Preservation of information related to genomic DNA methylation

• US 8,895,268 B2
• Filed: 09/06/2013
• Issued: 11/25/2014
• 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:
  US14/020,241
• Publication Number:
  US 20140038851A1
• Time in Patent Office: 445 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. An isolated nucleic acid pair comprising:

a template nucleic acid comprising a cytosine residue;

a complementary copy of said template nucleic acid having every cytosine replaced by a bisulfite-resistant cytosine analog; and

a tag capable of identifying said template nucleic acid and said complementary copy of said template nucleic acid as members of said nucleic acid pair, wherein said template nucleic acid and said complementary copy of said template nucleic acid are coupled to said tag.

2. The nucleic acid pair of claim 1, wherein said template nucleic acid is DNA.

3. The nucleic acid pair of claim 1, wherein said template nucleic acid is single-stranded.

4. The nucleic acid pair of claim 2, wherein said template nucleic acid is single-stranded and said complementary copy is single-stranded.

5. The nucleic acid pair of claim 1, wherein the bisulfite-resistant cytosine analog is 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.

6. The nucleic acid pair of claim 1, wherein the bisulfite-resistant cytosine analog is 5-methyl cytosine.

7. The nucleic acid pair of claim 4, wherein said tag is a molecule disposed between said template nucleic acid and said complementary copy.

8. The nucleic acid pair of claim 5, wherein said molecule comprises an oligonucleotide comprising a hairpin loop.

9. The nucleic acid pair of claim 4, wherein said tag comprises a first and second oligonucleotide comprising an identical nucleotide sequence, wherein said first oligonucleotide is coupled to said template nucleic acid and said second oligonucleotide is coupled to said complementary copy.

10. The nucleic acid pair of claim 4, wherein said tag comprises a first and second oligonucleotide comprising complementary nucleotide sequence, wherein said first oligonucleotide is coupled to said template nucleic acid and said second oligonucleotide is coupled to said complementary copy.

11. An isolated vector comprising the nucleic acid pair of claim 1.

12. A population of isolated nucleic acid pairs, comprising:

template nucleic acids and complementary copies of said template nucleic acids attached to each other as covalent pairs via a nucleic acid loop,

wherein said template nucleic acids each comprise at least one cytosine residue,

wherein every cytosine of said complementary copies is replaced by a bisulfite-resistant cytosine analog; and

wherein different nucleic acid pairs in said population have different sequences for said template nucleic acids and the same sequence for said nucleic acid loop.

13. The population of nucleic acid pairs of claim 12, wherein said template nucleic acids are DNA.

14. The population of nucleic acid pairs of claim 12, wherein said template nucleic acids are single-stranded.

15. The population of nucleic acid pairs of claim 13, wherein said template nucleic acids are single-stranded and said complementary copies are single-stranded.

16. The population of nucleic acid pairs of claim 12, wherein the bisulfite-resistant cytosine analog is 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.

17. The population of nucleic acid pairs of claim 12, wherein the bisulfite-resistant cytosine analog is 5-methyl cytosine.

18. An array comprising:

a solid support with a plurality of sites,

a bisulfite-converted template nucleic acid, and

a non-converted complementary copy of said template nucleic acid,

wherein said template nucleic acid is coupled to at least one of said plurality of sites and said non-converted complementary copy is coupled to at least one of said plurality of sites.

19. The array of claim 18, wherein said template nucleic acid is annealed to at least one of said plurality of sites and said non-converted complementary copy is annealed to at least one of said plurality of sites.

20. The array of claim 18, wherein said template nucleic acid and said non-converted complementary copy are annealed to the same site.