Massive parallel method for decoding DNA and RNA

• US 7,790,869 B2
• Filed: 06/05/2007
• Issued: 09/07/2010
• Est. Priority Date: 10/06/2000
• Status: Expired due to Fees

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Ju, Jingyue, Li, Zengmin, Edwards, John Robert, Itagaki, Yasuhiro
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US11/810,509
• Publication Number:
  US 20080319179A1
• Time in Patent Office: 1,190 Days
• Field of Search: 435/6, 536/23.1, 536/25.3, 536/26.6
• US Class Current: 536/23.1
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/14 Pyrrolo-pyrimidine radicals
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• 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 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures

Abstract

This invention provides methods for attaching a nucleic acid to a solid surface and for sequencing nucleic acid by detecting the identity of each nucleotide analogue after the nucleotide analogue is incorporated into a growing strand of DNA in a polymerase reaction. The invention also provides nucleotide analogues which comprise unique labels attached to the nucleotide analogue through a cleavable linker, and a cleavable chemical group to cap the —OH group at the 3′-position of the deoxyribose.

Claims

1. A nucleotide having a base that is attached to a detectable label through a cleavable linker, wherein the nucleotide has a deoxyribose comprising a cleavable chemical group capping the 3′

OH group of the deoxyribose and wherein both the cleavable linker and the capping cleavable chemical group are cleavable by light.

2. The nucleotide of claim 1, wherein the base is a purine or a pyrimidine.

3. The nucleotide of claim 2, wherein the base is a deazapurine.

4. The nucleotide of claim 1, wherein the nucleotide is a deoxyribonucleotide triphosphate.

5. The nucleotide of claim 1, wherein the detectable label is a fluorophore.

6. A nucleotide having a base that is linked to a detectable label via a cleavable linker, wherein the nucleotide has a deoxyribose sugar moiety comprising a protecting or capping group attached via the 3′

oxygen and the cleavable linker and the protecting or capping group are cleavable by light.

7. The nucleotide of claim 6, wherein the base is a purine or a pyrimidine.

8. The nucleotide of claim 7, wherein the base is a deazapurine.

9. The nucleotide of claim 6, that is a deoxyribonucleotide triphosphate.

10. The molecule of claim 6, wherein the detectable label is a fluorophore.

11. The molecule of claim 6, wherein the linker is photolabile or photocleavable.

12. A nucleotide having a base that is attached to a detectable label through a cleavable linker, wherein the nucleotide has a deoxyribose comprising a cleavable chemical group capping the 3′

OH group, wherein the cleavable linker is cleaved by a means selected from the group consisting of one or more of a physical means, a chemical means, a physical chemical means, heat, and light, and wherein the cleavable chemical group capping the 3′

OH group is cleaved by a means selected from the group consisting of one or more of a physical means, a chemical means, a physical chemical means, heat, and light.

13. The nucleotide of claim 12, wherein the cleavable linker is cleaved by chemical means, and wherein the cleavable chemical group capping the 3′

OH group is cleaved by chemical means.

14. The nucleotide of claim 12, wherein the cleavable linker is cleaved by light, and wherein the cleavable chemical group capping the 3′

OH group is cleaved by light.

15. The nucleotide of claim 12, wherein the base is a deazapurine.

16. The nucleotide of claim 15, wherein said deazapurine is selected from the group consisting of deaza-adenine and deaza-quanine, each comprising a unique label attached through a cleavable linker to a 7-position of deaza-adenine or deaza-guanine.

17. The nucleotide of claim 12, wherein the detectable label is a fluorophore.

18. The nucleotide of claim 12, wherein said cleavable chemical group comprises —

CH₂—

OCH₃.

19. The nucleotide of claim 12, wherein said cleavable chemical group comprises —

CH₂CH═

CH₂.

20. The nucleotide of claim 12, wherein said cleavable chemical group has a molecular weight of 300 Daltons or less.

21. The nucleotide of claim 12 wherein said nucleotide is an analog of adenosine, guanosine, cytosine, or thymidine.

22. The nucleotide of claim 21, wherein said nucleotide analog is incorporated in a primer.

23. The nucleotide of claim 22, wherein said primer is in a complex with a nucleic acid template.

24. The nucleotide of claim 23, wherein said template is immobilized on a solid surface.

25. The nucleotide of claim 23, wherein said template is part of array.

26. The nucleotide of claim 23, wherein said nucleic acid template is derived from genomic nucleic acid.

27. The nucleotide of claim 24, wherein said solid surface is a bead.

28. The nucleotide of claim 12, wherein said cleavable chemical group does not interfere with the recognition of the nucleotide by a polymerase.

29. The nucleotide of claim 23, wherein said complex further comprises a polymerase.

30. The nucleotide of claim 23, wherein the nucleic acid template is labeled with an azido-group via a polymerase chain reaction.

31. The nucleotide of claim 12, wherein the cleavable chemical group capping the 3′

OH group is a small chemical moiety.

32. The nucleotide of claim 31, wherein the cleavable chemical group capping the 3′

OH group comprises two carbon atoms or three carbon atoms.

33. The nucleotide of claim 24, wherein the template is attached to the solid surface via an attachment group.