Preservation of information related to genomic DNA methylation

• US 10,174,372 B2
• Filed: 03/27/2017
• Issued: 01/08/2019
• 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:
  US15/469,811
• Publication Number:
  US 20170260581A1
• Time in Patent Office: 652 Days
• Field of Search: 435 912
• 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. An array comprising:

a) a solid support with a plurality of sites configured for single molecule detection; and

b) a plurality of nucleic acid pairs coupled to the plurality of sites such that different pairs are spatially resolved from each other on the solid support, wherein each nucleic acid pair comprises a template nucleic acid and a complementary copy of said template nucleic acid, the template nucleic acid and complementary copy being physically tethered by an oligonucleotide capable of forming a hairpin loop.

2. The array of claim 1, wherein the solid support comprises a material selected from the group consisting of:

plastic, ceramic, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, thoria sol, carbon graphite, titanium dioxide, sepharose, cellulose, nylon, cross-linked micelles and teflon.

3. The array of claim 1, wherein the orientation of the template nucleic acid and the complementary copy of the nucleic acid pair forms an imperfect inverted repeat.

4. The array of claim 1, wherein each nucleic acid pair comprises a free 3′

end and a free 5′

end.

5. The array of claim 4, wherein the free 3′

end comprises a 3′

adapter sequence.

6. The array of claim 5, wherein the 3′

adapter sequence comprises a sequence complementary to a capture probe.

7. The array of claim 4, wherein the free 5′

end comprises a 5′

adapter sequence.

8. The array of claim 7, wherein the 5′

adapter sequence comprises a sequence complementary to a capture probe.

9. The array of claim 1, wherein the template nucleic acid comprises a cytosine residue.

10. The array of claim 1, wherein the template nucleic acid comprises a bisulfate-converted polynucleotide.

11. The array of claim 10, wherein the bisulfite-converted polynucleotide comprises a uracil residue.

12. The array of claim 1, wherein the complementary copy of said template nucleic acid comprises a bisulfite-resistant polynucleotide.

13. The array of claim 12, wherein the bisulfite-resistant polynucleotide comprises a bisulfite-resistant cytosine analog selected from the group consisting of 5-ethyl cytosine, 5-methyl cytosine, 5-fluoro cytosine, 5-bromo cytosine, 5-iodo cytosine, 5-chloro cytosine, 5-trifluoromethyl cytosine, and 5-aza cytosine.

14. The array of claim 12, wherein the bisulfite-resistant cytosine analog is 5-methyl cytosine.

15. The array of claim 1, wherein the template nucleic acid is single-stranded.

16. The array of claim 1, wherein the complementary copy is single-stranded.

17. The array of claim 1, wherein the template nucleic acid comprises DNA.

18. An array comprising:

a) a solid support with a plurality of sites; and

b) a plurality of nucleic acid pairs coupled to the plurality of sites, wherein each nucleic acid pair comprises;

(i) a template nucleic acid comprising a cytosine residue,

(ii) a complementary copy of said template nucleic acid comprising a bisulfite-resistant cytosine analog, and

(iii) an oligonucleotide capable of forming a hairpin loop physically tethering the template nucleic acid and the complementary copy.

19. The array of claim 18, wherein the template nucleic acid is single-stranded.

20. The array of claim 18, wherein the plurality of sites comprises a plurality of sites configured for single molecule detection.