpG target sequence L bases in length, wherein L is between 4 and 10, wherein at least 60% of the collective bases in the CpG target sequences of the capture pool are cytosine or guanine. 
     
     
         28 . The oligonucleotide capture pool of  claim 27 , wherein the CpG target sequence of each capture oligonucleotide in the capture pool is between 5 to 8 bases in length. 
     
     
         29 . The oligonucleotide capture pool of  claim 27 , wherein the collective percentage of cytosine and guanine bases in the capture oligonucleotide in the capture pool is selected from the group consisting of 70%, 80%, 90%, 95%, and 100%. 
     
     
         30 . The oligonucleotide capture pool of  claim 27 , wherein the number (P) of different capture oligonucleotides in the capture pool is defined by the formula P=2 L , wherein L is the number of bases of the CpG target sequence, wherein L=4, 5, 6, 7, 8, 9, or 10. 
     
     
         31 . The oligonucleotide capture pool of  claim 27 , wherein each capture oligonucleotide in the capture pool is cytosine or guanine, wherein the 3′-terminal dinucleotide in each capture oligonucleotide is CG, and wherein the CpG target sequence of each capture oligonucleotide is between 5 to 8 bases in length. 
     
     
         32 . The oligonucleotide capture pool of  claim 27 , wherein each capture oligonucleotide in the capture pool comprises a consecutive run of cytosine or guanine residues spanning the middle of the capture oligonucleotide sequence. 
     
     
         33 . The oligonucleotide capture pool of  claim 27 , wherein the capture oligonucleotides in the capture pool are designed in accordance with a progressive scanning analysis of CpG island DNA sequences in a given species genome, wherein the target sequences reflect their composition in the CpG island DNA sequences. 
     
     
         34 . The oligonucleotide capture pool of  claim 27 , wherein each capture oligonucleotide in the capture pool comprises at least one CG dinucleotide, wherein the cytosine nucleotide in the CG dinucleotide is positioned in the middle of the capture oligonucleotide or in a position immediately 5′ to the middle of the capture oligonucleotide. 
     
     
         35 . The oligonucleotide capture pool of  claim 27 , wherein each capture oligonucleotide in the capture pool contains a CG dinucleotide at the 3′-end. 
     
     
         36 . The oligonucleotide capture pool of  claim 27 , wherein each capture oligonucleotide in the capture pool comprises a cytosine or guanine residue, and wherein the scanning analysis selects GC-restricted oligonucleotide target sequences for the capture pool that represent the top 50%, the top 30%, or the top 10% of GC-restricted oligonucleotides L bases in length that are present in the CpG islands polynucleotide fragments. 
     
     
         37 . The oligonucleotide capture pool of  claim 27 , wherein each capture oligonucleotide in the capture pool includes at least one nucleotide comprising a member of the group consisting of DNA, RNA, LNA, and PNA. 
     
     
         38 . The oligonucleotide capture pool of  claim 27 , wherein the capture tag comprises a nanoparticle, biotin, or combination thereof. 
     
     
         39 . The oligonucleotide capture pool of  claim 27 , wherein each capture oligonucleotides in the capture pool comprises a spacer. 
     
     
         40 . The oligonucleotide capture pool of  claim 27 , wherein bound capture oligonucleotides are subjected to primer extension.