Methylation-sensitive restriction enzyme endonuclease method of whole genome methylation analysis
AssigneeIllumina, Inc.
InventorKevin Gunderson
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Abstract
The invention provides methods of identifying a plurality of reactive recognition sites for a restriction endonuclease in genomic DNA. In particular embodiments, the methods can be used to identify methylation state of a plurality of CpG target sites in genomic DNA. The method can include steps of treating genomic DNA with a restriction endonuclease, thereby producing genomic DNA fragments; ligating the fragments, thereby forming a concatenated DNA; and identifying sequence portions of the concatenated DNA that are re-ordered compared to the genomic DNA.
Claims19
A method of identifying a plurality of reactive recognition sites for a restriction endonuclease in genomic DNA, comprising
- (a) providing an isolated native genomic DNA comprising a first sequence,
- wherein said genomic DNA comprises a plurality of reactive recognition sites for a restriction endonuclease;
- (b) cleaving said genomic DNA at said recognition sites with said restriction endonuclease, thereby producing genomic DNA fragments comprising portions of said first sequence;
- (c) ligating said fragments, thereby forming a concatenated DNA comprising said portions in a second sequence, wherein said portions are re-ordered in said second sequence compared to said first sequence;
- and
- (d) identifying a plurality of said portions that are re-ordered, thereby identifying a plurality of reactive recognition sites for said restriction endonuclease in said genomic DNA.
A method of identifying a reactivity state of a plurality of recognition sites for a restriction endonuclease in genomic DNA, comprising
- (a) providing an isolated native genomic DNA comprising a first sequence,
- wherein said genomic DNA comprises a plurality of recognition sites for a restriction endonuclease, said recognition sites having recognition sequences in said first sequence,
- wherein said recognition sites comprise a first reactivity state or second reactivity state,
- wherein said genomic DNA comprises portions of said first sequence that are adjacent to said recognition sequences;
- (b) cleaving said genomic DNA at said recognition sites with said restriction endonuclease, thereby producing genomic DNA fragments comprising said portions of said first sequence;
- (c) ligating said fragments, thereby forming a concatenated DNA comprising a second sequence, wherein said portions are re-ordered in said second sequence compared to said first sequence if said recognition sites are in said first reactivity state and wherein said portions are ordered the same in said second sequence compared to said first sequence if said recognition sites are in said second reactivity state;
- and
- (d) identifying the order for a plurality of said portions, thereby identifying said reactivity state of said plurality of recognition sites for said restriction endonuclease in said genomic DNA.
A method of identifying methylation state of a plurality of CpG target sites in genomic DNA, comprising
- (a) providing an isolated native genomic DNA comprising a first sequence,
- wherein said genomic DNA comprises CpG target sites at a plurality of different locations,
- wherein said CpG target sites comprise a cytosine having a 5-methyl moiety or a 5-hydrogen moiety,
- wherein said genomic DNA comprises portions that are adjacent to said CpG target sites;
- (b) cleaving said genomic DNA at said CpG target sites that comprise said 5-methyl cytosine, thereby producing genomic DNA fragments comprising said portions;
- (c) ligating said fragments, thereby forming a concatenated DNA comprising said portions in a second sequence, wherein said portions are re-ordered in said second sequence compared to said first sequence if said CpG target sites comprise cytosine having said 5-methyl moiety and wherein said portions are ordered the same in said second sequence compared to said first sequence if said CpG target sites comprise cytosine having said 5-hydrogen moiety;
- and
- (d) identifying the order for a plurality of said portions, thereby identifying methylation state of said plurality of CpG target sites in said genomic DNA.
Dependencies are inferred from the claim text.