Massive parallel method for decoding DNA and RNA

• US 7,635,578 B2
• Filed: 08/20/2007
• Issued: 12/22/2009
• 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, Edwards, John Robert, Li, Zengmin, Itagaki, Yasuhiro
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US11/894,808
• Publication Number:
  US 20080199868A1
• Time in Patent Office: 855 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.3, 536/25.3, 536/26.6
• US Class Current: 435/91.1
• 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 method of detecting a fluorescently labeled nucleotide incorporated into a DNA coupled to a phosphine moiety, comprising:

a) reacting a DNA comprising an azido group with a compound comprising a phosphine moiety to produce the DNA coupled to the phosphine moiety;

b) contacting the DNA coupled to the phosphine moiety of step a) with a polymerase and a fluorescently labeled nucleotide so as to incorporate the fluorescently labeled nucleotide into the DNA coupled to the phosphine moiety; and

c) detecting the fluorescently labeled nucleotide incorporated into the DNA coupled to the phosphine moiety.

2. The method of claim 1, wherein the DNA comprising an azido group is a DNA template capable of self-priming in the presence of the polymerase thereby permitting the fluorescently labeled nucleotide to be incorporated into the DNA coupled to the phosphine moiety prior to step c).

3. The method of claim 1, wherein the DNA comprising an azido group is not capable of self-priming and wherein a primer is provided in step b) such that the fluorescently labeled nucleotide is incorporated into the primer prior to step c).

4. The method of claim 1, wherein the azido group is reacted with the phosphine moiety in step a) in an aqueous solution.

5. The method of claim 1, wherein the phosphine moiety is a triarylphosphine.

6. The method of claim 5, wherein the triarylphosphine is on a surface.

7. The method of claim 6, wherein the reaction of the azido group with the triarylphosphine results in attachment of the DNA to the surface.

8. A method of detecting a fluorescently labeled nucleotide incorporated into a DNA fragment coupled to a phosphine moiety, comprising:

a) reacting a DNA fragment having an azido group attached thereto through a linker with a compound comprising a phosphine moiety to produce the DNA fragment coupled to the phosphine moiety;

b) contacting the DNA fragment coupled to the phosphine moiety of step a) with a polymerase and a fluorescently labeled nucleotide so as to thereby incorporate the fluorescently labeled nucleotide into the DNA fragment coupled to the phosphine moiety; and

c) detecting the fluorescently labeled nucleotide incorporated into the DNA fragment coupled to the phosphine moiety.

9. The method of claim 8, wherein the DNA fragment is a DNA template capable of self-priming in the presence of the polymerase such that the fluorescently labeled nucleotide is incorporated into the DNA fragment prior to step c).

10. The method of claim 8, wherein the DNA fragment is not capable of self-priming and a primer is provided in step b) such that the fluorescently labeled nucleotide is incorporated into the primer prior to step c).

11. The method of claim 8, wherein the azido group is reacted with the phosphine moiety in step a) in an aqueous solution.

12. The method of claim 8, wherein the phosphine moiety is a triarylphosphine.

13. The method of claim 12, wherein the triarylphosphine is on a surface.

14. The method of claim 13, wherein the reaction of the azido group with the triarylphosphine results in the attachment of the DNA fragment to the surface.

15. A method comprising:

a) providing a nucleic acid template hybridized to a primer, the primer comprising an azido group; and

b) reacting said azido group with a phosphine.

16. A complex comprising a primer hybridized to a nucleic acid template, the primer comprising an azido group, and the nucleic acid template derived from genomic nucleic acid.

17. The complex of claim 16 further comprising a polymerase, wherein the primer comprises a deoxribonucleotide and the azido group is attached to a deoxyribose of the deoxyribonucleotide.

18. The complex of claim 17, wherein the azido group is attached to the deoxyribose at a 5′

position.

19. The complex of claim 17, wherein the azido group is attached to the deoxyribose via a linker.

20. The complex of claim 17 further comprising a fluorescently labeled nucleotide.

21. The complex of claim 20, wherein the nucleotide is a deaza analogue.

22. The complex of claim 20, wherein the fluorescently labeled nucleotide comprises a fluorescent label attached to the nucleotide through a cleavable linkage between the label and the base of the nucleotide.