# US 8,541,207 B2 · Preservation of information related to genomic DNA methylation > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,541,207 B2 - Document ID: US8541207B2 - Status: Granted - Kind: B2 - Application number: 13/125,419 - Filed: 2009-10-21 - Published: 2011-08-18 - Granted: 2013-09-24 - Earliest claimed: 2008-10-22 - Assignees: Illumina, Inc. - Applicants: Henri A. Kester - Inventors: Henri A. Kester - Web page: https://patentgrep.com/patent/US8541207B2 ## CPC - C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation - C12Q 1/6855: Ligating adaptors - C12Q 1/6858: Allele-specific amplification - C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes - C12Q 2600/156: Polymorphic or mutational markers - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract The present invention relates to compositions, methods and systems for analyzing the methylation state of nucleic acids. Some embodiments relate to a compositions, methods and systems for analyzing the methylation state of DNA with a gene array. ## Claims ### Claim 1 A method of sequencing nucleic acid comprising deaminated cytosine, said method comprising: providing a sample comprising a template nucleic acid; generating a complementary copy of said template nucleic acid, said generating being directed by an oligonucleotide primer using a nucleic acid polymerase in the presence of a bisulfite-resistant cytosine analog, wherein said generating produces a complementary copy of said template nucleic acid such that cytosine residues in said complementary copy are bisulfite-resistant; subjecting said template nucleic acid and said complementary copy to bisulfite treatment to convert unmethylated cytosine residues in said template nucleic acid into uracil residues, resulting in a bisulfite-converted template nucleic acid and a non-converted complementary copy; and determining the nucleotide sequence of said bisulfite-converted template nucleic acid and said non-converted complementary copy. ### Claim 2 (depends on 1) The method of claim 1, wherein the bisulfite-resistant cytosine analog is selected from the group consisting of: 5-ethyl dCTP, 5-methyl dCTP, 5-fluoro dCTP, 5-bromo dCTP, 5-iodo dCTP, 5-chloro dCTP, 5-trifluoromethyl dCTP, and 5-aza dCTP. ### Claim 3 (depends on 1) The method of claim 1, wherein said oligonucleotide primer is capable of forming a hairpin loop. ### Claim 4 (depends on 1) The method of claim 1, wherein said complementary copy is covalently coupled to said template nucleic acid. ### Claim 5 (depends on 4) The method of claim 4, wherein said oligonucleotide primer is ligated to said template nucleic acid prior to said generating step. ### Claim 6 (depends on 1) The method of claim 1, further comprising the step of ligating a second oligonucleotide primer to said complementary copy prior to bisulfite treatment. ### Claim 7 (depends on 1) The method of claim 1, wherein said oligonucleotide primer is covalently coupled to said complementary copy prior to bisulfite treatment, but not to said template nucleic acid. ### Claim 8 (depends on 1) The method of claim 1, wherein said template nucleic acid is covalently coupled to a partner oligonucleotide, said oligonucleotide primer and said partner oligonucleotide comprising a unique tag sufficient to identify said template nucleic acid and said complementary copy. ### Claim 9 (depends on 1) The method of claim 1, further comprising pairing said non-converted complementary copy and said bisulfite-converted template nucleic acid. ### Claim 10 (depends on 9) The method of claim 9, wherein said pairing is accomplished via a physical tether between said complementary copy and said bisulfite-converted template nucleic acid. ### Claim 11 (depends on 9) The method of claim 9, wherein said pairing is accomplished via tag molecules which identify said complementary copy and said bisulfite-converted template nucleic acid as members of a nucleic acid pair. ### Claim 12 A method of identifying methylated cytosines in a plurality of nucleic acids, said method comprising: providing a sample comprising a plurality of template nucleic acids; generating complementary copies of said template nucleic acids, said generating being directed by an oligonucleotide primer using a nucleic acid polymerase in the presence of a bisulfite-resistant cytosine analog, wherein said generating produces a complementary copy of each of said template nucleic acids such that cytosine residues in each of said complementary copy are bisulfite-resistant, and wherein each complementary copy is coupled to one of said template nucleic acids; subjecting said template nucleic acids and said complementary copies to bisulfite treatment to convert unmethylated cytosine residues in said template nucleic acids into uracil residues, resulting in each bisulfite-converted template nucleic acid being coupled to a non-converted complementary copy; determining the nucleotide sequence of said bisulfite-converted template nucleic acids and said non-converted complementary copies; and comparing said nucleotide sequence of said bisulfite-converted template nucleic acids to the nucleotide sequence of said non-converted complementary copies for each of said bisulfite-converted template nucleic acids coupled to non-converted complementary copies, thereby determining the methylation status of the template nucleic acids prior to bisulfite conversion. ### Claim 13 (depends on 12) The method of claim 12, wherein said plurality of nucleic acids comprises greater than 1,000 nucleic acids having different sequences. ### Claim 14 (depends on 12) The method of claim 12, wherein said different templates comprise a universal priming site and the same oligonucleotide primer sequence is used to generate complementary copies of said different template nucleic acids. ### Claim 15 (depends on 12) The method of claim 12, wherein the bisulfite-resistant cytosine analog is selected from the group consisting of: 5-ethyl dCTP, 5-methyl dCTP, 5-fluoro dCTP, 5-bromo dCTP, 5-iodo dCTP, 5-chloro dCTP, 5-trifluoromethyl dCTP, and 5-aza dCTP. ### Claim 16 (depends on 12) The method of claim 12, wherein said oligonucleotide primer is capable of forming a hairpin loop. ### Claim 17 (depends on 12) The method of claim 12, wherein said complementary copies are covalently coupled to said different template nucleic acids. ### Claim 18 (depends on 17) The method of claim 17, wherein said oligonucleotide primer is ligated to said template nucleic acid prior to said generating step. ### Claim 19 (depends on 12) The method of claim 12, further comprising the step of ligating a second oligonucleotide primer to each of said complementary copies prior to bisulfite treatment. ### Claim 20 (depends on 12) The method of claim 12, wherein each said oligonucleotide primer is covalently coupled to each said complementary copy prior to bisulfite treatment, but not to each said template nucleic acids. ### Claim 21 (depends on 20) The method of claim 20, wherein each said template nucleic acid is covalently coupled to a partner oligonucleotide, said oligonucleotide primer and said partner oligonucleotide comprising a unique tag sufficient to identify each said template nucleic acid and each said complementary copy. ### Claim 22 (depends on 12) The method of claim 12, further comprising pairing each of said non-converted complementary copies with its corresponding bisulfite-converted template nucleic acid. ### Claim 23 (depends on 22) The method of claim 22, wherein said pairing is accomplished via a physical tether between each said complementary copy and each corresponding bisulfite-converted template nucleic acid. ### Claim 24 (depends on 22) The method of claim 22, wherein said pairing is accomplished via tag molecules which identify each said complementary copy and each corresponding bisulfite-converted template nucleic acid as members of a nucleic acid pair.