Preservation of information related to genomic DNA methylation

• US 8,541,207 B2
• Filed: 10/21/2009
• Issued: 09/24/2013
• Est. Priority Date: 10/22/2008
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Kester, Henri A.
• Primary Examiner(s): WILDER, CYNTHIA B
• Application Number:
  US13/125,419
• Publication Number:
  US 20110201524A1
• Time in Patent Office: 1,434 Days
• Field of Search: 435/91.2, 435/6.1
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2535/119 Double strand sequencing
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

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

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.

2. 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.

3. The method of claim 1, wherein said oligonucleotide primer is capable of forming a hairpin loop.

4. The method of claim 1, wherein said complementary copy is covalently coupled to said template nucleic acid.

5. The method of claim 4, wherein said oligonucleotide primer is ligated to said template nucleic acid prior to said generating step.

6. The method of claim 1, further comprising the step of ligating a second oligonucleotide primer to said complementary copy prior to bisulfite treatment.

7. 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.

8. 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.

9. The method of claim 1, further comprising pairing said non-converted complementary copy and said bisulfite-converted template nucleic acid.

10. 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.

11. 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.

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.

13. The method of claim 12, wherein said plurality of nucleic acids comprises greater than 1,000 nucleic acids having different sequences.

14. 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.

15. 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.

16. The method of claim 12, wherein said oligonucleotide primer is capable of forming a hairpin loop.

17. The method of claim 12, wherein said complementary copies are covalently coupled to said different template nucleic acids.

18. The method of claim 17, wherein said oligonucleotide primer is ligated to said template nucleic acid prior to said generating step.

19. 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.

20. 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.

21. 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.

22. The method of claim 12, further comprising pairing each of said non-converted complementary copies with its corresponding bisulfite-converted template nucleic acid.

23. 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.

24. 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.